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Carter-Su C, Argetsinger LS, Svezhova N. 2022 Cannon lecture: an ode to signal transduction: how the growth hormone pathway revealed insight into height, malignancy, and obesity. Am J Physiol Endocrinol Metab 2023; 325:E425-E437. [PMID: 37672248 PMCID: PMC10874654 DOI: 10.1152/ajpendo.00265.2023] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 08/21/2023] [Accepted: 08/21/2023] [Indexed: 09/07/2023]
Abstract
Walter Cannon was a highly regarded American neurologist and physiologist with extremely broad interests. In the tradition of Cannon and his broad interests, we discuss our laboratory's multifaceted work in signal transduction over the past 40+ years. We show how our questioning of how growth hormone (GH) in the blood communicates with cells throughout the body to promote body growth and regulate body metabolism led to insight into not only body height but also important regulators of malignancy and body weight. Highlights include finding that 1) A critical initiating step in GH signal transduction is GH activating the GH receptor-associated tyrosine kinase JAK2; 2) GH activation of JAK2 leads to activation of a number of signaling proteins, including STAT transcription factors; 3) JAK2 is autophosphorylated on multiple tyrosines that regulate the activity of JAK2 and recruit signaling proteins to GH/GH receptor/JAK2 complexes; 4) Constitutively activated STAT proteins are associated with cancer; 5) GH activation of JAK2 recruits the adapter protein SH2B1 to GH/GH receptor/JAK2 complexes where it facilitates GH regulation of the actin cytoskeleton and motility; and 6) SH2B1 is recruited to other receptors in the brain, where it enhances satiety, most likely in part by regulating leptin action and neuronal connections of appetite-regulating neurons. These findings have led to increased understanding of how GH functions, as well as therapeutic interventions for certain cancer and obese individuals, thereby reinforcing the great importance of supporting basic research since one never knows ahead of time what important insight it can provide.
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Affiliation(s)
- Christin Carter-Su
- University of Michigan Medical School, Ann Arbor, Michigan, United States
| | | | - Nadezhda Svezhova
- University of Michigan Medical School, Ann Arbor, Michigan, United States
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LNK promotes the growth and metastasis of triple negative breast cancer via activating JAK/STAT3 and ERK1/2 pathway. Cancer Cell Int 2020; 20:124. [PMID: 32322171 PMCID: PMC7160949 DOI: 10.1186/s12935-020-01197-9] [Citation(s) in RCA: 8] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/11/2019] [Accepted: 03/31/2020] [Indexed: 11/17/2022] Open
Abstract
Background LNK adaptor protein is a crucial regulator of normal hematopoiesis, which down-regulates activated tyrosine kinases at the cell surface resulting in an antitumor effect. To date, little studies have examined activities of LNK in solid tumors except ovarian cancer. Methods Clinical tissue chips were obtained from 16 clinical patients after surgery. Western blotting assay and quantitative real time PCR was performed to measure the expression of LNK. We investigate the in vivo and vitro effect of LNK in Triple Negative Breast Cancer by using cell proliferation、migration assays and an in vivo murine xenograft model. Western blotting assay was performed to investigate the mechanism of LNK in triple negative breast cancer. Results We found that the levels of LNK expression were elevated in high grade triple-negative breast cancer through Clinical tissue chips. Remarkably, overexpression of LNK can promote breast cancer cell proliferation and migration in vivo and vitro, while silencing of LNK show the opposite phenomenon. We also found that LNK can promote breast cancer cell to proliferate and migrate via activating JAK/STAT3 and ERK1/2 pathway. Conclusions Our results suggest that the adaptor protein LNK acts as a positive signal transduction modulator in TNBC.
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Flores A, Argetsinger LS, Stadler LKJ, Malaga AE, Vander PB, DeSantis LC, Joe RM, Cline JM, Keogh JM, Henning E, Barroso I, Mendes de Oliveira E, Chandrashekar G, Clutter ES, Hu Y, Stuckey J, Farooqi IS, Myers MG, Carter-Su C. Crucial Role of the SH2B1 PH Domain for the Control of Energy Balance. Diabetes 2019; 68:2049-2062. [PMID: 31439647 PMCID: PMC6804625 DOI: 10.2337/db19-0608] [Citation(s) in RCA: 9] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 06/21/2019] [Accepted: 08/12/2019] [Indexed: 12/13/2022]
Abstract
Disruption of the adaptor protein SH2B1 (SH2-B, PSM) is associated with severe obesity, insulin resistance, and neurobehavioral abnormalities in mice and humans. Here, we identify 15 SH2B1 variants in severely obese children. Four obesity-associated human SH2B1 variants lie in the Pleckstrin homology (PH) domain, suggesting that the PH domain is essential for SH2B1's function. We generated a mouse model of a human variant in this domain (P322S). P322S/P322S mice exhibited substantial prenatal lethality. Examination of the P322S/+ metabolic phenotype revealed late-onset glucose intolerance. To circumvent P322S/P322S lethality, mice containing a two-amino acid deletion within the SH2B1 PH domain (ΔP317, R318 [ΔPR]) were studied. Mice homozygous for ΔPR were born at the expected Mendelian ratio and exhibited obesity plus insulin resistance and glucose intolerance beyond that attributable to their increased adiposity. These studies demonstrate that the PH domain plays a crucial role in how SH2B1 controls energy balance and glucose homeostasis.
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Affiliation(s)
- Anabel Flores
- Cell and Molecular Biology Graduate Program, University of Michigan, Ann Arbor, MI
| | - Lawrence S Argetsinger
- Department of Molecular and Integrative Physiology, University of Michigan, Ann Arbor, MI
| | - Lukas K J Stadler
- University of Cambridge Metabolic Research Laboratories and NIHR Cambridge Biomedical Research Centre, Wellcome Trust-MRC Institute of Metabolic Science, Addenbrooke's Hospital, Cambridge, U.K
| | - Alvaro E Malaga
- Department of Molecular and Integrative Physiology, University of Michigan, Ann Arbor, MI
| | - Paul B Vander
- Department of Molecular and Integrative Physiology, University of Michigan, Ann Arbor, MI
| | - Lauren C DeSantis
- Department of Molecular and Integrative Physiology, University of Michigan, Ann Arbor, MI
| | - Ray M Joe
- Cell and Molecular Biology Graduate Program, University of Michigan, Ann Arbor, MI
- Department of Molecular and Integrative Physiology, University of Michigan, Ann Arbor, MI
| | - Joel M Cline
- Department of Molecular and Integrative Physiology, University of Michigan, Ann Arbor, MI
| | - Julia M Keogh
- University of Cambridge Metabolic Research Laboratories and NIHR Cambridge Biomedical Research Centre, Wellcome Trust-MRC Institute of Metabolic Science, Addenbrooke's Hospital, Cambridge, U.K
| | - Elana Henning
- University of Cambridge Metabolic Research Laboratories and NIHR Cambridge Biomedical Research Centre, Wellcome Trust-MRC Institute of Metabolic Science, Addenbrooke's Hospital, Cambridge, U.K
| | - Ines Barroso
- MRC Epidemiology Unit, Wellcome Trust-MRC Institute of Metabolic Science, Addenbrooke's Hospital, Cambridge, U.K
| | - Edson Mendes de Oliveira
- University of Cambridge Metabolic Research Laboratories and NIHR Cambridge Biomedical Research Centre, Wellcome Trust-MRC Institute of Metabolic Science, Addenbrooke's Hospital, Cambridge, U.K
| | - Gowri Chandrashekar
- Department of Molecular and Integrative Physiology, University of Michigan, Ann Arbor, MI
| | - Erik S Clutter
- Department of Molecular and Integrative Physiology, University of Michigan, Ann Arbor, MI
| | - Yixin Hu
- Department of Molecular and Integrative Physiology, University of Michigan, Ann Arbor, MI
| | - Jeanne Stuckey
- Life Sciences Institute and Departments of Biological Chemistry and Biophysics, University of Michigan, Ann Arbor, MI
| | - I Sadaf Farooqi
- University of Cambridge Metabolic Research Laboratories and NIHR Cambridge Biomedical Research Centre, Wellcome Trust-MRC Institute of Metabolic Science, Addenbrooke's Hospital, Cambridge, U.K
| | - Martin G Myers
- Cell and Molecular Biology Graduate Program, University of Michigan, Ann Arbor, MI
- Department of Molecular and Integrative Physiology, University of Michigan, Ann Arbor, MI
- Department of Internal Medicine, University of Michigan, Ann Arbor, MI
| | - Christin Carter-Su
- Cell and Molecular Biology Graduate Program, University of Michigan, Ann Arbor, MI
- Department of Molecular and Integrative Physiology, University of Michigan, Ann Arbor, MI
- Department of Internal Medicine, University of Michigan, Ann Arbor, MI
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Phosphorylation of the Unique C-Terminal Tail of the Alpha Isoform of the Scaffold Protein SH2B1 Controls the Ability of SH2B1α To Enhance Nerve Growth Factor Function. Mol Cell Biol 2018; 38:MCB.00277-17. [PMID: 29229648 DOI: 10.1128/mcb.00277-17] [Citation(s) in RCA: 5] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/19/2017] [Accepted: 12/06/2017] [Indexed: 11/20/2022] Open
Abstract
The scaffold protein SH2B1, a major regulator of body weight, is recruited to the receptors of multiple cytokines and growth factors, including nerve growth factor (NGF). The β isoform but not the α isoform of SH2B1 greatly enhances NGF-dependent neurite outgrowth of PC12 cells. Here, we asked how the unique C-terminal tails of the α and β isoforms modulate SH2B1 function. We compared the actions of SH2B1α and SH2B1β to those of the N-terminal 631 amino acids shared by both isoforms. In contrast to the β tail, the α tail inhibited the ability of SH2B1 to both cycle through the nucleus and enhance NGF-mediated neurite outgrowth, gene expression, phosphorylation of Akt and phospholipase C-gamma (PLC-γ), and autophosphorylation of the NGF receptor TrkA. These functions were restored when Tyr753 in the α tail was mutated to phenylalanine. We provide evidence that TrkA phosphorylates Tyr753 in SH2B1α, as well as tyrosines 439 and 55 in both SH2B1α and SH2B1β. Finally, coexpression of SH2B1α but not SH2B1α with a mutation of Y to F at position 753 (Y753F) inhibited the ability of SH2B1β to enhance neurite outgrowth. These results suggest that the C-terminal tails of SH2B1 isoforms are key determinants of the cellular role of SH2B1. Furthermore, the function of SH2B1α is regulated by phosphorylation of the α tail.
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Chen KW, Chang YJ, Yeh CM, Lian YL, Chan MWY, Kao CF, Chen L. SH2B1 modulates chromatin state and MyoD occupancy to enhance expressions of myogenic genes. BIOCHIMICA ET BIOPHYSICA ACTA-GENE REGULATORY MECHANISMS 2016; 1860:270-281. [PMID: 28039048 DOI: 10.1016/j.bbagrm.2016.12.007] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Subscribe] [Scholar Register] [Received: 09/05/2016] [Revised: 11/29/2016] [Accepted: 12/23/2016] [Indexed: 10/20/2022]
Abstract
As mesoderm-derived cell lineage commits to myogenesis, a spectrum of signaling molecules, including insulin growth factor (IGF), activate signaling pathways and ultimately instruct chromatin remodeling and the transcription of myogenic genes. MyoD is a key transcription factor during myogenesis. In this study, we have identified and characterized a novel myogenic regulator, SH2B1. Knocking down SH2B1 delays global chromatin condensation and decreases the formation of myotubes. SH2B1 interacts with histone H1 and is required for the removal of histone H1 from active transcription sites, allowing for the expressions of myogenic genes, IGF2 and MYOG. Chromatin immunoprecipitation assays suggest the requirement of SH2B1 for the induction of histone H3 lysine 4 trimethylation as well as the reduction of histone H3 lysine 9 trimethylation at the promoters and/or enhancers of IGF2 and MYOG genes during myogenesis. Furthermore, SH2B1 is required for the transcriptional activity of MyoD and MyoD occupancy at the enhancer/promoter regions of IGF2 and MYOG during myogenesis. Together, this study demonstrates that SH2B1 fine-tunes global-local chromatin states, expressions of myogenic genes and ultimately promotes myogenesis.
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Affiliation(s)
- Kuan-Wei Chen
- Institute of Molecular Medicine, National Tsing Hua University, Hsinchu, Taiwan, R.O.C
| | - Yu-Jung Chang
- Institute of Molecular Medicine, National Tsing Hua University, Hsinchu, Taiwan, R.O.C
| | - Chia-Ming Yeh
- Department of Life Science, National Chung Cheng University, Chia-yi, Taiwan, R.O.C
| | - Yen-Ling Lian
- Institute of Molecular Medicine, National Tsing Hua University, Hsinchu, Taiwan, R.O.C
| | - Michael W Y Chan
- Department of Life Science, National Chung Cheng University, Chia-yi, Taiwan, R.O.C
| | - Cheng-Fu Kao
- Institute of Cellular and Organismic Biology, Academia Sinica, Taipei, Taiwan, R.O.C
| | - Linyi Chen
- Institute of Molecular Medicine, National Tsing Hua University, Hsinchu, Taiwan, R.O.C.; Department of Medical Science, National Tsing Hua University, Hsinchu, Taiwan, R.O.C..
