Phosphorylation of tyrosine 256 facilitates nuclear import of atypical protein kinase C.
Herein, we employed a combined approach of molecular modeling and site-directed mutagenesis to address the role of tyrosine phosphorylation in transport of atypical protein kinase C (aPKC) into the nucleus. Computer modeling of the three-dimensional structure of the aPKC catalytic core, reveals that tyrosine 256 (Tyr256) is located at the lip of the activation loop and is conserved among members of the aPKC family, iota/lambda and zeta. Based on these findings, we examined whether tyrosine phosphorylation of aPKC on the activation lip may facilitate nuclear import. An antiserum was generated that selectively recognizes the phosphorylated Tyr256 residue in aPKC. By isolating nuclei of PC12 cells and immunoprecipitating aPKC with Ab-PY256, we observed that Tyr256 is rapidly phosphorylated upon NGF treatment prior to entry of aPKC into the nucleus. aPKC was observed to exclusively bind to importin-beta. The interaction between importin-beta and aPKC was enhanced upon tyrosine phosphorylation of aPKC and binding was abrogated when Tyr256 was mutated to phenylalanine. We propose that phosphorylation of aPKC at Tyr256 induces a conformation, whereby, the arginine-rich NLS is exposed, which then binds importin-beta leading to import of aPKC into the nucleus. Altogether, these findings document a novel role for the tyrosine phosphorylation in regulating import of atypical PKC into the nucleus.
White
Wendy O
WO
Department of Biological Sciences, Auburn University, Auburn, Alabama 36849, USA.
Seibenhener
M Lamar
ML
Wooten
Marie W
MW
D021581:Active Transport, Cell Nucleus / Q000502:physiology; D000818:Animals; D000906:Antibodies / Q000032:analysis / Q000276:immunology; D001665:Binding Sites; D020134:Catalytic Domain / Q000502:physiology; D002460:Cell Line; D002467:Cell Nucleus / Q000378:metabolism*; D006801:Humans; D008958:Models, Molecular; D009036:Moths; D016297:Mutagenesis, Site-Directed; D020932:Nerve Growth Factor / Q000494:pharmacology; D019913:Nuclear Localization Signals / Q000378:metabolism; D016716:PC12 Cells / Q000187:drug effects; D010766:Phosphorylation; D011487:Protein Conformation; D011493:Protein Kinase C / Q000276:immunology / Q000378:metabolism*; D051381:Rats; D014443:Tyrosine / Q000378:metabolism*; D028961:beta Karyopherins / Q000378:metabolism; D019061:src-Family Kinases / Q000378:metabolism
D000906:Antibodies; D019913:Nuclear Localization Signals; D028961:beta Karyopherins; D014443:Tyrosine; D020932:Nerve Growth Factor; D019061:src-Family Kinases; C115015:PKC-3 protein; D011493:Protein Kinase C