Nerve growth factor stimulates multisite tyrosine phosphorylation and activation of the atypical protein kinase C's via a src kinase pathway.
Atypical protein kinase C (PKC) isoforms are required for nerve growth factor (NGF)-initiated differentiation of PC12 cells. In the present study, we report that PKC-iota becomes tyrosine phosphorylated in the membrane coincident with activation posttreatment with nerve growth factor. Tyrosine phosphorylation and activation of PKC-iota were inhibited in a dose-dependent manner by both PP2 and K252a, src and TrkA kinase inhibitors. Purified src was observed to phosphorylate and activate PKC-iota in vitro. In PC12 cells deficient in src kinase activity, both NGF-induced tyrosine phosphorylation and activation of PKC-iota were also diminished. Furthermore, we demonstrate activation of src by NGF along with formation of a signal complex including the TrkA receptor, src, and PKC-iota. Recruitment of PKC-iota into the complex was dependent on the tyrosine phosphorylation state of PKC-iota. The association of src and PKC-iota was constitutive but was enhanced by NGF treatment, with the src homology 3 domain interacting with a PXXP sequence within the regulatory domain of PKC-iota (amino acids 98 to 114). Altogether, these findings support a role for src in regulation of PKC-iota. Tyrosine 256, 271, and 325 were identified as major sites phosphorylated by src in the catalytic domain. Y256F and Y271F mutations did not alter src-induced activation of PKC-iota, whereas the Y325F mutation significantly reduced src-induced activation of PKC-iota. The functional relevance of these mutations was tested by determining the ability of each mutant to support TRAF6 activation of NF-kappaB, with significant impairment by the Y325F PKC-iota mutant. Moreover, when the Y352F mutant was expressed in PC12 cells, NGF's ability to promote survival in serum-free media was reduced. In summary, we have identified a novel mechanism for NGF-induced activation of atypical PKC involving tyrosine phosphorylation by c-Src.
Wooten
M W
MW
Department of Biological Sciences, Auburn University, 331 Funchess Hall, Auburn, AL 36849, USA. mwwooten@acesag.auburn.edu
Vandenplas
M L
ML
Seibenhener
M L
ML
Geetha
T
T
Diaz-Meco
M T
MT
D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D000818:Animals; D002454:Cell Differentiation; D002470:Cell Survival; D004305:Dose-Response Relationship, Drug; D004789:Enzyme Activation; D017930:Genes, Reporter; D015151:Immunoblotting; D008954:Models, Biological; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D016328:NF-kappa B / Q000378:metabolism; D020932:Nerve Growth Factor / Q000378:metabolism*; D016716:PC12 Cells; D010766:Phosphorylation; D011233:Precipitin Tests; D011392:Proline / Q000737:chemistry; D011485:Protein Binding; D011493:Protein Kinase C / Q000378:metabolism*; D017434:Protein Structure, Tertiary; D051381:Rats; D015398:Signal Transduction; D013347:Subcellular Fractions; D013997:Time Factors; D014443:Tyrosine / Q000737:chemistry / Q000378:metabolism*; D019061:src-Family Kinases / Q000378:metabolism*
D016328:NF-kappa B; D014443:Tyrosine; D020932:Nerve Growth Factor; D011392:Proline; D019061:src-Family Kinases; D011493:Protein Kinase C