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Rui L. SH2B1 regulation of energy balance, body weight, and glucose metabolism. World J Diabetes 2014; 5:511-526. [PMID: 25126397 PMCID: PMC4127586 DOI: 10.4239/wjd.v5.i4.511] [Citation(s) in RCA: 64] [Impact Index Per Article: 5.8] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 11/25/2013] [Revised: 03/06/2014] [Accepted: 06/03/2014] [Indexed: 02/05/2023] Open
Abstract
The Src homology 2B (SH2B) family members (SH2B1, SH2B2 and SH2B3) are adaptor signaling proteins containing characteristic SH2 and PH domains. SH2B1 (also called SH2-B and PSM) and SH2B2 (also called APS) are able to form homo- or hetero-dimers via their N-terminal dimerization domains. Their C-terminal SH2 domains bind to tyrosyl phosphorylated proteins, including Janus kinase 2 (JAK2), TrkA, insulin receptors, insulin-like growth factor-1 receptors, insulin receptor substrate-1 (IRS1), and IRS2. SH2B1 enhances leptin signaling by both stimulating JAK2 activity and assembling a JAK2/IRS1/2 signaling complex. SH2B1 promotes insulin signaling by both enhancing insulin receptor catalytic activity and protecting against dephosphorylation of IRS proteins. Accordingly, genetic deletion of SH2B1 results in severe leptin resistance, insulin resistance, hyperphagia, obesity, and type 2 diabetes in mice. Neuron-specific overexpression of SH2B1β transgenes protects against diet-induced obesity and insulin resistance. SH2B1 in pancreatic β cells promotes β cell expansion and insulin secretion to counteract insulin resistance in obesity. Moreover, numerous SH2B1 mutations are genetically linked to leptin resistance, insulin resistance, obesity, and type 2 diabetes in humans. Unlike SH2B1, SH2B2 and SH2B3 are not required for the maintenance of normal energy and glucose homeostasis. The metabolic function of the SH2B family is conserved from insects to humans.
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Hsu YC, Chen SL, Wang YJ, Chen YH, Wang DY, Chen L, Chen CH, Chen HH, Chiu IM. Signaling adaptor protein SH2B1 enhances neurite outgrowth and accelerates the maturation of human induced neurons. Stem Cells Transl Med 2014; 3:713-22. [PMID: 24736401 DOI: 10.5966/sctm.2013-0111] [Citation(s) in RCA: 11] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/20/2022] Open
Abstract
Recent advances in somatic cell reprogramming have highlighted the plasticity of the somatic epigenome, particularly through demonstrations of direct lineage reprogramming of adult mouse and human fibroblasts to induced pluripotent stem cells (iPSCs) and induced neurons (iNs) under defined conditions. However, human cells appear to be less plastic and have a higher epigenetic hurdle for reprogramming to both iPSCs and iNs. Here, we show that SH2B adaptor protein 1β (SH2B1) can enhance neurite outgrowth of iNs reprogrammed from human fibroblasts as early as day 14, when combined with miR124 and transcription factors BRN2 and MYT1L (IBM) under defined conditions. These SH2B1-enhanced iNs (S-IBM) showed canonical neuronal morphology, and expressed multiple neuronal markers, such as TuJ1, NeuN, and synapsin, and functional proteins for neurotransmitter release, such as GABA, vGluT2, and tyrosine hydroxylase. Importantly, SH2B1 accelerated mature process of functional neurons and exhibited action potentials as early as day 14; without SH2B1, the IBM iNs do not exhibit action potentials until day 21. Our data demonstrate that SH2B1 can enhance neurite outgrowth and accelerate the maturation of human iNs under defined conditions. This approach will facilitate the application of iNs in regenerative medicine and in vitro disease modeling.
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Affiliation(s)
- Yi-Chao Hsu
- Division of Regenerative Medicine, Institute of Cellular and System Medicine, and Center for Neuropsychiatric Research, National Health Research Institutes, Miaoli, Taiwan, Republic of China; Graduate Program of Biotechnology in Medicine, Institute of Biotechnology and Department of Life Science, Institute of Molecular Medicine, and Department of Medical Science, National Tsing Hua University, Hsinchu, Taiwan, Republic of China; Department of Psychiatry, Chang Gung Memorial Hospital at Linkou and Chang Gung University, Gueishan, Taoyuan, Taiwan, Republic of China; Department of Pharmacology, Tzu Chi University, Hualien, Taiwan, Republic of China; Department of Life Sciences, National Chung Hsing University, Taichung, Taiwan, Republic of China
| | - Su-Liang Chen
- Division of Regenerative Medicine, Institute of Cellular and System Medicine, and Center for Neuropsychiatric Research, National Health Research Institutes, Miaoli, Taiwan, Republic of China; Graduate Program of Biotechnology in Medicine, Institute of Biotechnology and Department of Life Science, Institute of Molecular Medicine, and Department of Medical Science, National Tsing Hua University, Hsinchu, Taiwan, Republic of China; Department of Psychiatry, Chang Gung Memorial Hospital at Linkou and Chang Gung University, Gueishan, Taoyuan, Taiwan, Republic of China; Department of Pharmacology, Tzu Chi University, Hualien, Taiwan, Republic of China; Department of Life Sciences, National Chung Hsing University, Taichung, Taiwan, Republic of China
| | - Ya-Jean Wang
- Division of Regenerative Medicine, Institute of Cellular and System Medicine, and Center for Neuropsychiatric Research, National Health Research Institutes, Miaoli, Taiwan, Republic of China; Graduate Program of Biotechnology in Medicine, Institute of Biotechnology and Department of Life Science, Institute of Molecular Medicine, and Department of Medical Science, National Tsing Hua University, Hsinchu, Taiwan, Republic of China; Department of Psychiatry, Chang Gung Memorial Hospital at Linkou and Chang Gung University, Gueishan, Taoyuan, Taiwan, Republic of China; Department of Pharmacology, Tzu Chi University, Hualien, Taiwan, Republic of China; Department of Life Sciences, National Chung Hsing University, Taichung, Taiwan, Republic of China
| | - Yun-Hsiang Chen
- Division of Regenerative Medicine, Institute of Cellular and System Medicine, and Center for Neuropsychiatric Research, National Health Research Institutes, Miaoli, Taiwan, Republic of China; Graduate Program of Biotechnology in Medicine, Institute of Biotechnology and Department of Life Science, Institute of Molecular Medicine, and Department of Medical Science, National Tsing Hua University, Hsinchu, Taiwan, Republic of China; Department of Psychiatry, Chang Gung Memorial Hospital at Linkou and Chang Gung University, Gueishan, Taoyuan, Taiwan, Republic of China; Department of Pharmacology, Tzu Chi University, Hualien, Taiwan, Republic of China; Department of Life Sciences, National Chung Hsing University, Taichung, Taiwan, Republic of China
| | - Dan-Yen Wang
- Division of Regenerative Medicine, Institute of Cellular and System Medicine, and Center for Neuropsychiatric Research, National Health Research Institutes, Miaoli, Taiwan, Republic of China; Graduate Program of Biotechnology in Medicine, Institute of Biotechnology and Department of Life Science, Institute of Molecular Medicine, and Department of Medical Science, National Tsing Hua University, Hsinchu, Taiwan, Republic of China; Department of Psychiatry, Chang Gung Memorial Hospital at Linkou and Chang Gung University, Gueishan, Taoyuan, Taiwan, Republic of China; Department of Pharmacology, Tzu Chi University, Hualien, Taiwan, Republic of China; Department of Life Sciences, National Chung Hsing University, Taichung, Taiwan, Republic of China
| | - Linyi Chen
- Division of Regenerative Medicine, Institute of Cellular and System Medicine, and Center for Neuropsychiatric Research, National Health Research Institutes, Miaoli, Taiwan, Republic of China; Graduate Program of Biotechnology in Medicine, Institute of Biotechnology and Department of Life Science, Institute of Molecular Medicine, and Department of Medical Science, National Tsing Hua University, Hsinchu, Taiwan, Republic of China; Department of Psychiatry, Chang Gung Memorial Hospital at Linkou and Chang Gung University, Gueishan, Taoyuan, Taiwan, Republic of China; Department of Pharmacology, Tzu Chi University, Hualien, Taiwan, Republic of China; Department of Life Sciences, National Chung Hsing University, Taichung, Taiwan, Republic of China
| | - Chia-Hsiang Chen
- Division of Regenerative Medicine, Institute of Cellular and System Medicine, and Center for Neuropsychiatric Research, National Health Research Institutes, Miaoli, Taiwan, Republic of China; Graduate Program of Biotechnology in Medicine, Institute of Biotechnology and Department of Life Science, Institute of Molecular Medicine, and Department of Medical Science, National Tsing Hua University, Hsinchu, Taiwan, Republic of China; Department of Psychiatry, Chang Gung Memorial Hospital at Linkou and Chang Gung University, Gueishan, Taoyuan, Taiwan, Republic of China; Department of Pharmacology, Tzu Chi University, Hualien, Taiwan, Republic of China; Department of Life Sciences, National Chung Hsing University, Taichung, Taiwan, Republic of China
| | - Hwei-Hsien Chen
- Division of Regenerative Medicine, Institute of Cellular and System Medicine, and Center for Neuropsychiatric Research, National Health Research Institutes, Miaoli, Taiwan, Republic of China; Graduate Program of Biotechnology in Medicine, Institute of Biotechnology and Department of Life Science, Institute of Molecular Medicine, and Department of Medical Science, National Tsing Hua University, Hsinchu, Taiwan, Republic of China; Department of Psychiatry, Chang Gung Memorial Hospital at Linkou and Chang Gung University, Gueishan, Taoyuan, Taiwan, Republic of China; Department of Pharmacology, Tzu Chi University, Hualien, Taiwan, Republic of China; Department of Life Sciences, National Chung Hsing University, Taichung, Taiwan, Republic of China
| | - Ing-Ming Chiu
- Division of Regenerative Medicine, Institute of Cellular and System Medicine, and Center for Neuropsychiatric Research, National Health Research Institutes, Miaoli, Taiwan, Republic of China; Graduate Program of Biotechnology in Medicine, Institute of Biotechnology and Department of Life Science, Institute of Molecular Medicine, and Department of Medical Science, National Tsing Hua University, Hsinchu, Taiwan, Republic of China; Department of Psychiatry, Chang Gung Memorial Hospital at Linkou and Chang Gung University, Gueishan, Taoyuan, Taiwan, Republic of China; Department of Pharmacology, Tzu Chi University, Hualien, Taiwan, Republic of China; Department of Life Sciences, National Chung Hsing University, Taichung, Taiwan, Republic of China
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Abstract
LNK (SH2B3) is an adaptor protein studied extensively in normal and malignant hematopoietic cells. In these cells, it downregulates activated tyrosine kinases at the cell surface resulting in an antiproliferative effect. To date, no studies have examined activities of LNK in solid tumors. In this study, we found by in silico analysis and staining tissue arrays that the levels of LNK expression were elevated in high-grade ovarian cancer. To test the functional importance of this observation, LNK was either overexpressed or silenced in several ovarian cancer cell lines. Remarkably, overexpression of LNK rendered the cells resistant to death induced by either serum starvation or nutrient deprivation, and generated larger tumors using a murine xenograft model. In contrast, silencing of LNK decreased ovarian cancer cell growth in vitro and in vivo. Western blot studies indicated that overexpression of LNK upregulated and extended the transduction of the mitogenic signal, whereas silencing of LNK produced the opposite effects. Furthermore, forced expression of LNK reduced cell size, inhibited cell migration and markedly enhanced cell adhesion. Liquid chromatography-mass spectroscopy identified 14-3-3 as one of the LNK-binding partners. Our results suggest that in contrast to the findings in hematologic malignancies, the adaptor protein LNK acts as a positive signal transduction modulator in ovarian cancers.
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Youssef G, Gillett C, Agbaje O, Crompton T, Montano X. Phosphorylation of NTRK1 at Y674/Y675 induced by TP53-dependent repression of PTPN6 expression: a potential novel prognostic marker for breast cancer. Mod Pathol 2014; 27:361-74. [PMID: 23948750 DOI: 10.1038/modpathol.2013.129] [Citation(s) in RCA: 11] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/12/2013] [Revised: 05/29/2013] [Accepted: 05/30/2013] [Indexed: 12/29/2022]
Abstract
We have identified a ligand-independent mechanism whereby the tumor suppressor, TP53, induces nerve growth factor receptor, NTRK1, phosphorylation at Y674/Y675 (NTRK1-pY674/pY675), via the repression of the NTRK1-phosphatase, PTPN6. This results in suppression of breast cancer cell proliferation. In this investigation, we aimed to establish whether perturbation of the wild-type TP53-NTRK1-pY674/pY675-PTPN6 pathway has an impact on disease-free survival of breast cancer patients without neo-adjuvant treatment. A total of 308 tumor samples were stained for NTRK1, NTRK1-pY674/pY675, PTPN6, and TP53 expression. Association between expression levels and disease-free survival was determined by the univariate/multivariate and Kaplan-Meir methods of analysis. DNA from tumors was sequenced to identify mutant or wild-type TP53. Tumors expressing NTRK1-pY674/pY675 but with undetectable or low levels of PTPN6 and TP53 were associated with prolonged 5, 10, and 15 years' disease-free survival by 48%, 36%, and 37%, respectively, in the multivariate analysis (P<0.05). A similar result was observed in tumors expressing wild-type TP53, NTRK1-pY674/pY675, and low or undetectable levels of PTPN6. Given that estrogen receptor-positive breast cancers encode wild-type TP53, we analyzed this expression pattern in these tumors. Multivariate analysis showed that it was significantly and independently predictive of prolonged survival by 66%, 70%, and 84%, respectively, (P<0.05). The Kaplan-Meir method demonstrated that NTRK1-pY674/pY675 together with undetectable or low levels of PTPN6 correlated with 59% probability of disease-free survival (median survival 15 years), compared with 7% probability of disease-free survival (median survival 4.5 years) when absent. In luminal A tumors, the presence of this pattern was estimated to have a 61% probability of disease-free survival (median survival 15 years), compared with 6% probability of disease-free survival (median survival 3 years) when it was absent. These results strongly suggest that expression of NTRK1-pY674/pY675 together with wild-type TP53 and low levels of PTPN6 expression are predictors of improved disease-free survival and that they could be useful biomarkers to predict clinical outcome.
