Basic Study
Copyright ©The Author(s) 2017. Published by Baishideng Publishing Group Inc. All rights reserved.
World J Biol Chem. Aug 26, 2017; 8(3): 163-174
Published online Aug 26, 2017. doi: 10.4331/wjbc.v8.i3.163
Control of nuclear-cytoplasmic shuttling of Ankrd54 by PKCδ
Amy L Samuels, Alison Louw, Reza Zareie, Evan Ingley
Amy L Samuels, Alison Louw, Evan Ingley, Cell Signalling Group, Harry Perkins Institute of Medical Research and Centre for Medical Research, the University of Western Australia, Nedlands, WA 6009, Australia
Reza Zareie, Proteomics International Laboratories Ltd, Nedlands, WA 6009, Australia
Reza Zareie, Proteowa Pty Ltd, SABC, Murdoch University, Murdoch, WA 6150, Australia
Evan Ingley, Cell Signalling Group, School of Veterinary and Health Sciences, Murdoch University, Murdoch, WA 6150, Australia. evan.ingley@mudcoh.edu.au
Author contributions: Samuels AL and Louw A contributed equally to the manuscript and designed, supported and performed experiments, and analyzed data; Zareie R designed and performed experiments, and analysed data; Ingley E designed and supported the research, designed and undertook experiments, analyzed data and contributed to writing the manuscript.
Supported by the National Health and Medical Research Council, Nos. 403987, 513714 and 634352; the Medical Research Foundation of Royal Perth Hospital; and the Cancer Council of Western Australia; Evan Ingley received support from the Cancer Council of Western Australia, the Harry Perkins Institute of Medical Research; Sock-it-to-Sarcoma and the Hollywood Private Hospital Research Foundation; and the MACA Ride to Conquer Cancer.
Conflict-of-interest statement: Each author declares no conflict of interest.
Data sharing statement: All data are available upon request.
Open-Access: This article is an open-access article which was selected by an in-house editor and fully peer-reviewed by external reviewers. It is distributed in accordance with the Creative Commons Attribution Non Commercial (CC BY-NC 4.0) license, which permits others to distribute, remix, adapt, build upon this work non-commercially, and license their derivative works on different terms, provided the original work is properly cited and the use is non-commercial. See: http://creativecommons.org/licenses/by-nc/4.0/
Correspondence to: Dr. Evan Ingley, Professor, Cell Signalling Group, School of Veterinary and Health Sciences, Murdoch University, 90 South Street, Murdoch, WA 6150, Australia. evan.ingley@mudcoh.edu.au
Telephone: +61-8-61510738 Fax: +61-8-61510701
Received: January 25, 2017
Peer-review started: February 1, 2017
First decision: March 8, 2017
Revised: April 28, 2017
Accepted: May 12, 2017
Article in press: May 15, 2017
Published online: August 26, 2017
Processing time: 209 Days and 18.3 Hours
Abstract
AIM

To identify and characterize the effect of phosphorylation on the subcellular localization of Ankrd54.

METHODS

HEK293T cells were treated with calyculin A, staurosporin or phorbol 12-myristate 13-acetate (PMA). Cells were transfected with eGFP-tagged Ankrd54 with or without Lyn tyrosine kinase (wild-type, Y397F mutant, or Y508F mutant). The subcellular localization was assessed by immunofluorescence imaging of cells, immunoblotting of subcellular fractionations. The phosphorylation of Ankrd54 was monitored using Phos-tagTM gel retardation. Phosphorylated peptides were analysed by multiple-reaction-monitoring (MRM) proteomic analysis.

RESULTS

Activation of PKC kinases using PMA promoted nuclear export of Ankrd54 and correlated with increased Ankrd54 phosphorylation, assayed using Phos-tagTM gel retardation. Co-expression of an active form of the PKCδ isoform specifically promoted both phosphorylation and cytoplasmic localization of Ankrd54, while PKCδ, Akt and PKA did not. Alanine mutation of several serine residues in the amino-terminal region of Ankrd54 (Ser14, Ser17, Ser18, Ser19) reduced both PMA induced cytoplasmic localization and phosphorylation of Ankrd54. Using MRM proteomic analysis, phosphorylation of the Ser18 residue of Ankrd54 was readily detectable in response to PMA stimulation. PMA stimulation of cells co-expressing Ankrd54 and Lyn tyrosine kinase displayed increased co-immunoprecipitation and enhanced co-localization in the cytoplasm.

CONCLUSION

We identify phosphorylation by PKCδ as a major regulator of nuclear-cytoplasmic shuttling of Ankrd54, and its interaction with the tyrosine kinase Lyn.

Keywords: Ankrd54; PKCδ; Lyn; Btk; Phosphorylation

Core tip: Ankrd54 is a nuclear-cytoplasmic shuttling adaptor that interacts with Lyn, Btk, Txk, and HCLS1. Activation of PKC kinases promoted nuclear export and phosphorylation of Ankrd54, and increased interaction and cytoplasmic co-localization with Lyn. Co-expression of an active form of the PKCδ isoform specifically promoted both phosphorylation and cytoplasmic localization of Ankrd54. Alanine mutation of several serine residues in the amino-terminal region of Ankrd54 reduced both phorbol 12-myristate 13-acetate induced cytoplasmic localization and phosphorylation of Ankrd54. These results identify PKCδ as a major regulator of nuclear-cytoplasmic shuttling and interaction of Ankrd54 with Lyn, through its phosphorylation of at least the Ser18 residue.