Nerve growth factor stimulates multisite tyrosine phosphorylation and activation of the atypical protein kinase C's via a src kinase pathway.

TitleNerve growth factor stimulates multisite tyrosine phosphorylation and activation of the atypical protein kinase C's via a src kinase pathway.
Publication TypeJournal Article
Year of Publication2001
AuthorsWooten MW, Vandenplas ML, Seibenhener ML, Geetha T, Diaz-Meco MT
JournalMol Cell Biol
Date Published2001 Dec
KeywordsAmino Acid Motifs, Amino Acid Sequence, Animals, Cell Differentiation, Cell Survival, Dose-Response Relationship, Drug, Enzyme Activation, Genes, Reporter, Immunoblotting, Models, Biological, Molecular Sequence Data, Mutagenesis, Site-Directed, Mutation, Nerve Growth Factor, NF-kappa B, PC12 Cells, Phosphorylation, Precipitin Tests, Proline, Protein Binding, Protein Kinase C, Protein Structure, Tertiary, Rats, Signal Transduction, src-Family Kinases, Subcellular Fractions, Time Factors, Tyrosine

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.

Alternate JournalMol Cell Biol
PubMed ID11713277
PubMed Central IDPMC100005
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Maria Diaz-Meco Conde, Ph.D.

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