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Affiliation(s)
- Gehad Youssef
- 1] Immunobiology Unit, UCL, Institute of Child Health, London, UK [2] Molecular Hematology and Cancer Biology Unit, UCL, Institute of Child Health, London, UK
| | - Cheryl Gillett
- Breast Tissue & Data Bank, Division of Cancer Studies, King's College London, Guys Hospital, London, UK
| | - Orunsola Agbaje
- Cancer Epidemiology Group, Division of Cancer Studies, King's College London, Guys Hospital, London, UK
| | - Tessa Crompton
- Immunobiology Unit, UCL, Institute of Child Health, London, UK
| | - Ximena Montano
- 1] Immunobiology Unit, UCL, Institute of Child Health, London, UK [2] Molecular Hematology and Cancer Biology Unit, UCL, Institute of Child Health, London, UK [3] School of Health and Social Work, Department of Allied Health Professions and Midwifery University of Hertfordshire, Hatfield, UK
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SH2B1β interacts with STAT3 and enhances fibroblast growth factor 1-induced gene expression during neuronal differentiation. Mol Cell Biol 2014; 34:1003-19. [PMID: 24396070 DOI: 10.1128/mcb.00940-13] [Citation(s) in RCA: 12] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/20/2022] Open
Abstract
Neurite outgrowth is an essential process during neuronal differentiation as well as neuroregeneration. Thus, understanding the molecular and cellular control of neurite outgrowth will benefit patients with neurological diseases. We have previously shown that overexpression of the signaling adaptor protein SH2B1β promotes fibroblast growth factor 1 (FGF1)-induced neurite outgrowth (W. F. Lin, C. J. Chen, Y. J. Chang, S. L. Chen, I. M. Chiu, and L. Chen, Cell. Signal. 21:1060-1072, 2009). SH2B1β also undergoes nucleocytoplasmic shuttling and regulates a subset of neurotrophin-induced genes. Although these findings suggest that SH2B1β regulates gene expression, the nuclear role of SH2B1β was not known. In this study, we show that SH2B1β interacts with the transcription factor, signal transducer, and activator of transcription 3 (STAT3) in neuronal PC12 cells, cortical neurons, and COS7 fibroblasts. By affecting the subcellular distribution of STAT3, SH2B1β increased serine phosphorylation and the concomitant transcriptional activity of STAT3. As a result, overexpressing SH2B1β enhanced FGF1-induced expression of STAT3 target genes Egr1 and Cdh2. Chromatin immunoprecipitation assays further reveal that, in response to FGF1, overexpression of SH2B1β promotes the in vivo occupancy of STAT3-Sp1 heterodimers at the promoter of Egr1 and Cdh2. These findings establish a central role of SH2B1β in orchestrating signaling events to transcriptional activation through interacting and regulating STAT3-containing complexes during neuronal differentiation.
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Sharanova NE, Toropygin IY, Khriapova EV, Vasilyev AV, Gapparov MMG. Effect of coenzyme Q10 on proteomic profile of blood plasma and cytosolic and microsomal fractions of rat hepatocytes during ontogeny. Bull Exp Biol Med 2013; 154:30-3. [PMID: 23330083 DOI: 10.1007/s10517-012-1867-5] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 10/27/2022]
Abstract
The proteomic features of blood plasma and subcellular fractions of rat hepatocytes were studied during long-term dietary consumption of coenzyme Q10 as the endogenous mediator of antioxidant and energy homeostasis in the cell. Long-term coenzyme Q10 consumption was followed by the formation of specific nutriproteomes of the microsomal and cytosolic fractions of rat hepatocytes.
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Affiliation(s)
- N E Sharanova
- Institute of Nutrition, Russian Academy of Medical Sciences; Center of Collective Use Human Proteome, V. N. Orekhovich Institute of Biomedical Chemistry, Russian Academy of Medical Sciences, Moscow, Russia.
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12
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Volckmar AL, Bolze F, Jarick I, Knoll N, Scherag A, Reinehr T, Illig T, Grallert H, Wichmann HE, Wiegand S, Biebermann H, Krude H, Fischer-Posovszky P, Rief W, Wabitsch M, Klingenspor M, Hebebrand J, Hinney A. Mutation screen in the GWAS derived obesity gene SH2B1 including functional analyses of detected variants. BMC Med Genomics 2012; 5:65. [PMID: 23270367 PMCID: PMC3544595 DOI: 10.1186/1755-8794-5-65] [Citation(s) in RCA: 22] [Impact Index Per Article: 1.7] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/05/2012] [Accepted: 12/20/2012] [Indexed: 12/21/2022] Open
Abstract
Background The SH2B1 gene (Src-homology 2B adaptor protein 1 gene) is a solid candidate gene for obesity. Large scale GWAS studies depicted markers in the vicinity of the gene; animal models suggest a potential relevance for human body weight regulation. Methods We performed a mutation screen for variants in the SH2B1 coding sequence in 95 extremely obese children and adolescents. Detected variants were genotyped in independent childhood and adult study groups (up to 11,406 obese or overweight individuals and 4,568 controls). Functional implications on STAT3 mediated leptin signalling of the detected variants were analyzed in vitro. Results We identified two new rare mutations and five known SNPs (rs147094247, rs7498665, rs60604881, rs62037368 and rs62037369) in SH2B1. Mutation g.9483C/T leads to a non-synonymous, non-conservative exchange in the beta (βThr656Ile) and gamma (γPro674Ser) splice variants of SH2B1. It was additionally detected in two of 11,206 (extremely) obese or overweight children, adolescents and adults, but not in 4,506 population-based normal-weight or lean controls. The non-coding mutation g.10182C/A at the 3’ end of SH2B1 was only detected in three obese individuals. For the non-synonymous SNP rs7498665 (Thr484Ala) we observed nominal over-transmission of the previously described risk allele in 705 obesity trios (nominal p = 0.009, OR = 1.23) and an increased frequency of the same allele in 359 cases compared to 429 controls (nominal p = 0.042, OR = 1.23). The obesity risk-alleles at Thr484Ala and βThr656Ile/γPro674Ser had no effect on STAT3 mediated leptin receptor signalling in splice variants β and γ. Conclusion The rare coding mutation βThr656Ile/γPro674Ser (g.9483C/T) in SH2B1 was exclusively detected in overweight or obese individuals. Functional analyzes did not reveal impairments in leptin signalling for the mutated SH2B1.
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Affiliation(s)
- Anna-Lena Volckmar
- Department of Child and Adolescent Psychiatry, University Duisburg-Essen, Virchowstr, 171, D 45147, Essen, Germany
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13
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Abstract
Fms-like tyrosine kinase 3 (FLT3) is a receptor tyrosine kinase with important roles in hematopoietic progenitor cell survival and proliferation. It is mutated in approximately one-third of AML patients, mostly by internal tandem duplications (ITDs). Adaptor protein Lnk is a negative regulator of hematopoietic cytokine signaling. In the present study, we show that Lnk interacts physically with both wild-type FLT3 (FLT3-WT) and FLT3-ITD through the SH2 domains. We have identified the tyrosine residues 572, 591, and 919 of FLT3 as phosphorylation sites involved in direct binding to Lnk. Lnk itself was tyrosine phosphorylated by both FLT3 ligand (FL)-activated FLT3-WT and constitutively activated FLT3-ITD. Both shRNA-mediated depletion and forced overexpression of Lnk demonstrated that activation signals emanating from both forms of FLT3 are under negative regulation by Lnk. Moreover, Lnk inhibited 32D cell proliferation driven by different FLT3 variants. Analysis of primary BM cells from Lnk-knockout mice showed that Lnk suppresses the expansion of FL-stimulated hematopoietic progenitors, including lymphoid-primed multipotent progenitors. The results of the present study show that through direct binding to FLT3, Lnk suppresses FLT3-WT/ITD-dependent signaling pathways involved in the proliferation of hematopoietic cells. Therefore, modulation of Lnk expression levels may provide a unique therapeutic approach for FLT3-ITD-associated hematopoietic disease.
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Javadi M, Hofstätter E, Stickle N, Beattie BK, Jaster R, Carter-Su C, Barber DL. The SH2B1 adaptor protein associates with a proximal region of the erythropoietin receptor. J Biol Chem 2012; 287:26223-34. [PMID: 22669948 DOI: 10.1074/jbc.m112.382721] [Citation(s) in RCA: 13] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/12/2022] Open
Abstract
Gene targeting experiments have shown that the cytokine erythropoietin (EPO), its cognate erythropoietin receptor (EPO-R), and associated Janus tyrosine kinase, JAK2, are all essential for erythropoiesis. Structural-functional and murine knock-in experiments have suggested that EPO-R Tyr-343 is important in EPO-mediated mitogenesis. Although Stat5 binds to EPO-R phosphotyrosine 343, the initial Stat5-deficient mice did not have profound erythroid abnormalities suggesting that additional Src homology 2 (SH2) domain-containing effectors may bind to EPO-R Tyr-343 and couple to downstream signaling pathways. We have utilized cloning of ligand target (COLT) screening to demonstrate that EPO-R Tyr(P)-343 and Tyr(P)-401 bind to the SH2 domain-containing adaptor protein SH2B1β. Immunoprecipitation and in vitro mixing experiments reveal that EPO-R binds to SH2B1 in an SH2 domain-dependent manner and that the sequence that confers SH2B1 binding to the EPO-R is pYXXL. Previous studies have shown that SH2B1 binds directly to JAK2, but we show that in hematopoietic cells, SH2B1β preferentially associates with the EPO-R. SH2B1 is capable of constitutive association with EPO-R, which is necessary for its optimal SH2-dependent recruitment to EPO-R-Tyr(P)-343/Tyr(P)-401. We also demonstrate that SH2B1 is responsive to EPO stimulation and becomes phosphorylated, most likely on serines/threonines, in an EPO dose- and time-dependent manner. In the absence of SH2B1, we observe enhanced activation of signaling pathways downstream of the EPO-R, indicating that SH2B1 is a negative regulator of EPO signaling.
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Affiliation(s)
- Mojib Javadi
- Ontario Cancer Institute, Campbell Family Cancer Research Institute, Toronto, Ontario M5G 2M9, Canada
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15
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Siddle K. Molecular basis of signaling specificity of insulin and IGF receptors: neglected corners and recent advances. Front Endocrinol (Lausanne) 2012; 3:34. [PMID: 22649417 PMCID: PMC3355962 DOI: 10.3389/fendo.2012.00034] [Citation(s) in RCA: 111] [Impact Index Per Article: 8.5] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 11/15/2011] [Accepted: 02/13/2012] [Indexed: 12/15/2022] Open
Abstract
Insulin and insulin-like growth factor (IGF) receptors utilize common phosphoinositide 3-kinase/Akt and Ras/extracellular signal-regulated kinase signaling pathways to mediate a broad spectrum of "metabolic" and "mitogenic" responses. Specificity of insulin and IGF action in vivo must in part reflect expression of receptors and responsive pathways in different tissues but it is widely assumed that it is also determined by the ligand binding and signaling mechanisms of the receptors. This review focuses on receptor-proximal events in insulin/IGF signaling and examines their contribution to specificity of downstream responses. Insulin and IGF receptors may differ subtly in the efficiency with which they recruit their major substrates (IRS-1 and IRS-2 and Shc) and this could influence effectiveness of signaling to "metabolic" and "mitogenic" responses. Other substrates (Grb2-associated binder, downstream of kinases, SH2Bs, Crk), scaffolds (RACK1, β-arrestins, cytohesins), and pathways (non-receptor tyrosine kinases, phosphoinositide kinases, reactive oxygen species) have been less widely studied. Some of these components appear to be specifically involved in "metabolic" or "mitogenic" signaling but it has not been shown that this reflects receptor-preferential interaction. Very few receptor-specific interactions have been characterized, and their roles in signaling are unclear. Signaling specificity might also be imparted by differences in intracellular trafficking or feedback regulation of receptors, but few studies have directly addressed this possibility. Although published data are not wholly conclusive, no evidence has yet emerged for signaling mechanisms that are specifically engaged by insulin receptors but not IGF receptors or vice versa, and there is only limited evidence for differential activation of signaling mechanisms that are common to both receptors. Cellular context, rather than intrinsic receptor activity, therefore appears to be the major determinant of whether responses to insulin and IGFs are perceived as "metabolic" or "mitogenic."
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Affiliation(s)
- Kenneth Siddle
- University of Cambridge Metabolic Research Laboratories and Department of Clinical Biochemistry, Institute of Metabolic Science, Addenbrooke's Hospital Cambridge, UK.
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16
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Expression of Trk A and Src and their interaction with ERβ ligand binding domain show age and sex dependent alteration in mouse brain. Neurochem Res 2011; 37:448-53. [PMID: 22011838 DOI: 10.1007/s11064-011-0631-y] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/12/2011] [Revised: 10/01/2011] [Accepted: 10/05/2011] [Indexed: 10/16/2022]
Abstract
Following the binding of estrogen to estrogen receptor (ER)β ligand binding domain (LBD) and its interaction with the target genes, a host of nuclear proteins is recruited to regulate the expression of specific genes(s). It is not known which proteins interact with ERβLBD and whether they vary with age and sex in the brain. Therefore, using pull down assay, immunoprecipitation and immunoblotting, we report that cell signaling molecules Trk A and Src interacted with ERβLBD, and showed alteration in the level of interaction and expression in the brain of AKR strain young (6 weeks), adult (25 weeks) and old (70 weeks) mice of both sexes. Trk A showed decreasing interaction with age, and lower expression in adult as compared to young and old males, whereas female mice exhibited decline in both interaction and expression as a function of age. On the other hand, Src interaction with ERβLBD decreased, but its expression increased with age in males, whereas the interaction and expression was lower in adult but higher in old as compared to young females. These findings suggest the implication of Trk A and Src in ERβ mediated brain functions and related disorders during aging.
