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Control of nuclear-cytoplasmic shuttling of Ankrd54 by PKCδ

Samuels, A.L., Louw, A., Zareie, R. and Ingley, E. (2017) Control of nuclear-cytoplasmic shuttling of Ankrd54 by PKCδ. World Journal of Biological Chemistry, 8 (3). pp. 163-174.

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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.

Publication Type: Journal Article
Murdoch Affiliation: School of Veterinary and Life Sciences
Publisher: Baishideng Publishing Group Inc
Copyright: ©The Author(s) 2017.
URI: http://researchrepository.murdoch.edu.au/id/eprint/38660
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