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Wang TC, Chiu H, Chang YJ, Hsu TY, Chiu IM, Chen L. The adaptor protein SH2B3 (Lnk) negatively regulates neurite outgrowth of PC12 cells and cortical neurons. PLoS One 2011; 6:e26433. [PMID: 22028877 PMCID: PMC3196555 DOI: 10.1371/journal.pone.0026433] [Citation(s) in RCA: 17] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/22/2011] [Accepted: 09/27/2011] [Indexed: 12/11/2022] Open
Abstract
SH2B adaptor protein family members (SH2B1-3) regulate various physiological responses through affecting signaling, gene expression, and cell adhesion. SH2B1 and SH2B2 were reported to enhance nerve growth factor (NGF)-induced neuronal differentiation in PC12 cells, a well-established neuronal model system. In contrast, SH2B3 was reported to inhibit cell proliferation during the development of immune system. No study so far addresses the role of SH2B3 in the nervous system. In this study, we provide evidence suggesting that SH2B3 is expressed in the cortex of embryonic rat brain. Overexpression of SH2B3 not only inhibits NGF-induced differentiation of PC12 cells but also reduces neurite outgrowth of primary cortical neurons. SH2B3 does so by repressing NGF-induced activation of PLCγ, MEK-ERK1/2 and PI3K-AKT pathways and the expression of Egr-1. SH2B3 is capable of binding to phosphorylated NGF receptor, TrkA, as well as SH2B1β. Our data further demonstrate that overexpression of SH2B3 reduces the interaction between SH2B1β and TrkA. Consistent with this finding, overexpressing the SH2 domain of SH2B3 is sufficient to inhibit NGF-induced neurite outgrowth. Together, our data demonstrate that SH2B3, unlike the other two family members, inhibits neuronal differentiation of PC12 cells and primary cortical neurons. Its inhibitory mechanism is likely through the competition of TrkA binding with the positive-acting SH2B1 and SH2B2.
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Affiliation(s)
- Tien-Cheng Wang
- Institute of Molecular Medicine, National Tsing Hua University, Hsinchu, Taiwan
| | - Hsun Chiu
- Department of Life Science, National Tsing Hua University, Hsinchu, Taiwan
| | - Yu-Jung Chang
- Institute of Molecular Medicine, National Tsing Hua University, Hsinchu, Taiwan
| | - Tai-Yu Hsu
- Institute of Molecular Medicine, National Tsing Hua University, Hsinchu, Taiwan
| | - Ing-Ming Chiu
- Institute of Cellular and System Medicine, National Health Research Institutes, Miaoli, Taiwan
| | - Linyi Chen
- Institute of Molecular Medicine, National Tsing Hua University, Hsinchu, Taiwan
- Department of Medical Science, National Tsing Hua University, Hsinchu, Taiwan
- * E-mail:
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Gueller S, Hehn S, Nowak V, Gery S, Serve H, Brandts CH, Koeffler HP. Adaptor protein Lnk binds to PDGF receptor and inhibits PDGF-dependent signaling. Exp Hematol 2011; 39:591-600. [PMID: 21310211 DOI: 10.1016/j.exphem.2011.02.001] [Citation(s) in RCA: 21] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/28/2009] [Revised: 01/11/2011] [Accepted: 01/27/2011] [Indexed: 12/19/2022]
Abstract
OBJECTIVE Platelet-derived growth factor receptors α and β (PDGFRA, PDGFRB) are frequently expressed on hematopoietic cells and regulate cellular responses such as proliferation, differentiation, survival, and transformation. Stimulation by autocrine loops or activation by chromosomal translocation makes them important factors in development of hematopoietic disorders. Interaction with the ligand PDGF results in activation of the tyrosine kinase domain and phosphorylation of tyrosine residues, thereby creating binding sites for molecules containing Src homology 2 domains. We hypothesized that one such protein may be Lnk, a negative regulator of cytokine receptors, including Mpl, EpoR, c-Kit, and c-Fms. MATERIALS AND METHODS Interaction of Lnk with PDGFRA, PDGFRB, or leukemogenic FIP1L1-PDGFRA or TEL-PDGFRB was studied in cotransfected 293T cells. Effects of Lnk on PDGFR signaling were shown in 293T and NIH3T3 cells, whereas its influence on either PDGF-dependent or factor-independent growth was investigated using Ba/F3 or 32D cells expressing wild-type PDGFR, FIP1L1-PDGFRA, or TEL-PDGFRB. RESULTS We show that Lnk binds to PDGFR after exposure of cells to PDGF. Furthermore, Lnk can bind the FIP1L1-PDGFRA fusion protein. Mutation or deletion of the Lnk Src homology 2 domain completely abolished binding of Lnk to FIP1L1-PDGFRA, but just partly prevented binding to PDGFRA or PDGFRB. Expression of Lnk inhibited proliferation of PDGF-dependent Ba/F3 cells and diminished phosphorylation of Erk in PDGF-treated NIH3T3. 32D cells transformed by either FIP1L1-PDGFRA or TEL-PDGFRB stopped growing when Lnk was expressed. CONCLUSIONS Lnk is a negative regulator of PDGFR signaling. Development of Lnk mimetic drugs might provide a novel therapeutic strategy for myeloproliferative disorders.
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Affiliation(s)
- Saskia Gueller
- Department of Medicine, Hematology/Oncology, Johann Wolfgang Goethe University, Theodor-Stern-Kai 7, Frankfurt, Germany.
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Maures TJ, Su HW, Argetsinger LS, Grinstein S, Carter-Su C. Phosphorylation controls a dual-function polybasic nuclear localization sequence in the adapter protein SH2B1β to regulate its cellular function and distribution. J Cell Sci 2011; 124:1542-52. [PMID: 21486950 DOI: 10.1242/jcs.078949] [Citation(s) in RCA: 17] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/31/2022] Open
Abstract
An intriguing question in cell biology is what targets proteins to, and regulates their translocation between, specific cellular locations. Here we report that the polybasic nuclear localization sequence (NLS) required for nuclear entry of the adapter protein and candidate human obesity gene product SH2B1β, also localizes SH2B1β to the plasma membrane (PM), most probably via electrostatic interactions. Binding of SH2B1β to the PM also requires its dimerization domain. Phosphorylation of serine residues near this polybasic region, potentially by protein kinase C, releases SH2B1β from the PM and enhances nuclear entry. Release of SH2B1β from the PM and/or nuclear entry appear to be required for SH2B1β enhancement of nerve growth factor (NGF)-induced expression of urokinase plasminogen activator receptor gene and neurite outgrowth of PC12 cells. Taken together, our results provide strong evidence that the polybasic NLS region of SH2B1 serves the dual function of localizing SH2B1 to both the nucleus and the PM, the latter most probably through electrostatic interactions that are enhanced by SH2B1β dimerization. Cycling between the different cellular compartments is a consequence of the phosphorylation and dephosphorylation of serine residues near the NLS and is important for physiological effects of SH2B1, including NGF-induced gene expression and neurite outgrowth.
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Affiliation(s)
- Travis J Maures
- Department of Molecular and Integrative Physiology, University of Michigan Medical School, Ann Arbor, MI 48109-5622, USA
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20
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Morris DL, Cho KW, Rui L. Critical role of the Src homology 2 (SH2) domain of neuronal SH2B1 in the regulation of body weight and glucose homeostasis in mice. Endocrinology 2010; 151:3643-51. [PMID: 20484460 PMCID: PMC2940518 DOI: 10.1210/en.2010-0254] [Citation(s) in RCA: 36] [Impact Index Per Article: 2.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 03/04/2010] [Accepted: 04/23/2010] [Indexed: 01/19/2023]
Abstract
SH2B1 is an SH2 domain-containing adaptor protein that plays a key role in the regulation of energy and glucose metabolism in both rodents and humans. Genetic deletion of SH2B1 in mice results in obesity and type 2 diabetes. Single-nucleotide polymorphisms in the SH2B1 loci and chromosomal deletions of the SH2B1 loci associate with obesity and insulin resistance in humans. In cultured cells, SH2B1 promotes leptin and insulin signaling by binding via its SH2 domain to phosphorylated tyrosines in Janus kinase 2 and the insulin receptor, respectively. Here we generated three lines of mice to analyze the role of the SH2 domain of SH2B1 in the central nervous system. Transgenic mice expressing wild-type, SH2 domain-defective (R555E), or SH2 domain-alone (DeltaN503) forms of SH2B1 specifically in neurons were crossed with SH2B1 knockout mice to generate KO/SH2B1, KO/R555E, or KO/DeltaN503 compound mutant mice. R555E had a replacement of Arg(555) with Glu within the SH2 domain. DeltaN503 contained an intact SH2 domain but lacked amino acids 1-503. Neuron-specific expression of recombinant SH2B1, but not R555E or DeltaN503, corrected hyperphagia, obesity, glucose intolerance, and insulin resistance in SH2B1 null mice. Neuron-specific expression of R555E in wild-type mice promoted obesity and insulin resistance. These results indicate that in addition to the SH2 domain, N-terminal regions of neuronal SH2B1 are also required for the maintenance of normal body weight and glucose metabolism. Additionally, mutations in the SH2 domain of SH2B1 may increase the susceptibility to obesity and type 2 diabetes in a dominant-negative manner.
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Affiliation(s)
- David L Morris
- Department of Molecular and Integrative Physiology, University of Michigan Medical School, Ann Arbor, Michigan 48109-0622, USA
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21
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Montano X. Repression of SHP-1 expression by p53 leads to trkA tyrosine phosphorylation and suppression of breast cancer cell proliferation. Oncogene 2009; 28:3787-800. [PMID: 19749791 DOI: 10.1038/onc.2009.143] [Citation(s) in RCA: 28] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/30/2022]
Abstract
The nerve growth factor (NGF) receptor, trkA, the tumour suppressor p53 and the phosphatase SHP-1 are critical in cell proliferation and differentiation. SHP-1 is a trkA phosphatase that dephosphorylates trkA at tyrosines (Y) 674 and 675. p53 can induce trkA activation and tyrosine phosphorylation in the absence of NGF stimulation. In breast cancer tumours trkA expression is associated with increased patient survival. TrkA protein expression is higher in breast-cancer cell lines than in normal breast epithelia. In cell lines (but not in normal breast epithelia) trkA is functional and can be NGF-stimulated to promote cell proliferation. This study investigates the functional relationship between trkA, p53 and SHP-1 in breast-cancer, and reveals that in wild-type (wt) trkA expressing breast-cancer cells both endogenous wtp53, activated by therapeutic agents, and transfected wtp53 repress expression of SHP-1 through the proximal CCAAT sequence of the SHP-1-P1-promoter and the transcription factor NF-Y. In these cells trkA-Y674/Y675 phosphorylation is detected when SHP-1 protein levels decrease in a wtp53-dependent manner. Proliferation and cell-cycle assays, with cells expressing endogenous or transfected wt-trkA and a temperature-sensitive p53 grown at 32 degrees C (when p53 is in the wt configuration), show suppressed cell proliferation. Suppression is not detected when grown at 37 degrees C (when p53 is in the mutant configuration). A release from suppression is observed when these cells are transiently transfected with wt-SHP-1 and grown at 32 degrees C. Suppression is also detected when, as control, wt-trkA-expressing cells are transiently transfected with SHP-1-siRNA, but not when a dominant-negative (DN) mutant trkA is used to abolish wt-trkA activity. Importantly, suppression is not seen with control trkA-negative breast-cancer cells (expressing wtp53, wt-SHP-1 and undetectable trkA), transfected with Y674F/Y675F mutant-trkA. BrdU-incorporation experiments reveal lack of incorporation in cells expressing wt-trkA and wtp53, or wt-trkA and SHP-1-siRNA. However, BrdU is incorporated in the presence of Y674F/Y675F mutant trkA or DN mutant trkA. These results indicate that p53 repression of SHP-1 expression leads to trkA-Y674/Y675 phosphorylation and trkA-dependent suppression of breast-cancer cell proliferation. These data provide an explanation as to why high trkA levels are associated with favourable prognosis.
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Affiliation(s)
- X Montano
- Division of Cell and Molecular Biology, Molecular Signalling Group, Imperial College London, London SW7 2AZ, UK.
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Maures TJ, Chen L, Carter-Su C. Nucleocytoplasmic shuttling of the adapter protein SH2B1beta (SH2-Bbeta) is required for nerve growth factor (NGF)-dependent neurite outgrowth and enhancement of expression of a subset of NGF-responsive genes. Mol Endocrinol 2009; 23:1077-91. [PMID: 19372237 DOI: 10.1210/me.2009-0011] [Citation(s) in RCA: 25] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/19/2022] Open
Abstract
The adapter protein SH2B1 (SH2-B, PSM) is recruited to multiple ligand-activated receptor tyrosine kinases, including the receptors for nerve growth factor (NGF), insulin, and IGF-I as well as the cytokine receptor-associated Janus kinase family kinases. In this study, we examine SH2B1's function in NGF signaling. We show that depleting endogenous SH2B1 using short hairpin RNA against SH2B1 inhibits NGF-dependent neurite outgrowth, but not NGF-mediated phosphorylation of Akt or ERKs 1/2. SH2B1 has been hypothesized to localize and function at the plasma membrane. We identify a nuclear localization signal within SH2B1 and show that it is required for nuclear translocation of SH2B1beta. Mutation of the nuclear localization signal has no effect on NGF-induced activation of TrkA and ERKs 1/2 but prevents SH2B1beta from enhancing NGF-induced neurite outgrowth. Disruption of SH2B1beta nuclear import also prevents SH2B1beta from enhancing NGF-induced transcription of genes important for neuronal differentiation, including those encoding urokinase plasminogen activator receptor, and matrix metalloproteinases 3 and 10. Disruption of SH2B1beta nuclear export by mutation of its nuclear export sequence similarly prevents SH2B1beta enhancement of NGF-induced transcription of those genes. Nuclear translocation of the highly homologous family member SH2B2(APS) was not observed. Together, these data suggest that rather than simply acting as an adapter protein linking signaling proteins to the activated TrkA receptor at the plasma membrane, SH2B1beta must shuttle between the plasma membrane and nucleus to function as a critical component of NGF-induced gene expression and neuronal differentiation.
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Affiliation(s)
- Travis J Maures
- Program in Cellular and Molecular Biology, University of Michigan Medical School, Ann Arbor, Michigan 48109-5622, USA
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Lin WF, Chen CJ, Chang YJ, Chen SL, Chiu IM, Chen L. SH2B1beta enhances fibroblast growth factor 1 (FGF1)-induced neurite outgrowth through MEK-ERK1/2-STAT3-Egr1 pathway. Cell Signal 2009; 21:1060-72. [PMID: 19249349 DOI: 10.1016/j.cellsig.2009.02.009] [Citation(s) in RCA: 39] [Impact Index Per Article: 2.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/19/2008] [Revised: 02/05/2009] [Accepted: 02/17/2009] [Indexed: 11/27/2022]
Abstract
Genetic studies have established the crucial roles of FGF signaling, FGF-induced gene expression and morphogenesis during embryogenesis. In this study, we showed that overexpressing a signaling adaptor protein, SH2B1beta, enhanced FGF1-induced neurite outgrowth in PC12 cells. SH2B1beta has previously been shown to promote nerve growth factor (NGF) and glial cell line-derived neurotrophic factor (GDNF)-induced neurite outgrowth, in part, through prolonging NGF and GDNF-induced signaling. To delineate how SH2B1beta promotes FGF1-induced neurite outgrowth, we examined its role in FGF1-dependent signaling. Our data suggest that SH2B1beta enhances and prolongs FGF1-induced MEK-ERK1/2 and PI3K-AKT pathways. We also provided the first evidence that FGF1 induces the phosphorylation of signal transducer and activator of transcription 3 (STAT3) at serine 727 [pSTAT3(S727)] in PC12 cells. SH2B1beta enhances this phosphorylation and the expression of the immediate early gene, Egr1. Through inhibitor assays, we have further shown that MEK-ERK1/2 is required for FGF1-induced neurite outgrowth, pSTAT3(S727) and Egr1 expression. Moreover, inhibiting Rho kinase, ROCK, enhances FGF1-induced neurite outgrowth through pSTAT3(S727)-independent manner. Taken together, our results demonstrate, for the first time, that SH2B1beta enhances FGF1-induced neurite outgrowth in PC12 cells mainly through MEK-ERK1/2-STAT3-Egr1 pathway.
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Affiliation(s)
- Wei-Fan Lin
- Institute of Molecular Medicine, Department of Life Science and Brain Research Center, National Tsing Hua University, 101 Section 2 Kuang-Fu Road, Hsinchu, Taiwan
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Abstract
Neurotrophins were christened in consideration of their actions on the nervous system and, for a long time, they were the exclusive interest of neuroscientists. However, more recently, this family of proteins has been shown to possess essential cardiovascular functions. During cardiovascular development, neurotrophins and their receptors are essential factors in the formation of the heart and critical regulator of vascular development. Postnatally, neurotrophins control the survival of endothelial cells, vascular smooth muscle cells, and cardiomyocytes and regulate angiogenesis and vasculogenesis, by autocrine and paracrine mechanisms. Recent studies suggest the capacity of neurotrophins, via their tropomyosin-kinase receptors, to promote therapeutic neovascularization in animal models of hindlimb ischemia. Conversely, the neurotrophin low-affinity p75(NTR) receptor induces apoptosis of endothelial cells and vascular smooth muscle cells and impairs angiogenesis. Finally, nerve growth factor looks particularly promising in treating microvascular complications of diabetes or reducing cardiomyocyte apoptosis in the infarcted heart. These seminal discoveries have fuelled basic and translational research and thus opened a new field of investigation in cardiovascular medicine and therapeutics. Here, we review recent progress on the molecular signaling and roles played by neurotrophins in cardiovascular development, function, and pathology, and we discuss therapeutic potential of strategies based on neurotrophin manipulation.
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Affiliation(s)
- Andrea Caporali
- Division of Experimental Cardiovascular Medicine, University of Bristol, Bristol, UK
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Zhang M, Deng Y, Riedel H. PSM/SH2B1 splice variants: critical role in src catalytic activation and the resulting STAT3s-mediated mitogenic response. J Cell Biochem 2008; 104:105-18. [PMID: 18247337 DOI: 10.1002/jcb.21606] [Citation(s) in RCA: 11] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/16/2022]
Abstract
A role of PSM/SH2B1 had been shown in mitogenesis and extending to phenotypic cell transformation, however, the underlying molecular mechanism remained to be established. Here, four alternative PSM splice variants and individual functional protein domains were compared for their role in the regulation of Src activity. We found that elevated cellular levels of PSM variants resulted in phenotypic cell transformation and potentiated cell proliferation and survival in response to serum withdrawal. PSM variant activity presented a consistent signature pattern for any tested response of highest activity observed for gamma, followed by delta, alpha, and beta with decreasing activity. PSM-potentiated cell proliferation was sensitive to Src inhibitor herbimycin and PSM and Src were found in the same immune complex. PSM variants were substrates of the Src Tyr kinase and potentiated Src catalytic activity by increasing the V(max) and decreasing the K(m) for ATP with the signature pattern of variant activity. Dominant-negative PSM peptide mimetics including the SH2 or PH domains inhibited Src catalytic activity as well as Src-mediated phenotypic cell transformation. Activation of major Src substrate STAT3 was similarly potentiated by the PSM variants in a Src-dependent fashion or inhibited by PSM domain-specific peptide mimetics. Expression of a dominant-negative STAT3 mutant blocked PSM variant-mediated phenotypic cell transformation. Our results implicate an essential role of the PSM variants in the activation of the Src kinase and the resulting mitogenic response--extending to phenotypic cell transformation and involving the established Src substrate STAT3.
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Affiliation(s)
- Manchao Zhang
- Department of Biochemistry, West Virginia University, School of Medicine, Morgantown, West Virginia 26506-9142, USA
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26
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Yamanashi Y, Higuch O, Beeson D. Dok-7/MuSK signaling and a congenital myasthenic syndrome. ACTA MYOLOGICA : MYOPATHIES AND CARDIOMYOPATHIES : OFFICIAL JOURNAL OF THE MEDITERRANEAN SOCIETY OF MYOLOGY 2008; 27:25-9. [PMID: 19108574 PMCID: PMC2859609] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Subscribe] [Scholar Register] [Indexed: 05/27/2023]
Abstract
Skeletal muscle contraction is controlled by motor neurons, which contact the muscle at the neuromuscular junction (NMJ). The formation and maintenance of the NMJ, which includes the aggregation of densely packed clusters of acetylcholine receptor (AChR) opposite the motor nerve terminal, is orchestrated by muscle-specific receptor tyrosine kinase, MuSK. Recently, a MuSK-interacting cytoplasmic adaptor-like protein Dok-7 was identified and its localization at the postsynaptic region of the NMJ was revealed. Mice lacking Dok-7 have a phenotype indistinguishable from MuSK-deficient mice, and fail to form both AChR clusters and NMJs. In cultured myotubes, Dok-7 is required for MuSK activation and AChR clustering. Thus, Dok-7 is essential for neuromuscular synaptogenesis and it appears that the regulatory interaction of Dok-7 with MuSK is integrally involved in this process. In humans there are both autoimmune and genetic causes of defective neuromuscular transmission that gives rise to the fatigable muscle weakness known as myasthenia. DOK7 has been found to be a major locus for mutations that underlie a genetic form of myasthenia with a characteristic 'limb girdle' pattern of muscle weakness (DOK7 CMS). Patients with DOK7 CMS have small, simplified NMJs but normal AChR function. The most common mutation causes a COOH-terminal truncation, which greatly impairs Dok-7's ability to activate MuSK. Recently, a series of differing DOK7 mutations have been identified, which affect not only the COOH-terminal region but also the NH2-terminal moiety. The study of these mutations may help understand the underlying pathogenic mechanism of DOK7 CMS.
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Affiliation(s)
- Y Yamanashi
- Division of Genetics, Department of Cancer Biology, The Institute of Medical Science, The University of Tokyo, Japan.
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27
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Zhang M, Deng Y, Tandon R, Bai C, Riedel H. Essential role of PSM/SH2-B variants in insulin receptor catalytic activation and the resulting cellular responses. J Cell Biochem 2008; 103:162-81. [PMID: 17615553 DOI: 10.1002/jcb.21397] [Citation(s) in RCA: 14] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/12/2022]
Abstract
The positive regulatory role of PSM/SH2-B downstream of various mitogenic receptor tyrosine kinases or gene disruption experiments in mice support a role of PSM in the regulation of insulin action. Here, four alternative PSM splice variants and individual functional domains were compared for their role in the regulation of specific metabolic insulin responses. We found that individual PSM variants in 3T3-L1 adipocytes potentiated insulin-mediated glucose and amino acid transport, glycogenesis, lipogenesis, and key components in the metabolic insulin response including p70 S6 kinase, glycogen synthase, glycogen synthase kinase 3 (GSK3), Akt, Cbl, and IRS-1. Highest activity was consistently observed for PSM alpha, followed by beta, delta, and gamma with decreasing activity. In contrast, dominant-negative peptide mimetics of the PSM Pro-rich, pleckstrin homology (PH), or src homology 2 (SH2) domains inhibited any tested insulin response. Potentiation of the insulin response originated at the insulin receptor (IR) kinase level by PSM variant-specific regulation of the Km (ATP) whereas the Vmax remained unaffected. IR catalytic activation was inhibited by peptide mimetics of the PSM SH2 or dimerization domain (DD). Either peptide should disrupt the complex of a PSM dimer linked to IR via SH2 domains as proposed for PSM activation of tyrosine kinase JAK2. Either peptide abolished downstream insulin responses indistinguishable from PSM siRNA knockdown. Our results implicate an essential role of the PSM variants in the activation of the IR kinase and the resulting metabolic insulin response. PSM variants act as internal IR ligands that in addition to potentiating the insulin response stimulate IR catalytic activation even in the absence of insulin.
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Affiliation(s)
- Manchao Zhang
- Department of Biochemistry, West Virginia University, School of Medicine, Morgantown, WV 26506-9142, USA
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Chen L, Maures TJ, Jin H, Huo JS, Rabbani SA, Schwartz J, Carter-Su C. SH2B1beta (SH2-Bbeta) enhances expression of a subset of nerve growth factor-regulated genes important for neuronal differentiation including genes encoding urokinase plasminogen activator receptor and matrix metalloproteinase 3/10. Mol Endocrinol 2007; 22:454-76. [PMID: 17947375 DOI: 10.1210/me.2007-0384] [Citation(s) in RCA: 32] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/19/2022] Open
Abstract
Previous work showed that the adapter protein SH2B adapter protein 1beta (SH2B1) (SH2-B) binds to the activated form of the nerve growth factor (NGF) receptor TrkA and is critical for both NGF-dependent neurite outgrowth and maintenance. To identify SH2B1beta-regulated genes critical for neurite outgrowth, we performed microarray analysis of control PC12 cells and PC12 cells stably overexpressing SH2B1beta (PC12-SH2B1beta) or the dominant-negative SH2B1beta(R555E) [PC12-SH2B1beta(R555E)]. NGF-induced microarray expression of Plaur and Mmp10 genes was greatly enhanced in PC12-SH2B1beta cells, whereas NGF-induced Plaur and Mmp3 expression was substantially depressed in PC12-SH2B1beta(R555E) cells. Plaur, Mmp3, and Mmp10 are among the 12 genes most highly up-regulated after 6 h of NGF. Their protein products [urokinase plasminogen activator receptor (uPAR), matrix metalloproteinase 3 (MMP3), and MMP10] lie in the same pathway of extracellular matrix degradation; uPAR has been shown previously to be critical for NGF-induced neurite outgrowth. Quantitative real-time PCR analysis revealed SH2B1beta enhancement of NGF induction of all three genes and the suppression of NGF induction of all three when endogenous SH2B1 was reduced using short hairpin RNA against SH2B1 and in PC12-SH2B1beta(R555E) cells. NGF-induced levels of uPAR and MMP3/10 and neurite outgrowth through Matrigel (MMP3-dependent) were also increased in PC12-SH2B1beta cells. These results suggest that SH2B1beta stimulates NGF-induced neuronal differentiation at least in part by enhancing expression of a specific subset of NGF-sensitive genes, including Plaur, Mmp3, and/or Mmp10, required for neurite outgrowth.
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Affiliation(s)
- Linyi Chen
- Department of Molecular and Integrative Physiology, The University of Michigan Medical School, Ann Arbor, MI 48109-0622, USA
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Li Z, Zhou Y, Carter-Su C, Myers MG, Rui L. SH2B1 enhances leptin signaling by both Janus kinase 2 Tyr813 phosphorylation-dependent and -independent mechanisms. Mol Endocrinol 2007; 21:2270-81. [PMID: 17565041 DOI: 10.1210/me.2007-0111] [Citation(s) in RCA: 86] [Impact Index Per Article: 4.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/16/2023] Open
Abstract
Leptin controls body weight by activating its long form receptor (LEPRb). LEPRb binds to Janus kinase 2 (JAK2), a cytoplasmic tyrosine kinase that mediates leptin signaling. We previously reported that genetic deletion of SH2B1 (previously known as SH2-B), a JAK2-binding protein, results in severe leptin-resistant and obese phenotypes, indicating that SH2B1 is a key endogenous positive regulator of leptin sensitivity. Here we show that SH2B1 regulates leptin signaling by multiple mechanisms. In the absence of leptin, SH2B1 constitutively bound, via its non-SH2 domain region(s), to non-tyrosyl-phosphorylated JAK2, and inhibited JAK2. Leptin stimulated JAK2 phosphorylation on Tyr(813), which subsequently bound to the SH2 domain of SH2B1. Binding of the SH2 domain of SH2B1 to phospho-Tyr(813) in JAK2 enhanced leptin induction of JAK2 activity. JAK2 was required for leptin-stimulated phosphorylation of insulin receptor substrate 1 (IRS1), an upstream activator of the phosphatidylinositol 3-kinase pathway. Overexpression of SH2B1 enhanced both JAK2- and JAK2(Y813F)-mediated tyrosine phosphorylation of IRS1 in response to leptin, even though SH2B1 did not enhance JAK2(Y813F) activation. Leptin promoted the interaction of SH2B1 with IRS1. These data suggest that constitutive SH2B1-JAK2 interaction, mediated by the non-SH2 domain region(s) of SH2B1 and the non-Tyr(813) region(s) in JAK2, increases the local concentration of SH2B1 close to JAK2 and inhibits JAK2 activity. Leptin-stimulated SH2B1-JAK2 interaction, mediated by the SH2 domain of SH2B1 and phospho-Tyr(813) in JAK2, promotes JAK2 activation, thus globally enhancing leptin signaling. SH2B1-IRS1 interaction facilitates IRS1 phosphorylation by recruiting IRS1 to JAK2 and/or by protecting IRS1 from dephosphorylation, thus specifically enhancing leptin stimulation of the phosphatidylinositol 3-kinase pathway.
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Affiliation(s)
- Zhiqin Li
- Department of Molecular & Integrative Physiology, University of Michigan Medical School, Ann Arbor, Michigan 48109-0622, USA
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30
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Limpert AS, Karlo JC, Landreth GE. Nerve growth factor stimulates the concentration of TrkA within lipid rafts and extracellular signal-regulated kinase activation through c-Cbl-associated protein. Mol Cell Biol 2007; 27:5686-98. [PMID: 17548467 PMCID: PMC1952120 DOI: 10.1128/mcb.01109-06] [Citation(s) in RCA: 59] [Impact Index Per Article: 3.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/12/2023] Open
Abstract
Nerve growth factor (NGF) acts through its receptor, TrkA, to elicit the neuronal differentiation of PC12 cells through the action of extracellular signal-regulated kinase 1 (ERK1) and ERK2. Upon NGF binding, TrkA translocates and concentrates in cholesterol-rich membrane microdomains or lipid rafts, facilitating formation of receptor-associated signaling complexes, activation of downstream signaling pathways, and internalization into endosomes. We have investigated the mechanisms responsible for the localization of TrkA within lipid rafts and its ability to activate ERK1 and ERK2. We report that NGF treatment results in the translocation of activated forms of TrkA to lipid rafts, and this localization is important for efficient activation of the ERKs. TrkA is recruited and retained within lipid rafts through its association with flotillin, an intrinsic constituent of these membrane microdomains, via the adapter protein, c-Cbl associated protein (CAP). Mutant forms of CAP that lack protein interaction domains block TrkA localization to lipid rafts and attenuate ERK activation. Importantly, suppression of endogenous CAP expression inhibited NGF-stimulated neurite outgrowth from primary dorsal root ganglion neurons. These data provide a mechanism for the lipid raft localization of TrkA and establish the importance of the CAP adaptor protein for NGF activation of the ERKs and neuronal differentiation.
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Affiliation(s)
- Allison S Limpert
- Department of Neurosciences, Alzheimer Research Laboratory, Case Western Reserve University School of Medicine, Cleveland, OH 44106-4928, USA
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31
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Donatello S, Fiorino A, Degl'Innocenti D, Alberti L, Miranda C, Gorla L, Bongarzone I, Rizzetti MG, Pierotti MA, Borrello MG. SH2B1beta adaptor is a key enhancer of RET tyrosine kinase signaling. Oncogene 2007; 26:6546-59. [PMID: 17471236 DOI: 10.1038/sj.onc.1210480] [Citation(s) in RCA: 21] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/12/2022]
Abstract
The RET gene encodes two main isoforms of a receptor tyrosine kinase (RTK) implicated in various human diseases. Activating germ-line point mutations are responsible for multiple endocrine neoplasia type 2-associated medullary thyroid carcinomas, inactivating germ-line mutations for Hirschsprung's disease, while somatic rearrangements (RET/PTCs) are specific to papillary thyroid carcinomas. SH2B1beta, a member of the SH2B adaptors family, and binding partner for several RTKs, has been recently described to interact with proto-RET. Here, we show that both RET isoforms and its oncogenic derivatives bind to SH2B1beta through the SRC homology 2 (SH2) domain and a kinase activity-dependent mechanism. As a result, RET phosphorylates SH2B1beta, which in turn enhances its autophosphorylation, kinase activity, and downstream signaling. RET tyrosine residues 905 and 981 are important determinants for functional binding of the adaptor, as removal of both autophosphorylation sites displaces its recruitment. Binding of SH2B1beta appears to protect RET from dephosphorylation by protein tyrosine phosphatases, and might represent a likely mechanism contributing to its upregulation. Thus, overexpression of SH2B1beta, by enhancing phosphorylation/activation of RET transducers, potentiates the cellular differentiation and the neoplastic transformation thereby induced, and counteracts the action of RET inhibitors. Overall, our results identify SH2B1beta as a key enhancer of RET physiologic and pathologic activities.
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Affiliation(s)
- S Donatello
- Department of Experimental Oncology, Research Unit no. 3, Milan, Italy
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32
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Li M, Li Z, Morris DL, Rui L. Identification of SH2B2beta as an inhibitor for SH2B1- and SH2B2alpha-promoted Janus kinase-2 activation and insulin signaling. Endocrinology 2007; 148:1615-21. [PMID: 17204555 PMCID: PMC4710543 DOI: 10.1210/en.2006-1010] [Citation(s) in RCA: 33] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 12/18/2022]
Abstract
The SH2B family has three members (SH2B1, SH2B2, and SH2B3) that contain conserved dimerization (DD), pleckstrin homology, and SH2 domains. The DD domain mediates the formation of homo- and heterodimers between members of the SH2B family. The SH2 domain of SH2B1 (previously named SH2-B) or SH2B2 (previously named APS) binds to phosphorylated tyrosines in a variety of tyrosine kinases, including Janus kinase-2 (JAK2) and the insulin receptor, thereby promoting the activation of JAK2 or the insulin receptor, respectively. JAK2 binds to various members of the cytokine receptor family, including receptors for GH and leptin, to mediate cytokine responses. In mice, SH2B1 regulates energy and glucose homeostasis by enhancing leptin and insulin sensitivity. In this work, we identify SH2B2beta as a new isoform of SH2B2 (designated as SH2B2alpha) derived from the SH2B2 gene by alternative mRNA splicing. SH2B2beta has a DD and pleckstrin homology domain but lacks a SH2 domain. SH2B2beta bound to both SH2B1 and SH2B2alpha, as demonstrated by both the interaction of glutathione S-transferase-SH2B2beta fusion protein with SH2B1 or SH2B2alpha in vitro and coimmunoprecipitation of SH2B2beta with SH2B1 or SH2B2alpha in intact cells. SH2B2beta markedly attenuated the ability of SH2B1 to promote JAK2 activation and subsequent tyrosine phosphorylation of insulin receptor substrate-1 by JAK2. SH2B2beta also significantly inhibited SH2B1- or SH2B2alpha-promoted insulin signaling, including insulin-stimulated tyrosine phosphorylation of insulin receptor substrate-1. These data suggest that SH2B2beta is an endogenous inhibitor of SH2B1 and/or SH2B2alpha, negatively regulating insulin signaling and/or JAK2-mediated cellular responses.
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Affiliation(s)
- Minghua Li
- Department of Molecular and Integrative Physiology, University of Michigan Medical School, Ann Arbor, Michigan 48109-0622, USA
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33
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Deng Y, Xu H, Riedel H. PSM/SH2-B distributes selected mitogenic receptor signals to distinct components in the PI3-kinase and MAP kinase signaling pathways. J Cell Biochem 2007; 100:557-73. [PMID: 16960871 DOI: 10.1002/jcb.21030] [Citation(s) in RCA: 7] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/26/2022]
Abstract
The Pro-rich, PH, and SH2 domain containing mitogenic signaling adapter PSM/SH2-B has been implicated as a cellular partner of various mitogenic receptor tyrosine kinases and related signaling mechanisms. Here, we report in a direct comparison of three peptide hormones, that PSM participates in the assembly of distinct mitogenic signaling complexes in response to insulin or IGF-I when compared to PDGF in cultured normal fibroblasts. The complex formed in response to insulin or IGF-I involves the respective peptide hormone receptor and presumably the established components leading to MAP kinase activation. However, our data suggest an alternative link from the PDGF receptor via PSM directly to MEK1/2 and consequently also to p44/42 activation, possibly through a scaffold protein. At least two PSM domains participate, the SH2 domain anticipated to link PSM to the respective receptor and the Pro-rich region in an association with an unidentified downstream component resulting in direct MEK1/2 and p44/42 regulation. The PDGF receptor signaling complex formed in response to PDGF involves PI 3-kinase in addition to the same components and interactions as described for insulin or IGF-I. PSM associates with PI 3-kinase via p85 and in addition the PSM PH domain participates in the regulation of PI 3-kinase activity, presumably through membrane interaction. In contrast, the PSM Pro-rich region appears to participate only in the MAP kinase signal. Both pathways contribute to the mitogenic response as shown by cell proliferation, survival, and focus formation. PSM regulates p38 MAP kinase activity in a pathway unrelated to the mitogenic response.
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Affiliation(s)
- Youping Deng
- Department of Biological Sciences, The University of Southern Mississippi, Hattiesburg, MS 39406, USA
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34
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Nikolaidis N, Chalkia D, Watkins DN, Barrow RK, Snyder SH, van Rossum DB, Patterson RL. Ancient origin of the new developmental superfamily DANGER. PLoS One 2007; 2:e204. [PMID: 17301879 PMCID: PMC1784063 DOI: 10.1371/journal.pone.0000204] [Citation(s) in RCA: 14] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/13/2006] [Accepted: 01/14/2007] [Indexed: 01/15/2023] Open
Abstract
Developmental proteins play a pivotal role in the origin of animal complexity and diversity. We report here the identification of a highly divergent developmental protein superfamily (DANGER), which originated before the emergence of animals (∼850 million years ago) and experienced major expansion-contraction events during metazoan evolution. Sequence analysis demonstrates that DANGER proteins diverged via multiple mechanisms, including amino acid substitution, intron gain and/or loss, and recombination. Divergence for DANGER proteins is substantially greater than for the prototypic member of the superfamily (Mab-21 family) and other developmental protein families (e.g., WNT proteins). DANGER proteins are widely expressed and display species-dependent tissue expression patterns, with many members having roles in development. DANGER1A, which regulates the inositol trisphosphate receptor, promotes the differentiation and outgrowth of neuronal processes. Regulation of development may be a universal function of DANGER family members. This family provides a model system to investigate how rapid protein divergence contributes to morphological complexity.
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Affiliation(s)
- Nikolas Nikolaidis
- Biology Department, Pennsylvania State University, University Park, Pennsylvania, United States of America
- * To whom correspondence should be addressed. E-mail: (RP); (NN)
| | - Dimitra Chalkia
- Biology Department, Pennsylvania State University, University Park, Pennsylvania, United States of America
| | - D. Neil Watkins
- The Sidney Kimmel Cancer Institute, Johns Hopkins University, School of Medicine, Baltimore, Maryland, United States of America
| | - Roxanne K. Barrow
- The Solomon H. Snyder Department of Neuroscience, Johns Hopkins University, School of Medicine, Baltimore, Maryland, United States of America
| | - Solomon H. Snyder
- The Solomon H. Snyder Department of Neuroscience, Johns Hopkins University, School of Medicine, Baltimore, Maryland, United States of America
- Department of Pharmacology and Molecular Science, Johns Hopkins University, Baltimore, Maryland, United States of America
- Department of Psychiatry and Behavioral Sciences, Johns Hopkins University, Baltimore, Maryland, United States of America
| | - Damian B. van Rossum
- Biology Department, Pennsylvania State University, University Park, Pennsylvania, United States of America
| | - Randen L. Patterson
- Biology Department, Pennsylvania State University, University Park, Pennsylvania, United States of America
- * To whom correspondence should be addressed. E-mail: (RP); (NN)
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Kishi K, Mawatari K, Sakai-Wakamatsu K, Yuasa T, Wang M, Ogura-Sawa M, Nakaya Y, Hatakeyama S, Ebina Y. APS-mediated ubiquitination of the insulin receptor enhances its internalization, but does not induce its degradation. Endocr J 2007; 54:77-88. [PMID: 17102568 DOI: 10.1507/endocrj.k06-056] [Citation(s) in RCA: 16] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 11/23/2022] Open
Abstract
APS, a tyrosine kinase adaptor protein with pleckstrin homology and Src homology 2 domains, is rapidly and strongly tyrosine-phosphorylated by insulin receptor kinase upon insulin stimulation. We have previously shown that APS knockout mice have increased insulin sensitivity, and that this enhancement is possibly due to increased insulin-response on adipose tissues. However, the function of APS in insulin signaling has so far been controversial. Here, we report that APS enhanced ligand-dependent multi-ubiquitination of the insulin receptor (IR) in CHO cells overexpressing the IR. APS-mediated ubiquitination of the IR induced enhancement of the IR internalization, but did not affect the IR degradation. This finding shows one of the pleiotropic functions of APS in insulin signaling.
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Affiliation(s)
- Kazuhiro Kishi
- Division of Molecular Genetics, Institute for Enzyme Research, The University of Tokushima, Japan
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36
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Maures TJ, Kurzer JH, Carter-Su C. SH2B1 (SH2-B) and JAK2: a multifunctional adaptor protein and kinase made for each other. Trends Endocrinol Metab 2007; 18:38-45. [PMID: 17140804 DOI: 10.1016/j.tem.2006.11.007] [Citation(s) in RCA: 88] [Impact Index Per Article: 4.9] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 09/11/2006] [Revised: 11/13/2006] [Accepted: 11/20/2006] [Indexed: 12/28/2022]
Abstract
Src homology 2 (SH2) B adaptor protein 1 (SH2B1; originally named SH2-B) is a member of a family of adaptor proteins that influences a variety of signaling pathways mediated by Janus kinase (JAK) and receptor tyrosine kinases. Although SH2B1 performs classical adaptor functions, such as recruitment of specific proteins to activated receptors, it also demonstrates a unique ability to enhance the kinase activity of the cytokine receptor-associated tyrosine kinase JAK2, as well as that of several receptor tyrosine kinases. SH2B1 is also among a small number of adaptor proteins shown to undergo nucleocytoplasmic shuttling, although its exact role within the nucleus is not yet clear. Deletion of the SH2B1 gene results in severe obesity and both leptin and insulin resistance, as well as infertility, which might be a consequence of resistance to insulin-like growth factor I. Thus, knockout mice support a role for SH2B1 as a positive regulator of JAK2 signaling pathways initiated by leptin, as well as of pathways initiated by insulin and, potentially, by insulin-like growth factor I.
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Affiliation(s)
- Travis J Maures
- Graduate Program in Cellular and Molecular Biology, University of Michigan Medical School, Ann Arbor, MI 48109-0662, USA
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Abstract
Neurotrophins are a family of closely related proteins that were identified initially as survival factors for sensory and sympathetic neurons, and have since been shown to control many aspects of survival, development and function of neurons in both the peripheral and the central nervous systems. Each of the four mammalian neurotrophins has been shown to activate one or more of the three members of the tropomyosin-related kinase (Trk) family of receptor tyrosine kinases (TrkA, TrkB and TrkC). In addition, each neurotrophin activates p75 neurotrophin receptor (p75NTR), a member of the tumour necrosis factor receptor superfamily. Through Trk receptors, neurotrophins activate Ras, phosphatidyl inositol-3 (PI3)-kinase, phospholipase C-gamma1 and signalling pathways controlled through these proteins, such as the MAP kinases. Activation of p75NTR results in activation of the nuclear factor-kappaB (NF-kappaB) and Jun kinase as well as other signalling pathways. Limiting quantities of neurotrophins during development control the number of surviving neurons to ensure a match between neurons and the requirement for a suitable density of target innervation. The neurotrophins also regulate cell fate decisions, axon growth, dendrite growth and pruning and the expression of proteins, such as ion channels, transmitter biosynthetic enzymes and neuropeptide transmitters that are essential for normal neuronal function. Continued presence of the neurotrophins is required in the adult nervous system, where they control synaptic function and plasticity, and sustain neuronal survival, morphology and differentiation. They also have additional, subtler roles outside the nervous system. In recent years, three rare human genetic disorders, which result in deleterious effects on sensory perception, cognition and a variety of behaviours, have been shown to be attributable to mutations in brain-derived neurotrophic factor and two of the Trk receptors.
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Affiliation(s)
- Louis F Reichardt
- Neuroscience Program, Department of Physiology and Howard Hughes Medical Institute, University of California-San Francisco, 1550 Fourth Street, Rock Hall 284a, San Francisco, CA 94158, USA.
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38
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Kurzer JH, Saharinen P, Silvennoinen O, Carter-Su C. Binding of SH2-B family members within a potential negative regulatory region maintains JAK2 in an active state. Mol Cell Biol 2006; 26:6381-94. [PMID: 16914724 PMCID: PMC1592834 DOI: 10.1128/mcb.00570-06] [Citation(s) in RCA: 34] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/09/2023] Open
Abstract
The tyrosine kinase Janus kinase 2 (JAK2) transduces signaling for the majority of known cytokine receptor family members and is constitutively activated in some cancers. Here we examine the mechanisms by which the adapter proteins SH2-Bbeta and APS regulate the activity of JAK2. We show that like SH2-Bbeta, APS binds JAK2 at multiple sites and that binding to phosphotyrosine 813 is essential for APS to increase active JAK2 and to be phosphorylated by JAK2. Binding of APS to a phosphotyrosine 813-independent site inhibits JAK2. Both APS and SH2-Bbeta increase JAK2 activity independent of their N-terminal dimerization domains. SH2-Bbeta-induced increases in JAK2 dimerization require only the SH2 domain and only one SH2-Bbeta to be bound to a JAK2 dimer. JAK2 mutations and truncations revealed that amino acids 809 to 811 in JAK2 are a critical component of a larger regulatory region within JAK2, most likely including amino acids within the JAK homology 1 (JH1) and JH2 domains and possibly the FERM domain. Together, our data suggest that SH2-Bbeta and APS do not activate JAK2 as a consequence of their own dimerization, recruitment of an activator of JAK2, or direct competition with a JAK2 inhibitor for binding to JAK2. Rather, they most likely induce or stabilize an active conformation of JAK2.
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Affiliation(s)
- Jason H Kurzer
- Department of Molecular and Integrative Physiology, The University of Michigan Medical School, Ann Arbor, MI 48109-0622, USA
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Pyle AD, Lock LF, Donovan PJ. Neurotrophins mediate human embryonic stem cell survival. Nat Biotechnol 2006; 24:344-50. [PMID: 16444268 DOI: 10.1038/nbt1189] [Citation(s) in RCA: 199] [Impact Index Per Article: 10.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/02/2005] [Accepted: 12/06/2005] [Indexed: 12/30/2022]
Abstract
Growth of human embryonic stem (hES) cells as a pluripotent population requires a balance between survival, proliferation and self-renewal signals. Here we demonstrate that hES cells express receptors of the tropomyosin-related kinase (TRK) family, which mediate antiapoptotic signals. We show that three TRK ligands, brain-derived neurotrophic factor, neurotrophin 3 and neurotrophin 4, are survival factors for hES cells. Addition of neurotrophins to hES cell cultures effects a 36-fold improvement in their clonal survival. hES cell cultures maintained in medium containing neurotrophins remain diploid and retain full developmental potency. In the presence of neurotrophins, TRK receptors in hES cells are phosphorylated; TRK receptor inhibition leads to hES cell apoptosis. The survival activity of neurotrophins in hES cells is mediated by the phosphatidylinositol-3-kinase pathway but not the mitogen-activated protein kinase pathway. Neurotrophins improve hES cell survival and may facilitate their manipulation and the development of high-throughput screens to identify factors responsible for hES cell differentiation.
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Affiliation(s)
- April D Pyle
- Germ Cell and Stem Cell Group, Institute for Cell Engineering, Johns Hopkins University, School of Medicine, Broadway Research Building, Suite 759, 733 North Broadway, Baltimore, Maryland 21205, USA
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Yan C, Mirnics ZK, Portugal CF, Liang Y, Nylander KD, Rudzinski M, Zaccaro C, Saragovi HU, Schor NF. Cholesterol biosynthesis and the pro-apoptotic effects of the p75 nerve growth factor receptor in PC12 pheochromocytoma cells. ACTA ACUST UNITED AC 2006; 139:225-34. [PMID: 15967538 DOI: 10.1016/j.molbrainres.2005.05.025] [Citation(s) in RCA: 24] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/05/2004] [Revised: 04/22/2005] [Accepted: 05/12/2005] [Indexed: 11/15/2022]
Abstract
Neocarzinostatin (NCS), an enediyne antimitotic agent, induces cell death in both p75NTR neurotrophin receptor (NTR)-positive and p75NTR-negative PC12 cells in a concentration-dependent fashion. However, p75NTR-positive cells demonstrate a higher susceptibility to NCS-induced cell damage. Furthermore, treatment of p75NTR-positive cells with the p75NTR-specific ligand, MC192, resulted in apoptosis, while treatment of these cells with the TrkA-specific ligand, NGF-mAbNGF30, protected them from NCS-induced death, implying that both the naked and liganded p75NTR receptors have a pro-apoptotic effect on PC12 cells. Microarray studies aimed at examining differential gene expression between p75NTR-positive and p75NTR-negative cells suggested that enzymes of the cholesterol biosynthetic pathway are differentially expressed. We therefore tested the hypothesis that altered cholesterol biosynthesis contributes directly to the pro-apoptotic effects of p75NTR in this PC12 cell-NCS model. Subsequent Northern blotting studies confirmed that the expression of p75NTR is associated with the upregulation of cholesterol biosynthetic enzymes including 3-hydroxy-3-methylglutaryl CoA reductase (HMG CoA reductase), farnesyl-diphosphate synthase, and 7-dehydro-cholesterol reductase. Mevastatin, an HMG CoA reductase inhibitor, converts the apoptosis susceptibility of p75NTR-positive cells to that of p75NTR-negative cells. It does so at concentrations that do not themselves alter cell survival. These studies provide evidence that the pro-apoptotic effects of p75NTR in PC12 cells are related to the upregulation of cholesterol biosynthetic enzymes and consequent increased cholesterol biosynthesis.
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Affiliation(s)
- Chaohua Yan
- Division of Child Neurology, The Pediatric Center for Neuroscience, Children's Hospital of Pittsburgh, 3705 Fifth Avenue, Pittsburgh, PA 15213, USA
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Takizawa H, Kubo-Akashi C, Nobuhisa I, Kwon SM, Iseki M, Taga T, Takatsu K, Takaki S. Enhanced engraftment of hematopoietic stem/progenitor cells by the transient inhibition of an adaptor protein, Lnk. Blood 2005; 107:2968-75. [PMID: 16332975 DOI: 10.1182/blood-2005-05-2138] [Citation(s) in RCA: 37] [Impact Index Per Article: 1.9] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/20/2022] Open
Abstract
Hematopoietic stem cells (HSCs) are the key elements responsible for maintaining blood-cell production throughout life and for lymphohematopoietic reconstitution following bone marrow (BM) transplantation. Enhancement of the engrafting potential and expansion capabilities of HSCs as well as hematopoietic progenitor cells (HPCs) has been a long-time desire as a means of reducing the risks and difficulties that accompany BM transplantation. The ability of HSCs/HPCs to reconstitute the hematopoietic system of irradiated hosts is negatively regulated by an intracellular adaptor protein, Lnk. Here we have identified the functional domains of Lnk and developed a dominant-negative (DN) Lnk mutant that inhibits the functions of Lnk endogenously expressed in the HSCs/HPCs and thereby potentiates the HSCs/HPCs for engraftment. Importantly, even transient expression of DN-Lnk in HSCs/HPCs facilitated their engraftment under nonmyeloablative conditions and fully reconstituted the lymphoid compartments of immunodeficient host animals. HPCs expressing DN-Lnk were efficiently trapped by immobilized vascular cell adhesion molecule-1 (VCAM-1) in a transwell migration assay, suggesting involvement of Lnk in the regulation of cell mobility or cellular interaction in microenvironments. Transient inhibition of Lnk or Lnk-mediated pathways could be a potent approach to augment engraftment of HSCs/HPCs without obvious side effects.
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Affiliation(s)
- Hitoshi Takizawa
- Division of Immunology, Department of Microbiology and Immunology, Institute of Medical Science, University of Tokyo, 4-6-1 Shirokanedai, Minato-ku, Tokyo, 108-8639, Japan
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Lo KY, Chin WH, Ng YP, Cheng AW, Cheung ZH, Ip NY. SLAM-associated Protein as a Potential Negative Regulator in Trk Signaling. J Biol Chem 2005; 280:41744-52. [PMID: 16223723 DOI: 10.1074/jbc.m506554200] [Citation(s) in RCA: 14] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/06/2022] Open
Abstract
Neurotrophin signaling plays important roles in regulating the survival, differentiation, and maintenance of neurons in the nervous system. Binding of neurotrophins to their cognate receptors Trks induces transactivation and phosphorylation of the receptor at several tyrosine residues. These phosphorylated tyrosine residues then serve as crucial docking sites for adaptor proteins containing a Src homology 2 or phosphotyrosine binding domain, which upon association with the receptor initiates multiple signaling events to mediate the action of neurotrophins. Here we report the identification of a Src homology 2 domain-containing molecule, SLAM-associated protein (SAP), as an interacting protein of TrkB in a yeast two-hybrid screen. SAP was initially identified as an adaptor molecule in SLAM family receptor signaling for regulating interferon-gamma secretion. In the current study, we found that SAP interacted with TrkA, TrkB, and TrkC receptors in vitro and in vivo. Binding of SAP required Trk receptor activation and phosphorylation at the tyrosine 674 residue, which is located in the activation loop of the kinase domain. Overexpression of SAP with Trk attenuated tyrosine phosphorylation of the receptors and reduced the binding of SH2B and Shc to TrkB. Moreover, overexpression of SAP in PC12 cells suppressed the nerve growth factor-dependent activation of extracellular signal-regulated kinases 1/2 and phospholipase Cgamma, in addition to inhibiting neurite outgrowth. In summary, our findings demonstrated that SAP may serve as a negative regulator of Trk receptor activation and downstream signaling.
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Affiliation(s)
- Kin Yip Lo
- Department of Biochemistry, Biotechnology Research Institute and Molecular Neuroscience Center, Hong Kong University of Science and Technology, Clear Water Bay, Hong Kong, China
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Schramm A, Schulte JH, Astrahantseff K, Apostolov O, Limpt VV, Sieverts H, Kuhfittig-Kulle S, Pfeiffer P, Versteeg R, Eggert A. Biological effects of TrkA and TrkB receptor signaling in neuroblastoma. Cancer Lett 2005; 228:143-53. [PMID: 15921851 DOI: 10.1016/j.canlet.2005.02.051] [Citation(s) in RCA: 90] [Impact Index Per Article: 4.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/11/2005] [Accepted: 02/05/2005] [Indexed: 01/14/2023]
Abstract
The Trk family consists of three receptor tyrosine kinases, each of which can be activated by one or more of four neurotrophins-NGF, BDNF, NT3 and NT4. Neurotrophins mediate their multiple effects through a number of distinct intracellular signaling cascades regulating such diverse biological responses as cell survival, proliferation and differentiation in normal and neoplastic neuronal cells. Expression of Trk receptors also plays an important role in the biology and clinical behavior of neuroblastomas. High expression of TrkA is present in neuroblastomas with favorable biological features and highly correlated with patient survival, whereas TrkB is mainly expressed on unfavorable, aggressive neuroblastomas. This short review discusses recent data on the biological roles of TrkA and TrkB signaling in neuroblastoma.
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Affiliation(s)
- Alexander Schramm
- Division of Hematology/Oncology and Endocrinology, University Children's Hospital of Essen, Hufelandstrasse 55, 45122 Essen, Germany
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Abstract
The Grb proteins (growth factor receptor-bound proteins) Grb7, Grb10 and Grb14 constitute a family of structurally related multidomain adapters with diverse cellular functions. Grb10 and Grb14, in particular, have been implicated in the regulation of insulin receptor signalling, whereas Grb7 appears predominantly to be involved in focal adhesion kinase-mediated cell migration. However, at least in vitro, these adapters can bind to a variety of growth factor receptors. The highest identity within the Grb7/10/14 family occurs in the C-terminal SH2 (Src homology 2) domain, which mediates binding to activated receptors. A second well-conserved binding domain, BPS [between the PH (pleckstrin homology) and SH2 domains], can act to enhance binding to the IR (insulin receptor). Consistent with a putative adapter function, some non-receptor-binding partners, including protein kinases, have also been identified. Grb10 and Grb14 are widely, but not uniformly, expressed in mammalian tissues, and there are various isoforms of Grb10. Binding of Grb10 or Grb14 to autophosphorylated IR in vitro inhibits tyrosine kinase activity towards other substrates, but studies on cultured cell lines have been conflicting as to whether Grb10 plays a positive or negative role in insulin signalling. Recent gene knockouts in mice have established that Grb10 and Grb14 act as inhibitors of intracellular signalling pathways regulating growth and metabolism, although the phenotypes of the two knockouts are distinct. Ablation of Grb14 enhances insulin action in liver and skeletal muscle and improves whole-body tolerance, with little effect on embryonic growth. Ablation of Grb10 results in disproportionate overgrowth of the embryo and placenta involving unidentified pathways, and also impacts on hepatic glycogen synthesis, and probably on glucose homoeostasis. This review discusses the extent to which previous studies in vitro can account for the observed phenotype of knockout animals, and considers evidence that aberrant function of Grb10 or Grb14 may contribute to disorders of growth and metabolism in humans.
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Affiliation(s)
- Lowenna J Holt
- University of Cambridge, Department of Clinical Biochemistry, Addenbrooke's Hospital, Cambridge CB2 2QR, UK.
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Barrès R, Gonzalez T, Le Marchand-Brustel Y, Tanti JF. The interaction between the adaptor protein APS and Enigma is involved in actin organisation. Exp Cell Res 2005; 308:334-44. [PMID: 15946664 DOI: 10.1016/j.yexcr.2005.05.008] [Citation(s) in RCA: 22] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/08/2004] [Revised: 05/03/2005] [Accepted: 05/04/2005] [Indexed: 11/17/2022]
Abstract
APS (adaptor protein with PH and SH2 domains) is an adaptor protein phosphorylated by several tyrosine kinase receptors including the insulin receptor. To identify novel binding partners of APS, we performed yeast two-hybrid screening. We identified Enigma, a PDZ and LIM domain-containing protein that was previously shown to be associated with the actin cytoskeleton. In HEK 293 cells, Enigma interacted specifically with APS, but not with the APS-related protein SH2-B. This interaction required the NPTY motif of APS and the LIM domains of Enigma. In NIH-3T3 cells that express the insulin receptor, Enigma and APS were partially co-localised with F-actin in small ruffling structures. Insulin increased the complex formation between APS and Enigma and their co-localisation in large F-actin containing ruffles. While in NIH-3T3 and HeLa cells the co-expression of both Enigma and APS did not modify the actin cytoskeleton organisation, expression of Enigma alone led to the formation of F-actin clusters. Similar alteration in actin cytoskeleton organisation was observed in cells expressing both Enigma and APS with a mutation in the NPTY motif. These results identify Enigma as a novel APS-binding protein and suggest that the APS/Enigma complex plays a critical role in actin cytoskeleton organisation.
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Affiliation(s)
- Romain Barrès
- INSERM U568 and IFR 50, Faculté de Médecine, Avenue de Valombrose, 06107 Nice Cedex 02, France
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Chakrabarti K, Lin R, Schiller NI, Wang Y, Koubi D, Fan YX, Rudkin BB, Johnson GR, Schiller MR. Critical role for Kalirin in nerve growth factor signaling through TrkA. Mol Cell Biol 2005; 25:5106-18. [PMID: 15923627 PMCID: PMC1140581 DOI: 10.1128/mcb.25.12.5106-5118.2005] [Citation(s) in RCA: 42] [Impact Index Per Article: 2.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/24/2023] Open
Abstract
Kalirin is a multidomain guanine nucleotide exchange factor (GEF) that activates Rho proteins, inducing cytoskeletal rearrangement in neurons. Although much is known about the effects of Kalirin on Rho GTPases and neuronal morphology, little is known about the association of Kalirin with the receptor/signaling systems that affect neuronal morphology. Our experiments demonstrate that Kalirin binds to and colocalizes with the TrkA neurotrophin receptor in neurons. In PC12 cells, inhibition of Kalirin expression using antisense RNA decreased nerve growth factor (NGF)-induced TrkA autophosphorylation and process extension. Kalirin overexpression potentiated neurotrophin-stimulated TrkA autophosphorylation and neurite outgrowth in PC12 cells at a low concentration of NGF. Furthermore, elevated Kalirin expression resulted in catalytic activation of TrkA, as demonstrated by in vitro kinase assays and increased NGF-stimulated cellular activation of Rac, Mek, and CREB. Domain mapping demonstrated that the N-terminal Kalirin pleckstrin homology domain mediates the interaction with TrkA. The effects of Kalirin on TrkA provide a molecular basis for the requirement of Kalirin in process extension from PC12 cells and for previously observed effects on axonal extension and dendritic maintenance. The interaction of TrkA with the pleckstrin homology domain of Kalirin may be one example of a general mechanism whereby receptor/Rho GEF pairings play an important role in receptor tyrosine kinase activation and signal transduction.
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Affiliation(s)
- Kausik Chakrabarti
- University of Connecticut Health Center, Department of Neuroscience, 263 Farmington Ave., Farmington, CT 06030-4301, USA
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Iseki M, Kubo-Akashi C, Kwon SM, Yamaguchi A, Takatsu K, Takaki S. APS, an adaptor molecule containing PH and SH2 domains, has a negative regulatory role in B cell proliferation. Biochem Biophys Res Commun 2005; 330:1005-13. [PMID: 15809095 DOI: 10.1016/j.bbrc.2005.03.073] [Citation(s) in RCA: 8] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/03/2005] [Indexed: 11/18/2022]
Abstract
Adaptor molecule containing PH and SH2 domains (APS) is an intracellular adaptor protein that forms part of an adaptor family along with Lnk and SH2-B. APS transcripts are expressed in various tissues including brain, kidney, and muscle, as well as in splenic B cells but not in T cells. We investigated the functions of APS in B cell development and activation by generating APS-transgenic (APS-Tg) mice that overexpressed APS in lymphocytes. The number of B-1 cells in the peritoneal cavity was reduced in APS-Tg mice, as were B-2 cells in the spleen. B cell development in the bone marrow was partially impaired at the transition stage from proliferating large pre-B to small pre-B cells. B cell proliferation induced by B cell receptor (BCR) crosslinking but not by other B cell mitogens was also impaired in APS-Tg mice. APS co-localized with BCR complexes and filamentous actin in activated APS-Tg B cells. Thus, APS appears to play novel negative regulatory roles in BCR signaling, actin reorganization pathways, and control of compartment sizes of B-lineage cells.
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Affiliation(s)
- Masanori Iseki
- Division of Immunology, Department of Microbiology and Immunology, The Institute of Medical Science, The University of Tokyo, 4-6-1 Shirokanedai, Minato-ku, Tokyo 108-8639, Japan
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Liu HY, MacDonald JIS, Hryciw T, Li C, Meakin SO. Human Tumorous Imaginal Disc 1 (TID1) Associates with Trk Receptor Tyrosine Kinases and Regulates Neurite Outgrowth in nnr5-TrkA Cells. J Biol Chem 2005; 280:19461-71. [PMID: 15753086 DOI: 10.1074/jbc.m500313200] [Citation(s) in RCA: 28] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/06/2022] Open
Abstract
The human tumorous imaginal disc 1 (TID1) proteins including TID1(L) and TID1(S), members of the DnaJ domain protein family, are involved in multiple intracellular signaling pathways such as apoptosis induction, cell proliferation, and survival. Here we report that TID1 associates with the Trk receptor tyrosine kinases and regulates nerve growth factor (NGF)-induced neurite outgrowth in PC12-derived nnr5 cells. Binding assays and transfection studies showed that the carboxyl-terminal end of TID1 (residues 224-429) bound to Trk at the activation loop (Tyr(P)(683)-Tyr(684)(P)(684) in rat TrkA) and that TID1 was tyrosine phosphorylated by Trk both in yeast and in transfected cells. Moreover endogenous TID1 was also tyrosine phosphorylated by and co-immunoprecipitated with Trk in neurotrophin-stimulated primary rat hippocampal neurons. Overexpression studies showed that both TID1(L) and TID1(S) significantly facilitated NGF-induced neurite outgrowth in TrkA-expressing nnr5 cells possibly through a mechanism involving increased activation of mitogen-activated protein kinase. Consistently knockdown of endogenous TID1, mediated with specific short hairpin RNA, significantly reduced NGF-induced neurite growth in nnr5-TrkA cells. These data provide the first evidence that TID1 is a novel intracellular adaptor that interacts with the Trk receptor tyrosine kinases in an activity-dependent manner to facilitate Trk-dependent intracellular signaling.
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Affiliation(s)
- Hui-Yu Liu
- Cell Biology Group, Robarts Research Institute, London, Ontario, Canada
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Nishi M, Werner ED, Oh BC, Frantz JD, Dhe-Paganon S, Hansen L, Lee J, Shoelson SE. Kinase activation through dimerization by human SH2-B. Mol Cell Biol 2005; 25:2607-21. [PMID: 15767667 PMCID: PMC1061652 DOI: 10.1128/mcb.25.7.2607-2621.2005] [Citation(s) in RCA: 51] [Impact Index Per Article: 2.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/20/2022] Open
Abstract
The isoforms of SH2-B, APS, and Lnk form a family of signaling proteins that have been described as activators, mediators, or inhibitors of cytokine and growth factor signaling. We now show that the three alternatively spliced isoforms of human SH2-B readily homodimerize in yeast two-hybrid and cellular transfections assays, and this is mediated specifically by a unique domain in its amino terminus. Consistent with previous reports, we further show that the SH2 domains of SH2-B and APS bind JAK2 at Tyr813. These findings suggested a model in which two molecules of SH2-B or APS homodimerize with their SH2 domains bound to two JAK2 molecules, creating heterotetrameric JAK2-(SH2-B)2-JAK2 or JAK2-(APS)2-JAK2 complexes. We further show that APS and SH2-B isoforms heterodimerize. At lower levels of SH2-B or APS expression, dimerization approximates two JAK2 molecules to induce transactivation. At higher relative concentrations of SH2-B or APS, kinase activation is blocked. SH2-B or APS homodimerization and SH2-B/APS heterodimerization thus provide direct mechanisms for activating and inhibiting JAK2 and other kinases from the inside of the cell and for potentiating or attenuating cytokine and growth factor receptor signaling when ligands are present.
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Affiliation(s)
- Masahiro Nishi
- Joslin Diabetes Center, One Joslin Place, Boston, MA 02215, USA
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Duan C, Yang H, White MF, Rui L. Disruption of the SH2-B gene causes age-dependent insulin resistance and glucose intolerance. Mol Cell Biol 2004; 24:7435-43. [PMID: 15314154 PMCID: PMC506995 DOI: 10.1128/mcb.24.17.7435-7443.2004] [Citation(s) in RCA: 103] [Impact Index Per Article: 4.9] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/21/2022] Open
Abstract
Insulin regulates glucose homeostasis by binding and activating the insulin receptor, and defects in insulin responses (insulin resistance) induce type 2 diabetes. SH2-B, an Src homology 2 (SH2) and pleckstrin homology domain-containing adaptor protein, binds via its SH2 domain to insulin receptor in response to insulin; however, its physiological role remains unclear. Here we show that SH2-B was expressed in the liver, skeletal muscle, and fat. Systemic deletion of SH2-B impaired insulin receptor activation and signaling in the liver, skeletal muscle, and fat, including tyrosine phosphorylation of insulin receptor substrate 1 (IRS1) and IRS2 and activation of the phosphatidylinositol 3-kinase/Akt and the Erk1/2 pathways. Consequently, SH2-B-/- knockout mice developed age-dependent hyperinsulinemia, hyperglycemia, and glucose intolerance. Moreover, SH2-B directly enhanced autophosphorylation of insulin receptor and tyrosine phosphorylation of IRS1 and IRS2 in an SH2 domain-dependent manner in cultured cells. Our data suggest that SH2-B is a physiological enhancer of insulin receptor activation and is required for maintaining normal insulin sensitivity and glucose homeostasis during aging.
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Affiliation(s)
- Chaojun Duan
- Department of Molecular and Integrative Physiology, University of Michigan Medical School, Ann Arbor, MI 48109-0622, USA
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