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

Mol Cell Biol. 2001 Dec;21(24):8414-27. doi: 10.1128/MCB.21.24.8414-8427.2001.

Abstract

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.

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Amino 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
  • NF-kappa B / metabolism
  • Nerve Growth Factor / metabolism*
  • PC12 Cells
  • Phosphorylation
  • Precipitin Tests
  • Proline / chemistry
  • Protein Binding
  • Protein Kinase C / metabolism*
  • Protein Structure, Tertiary
  • Rats
  • Signal Transduction
  • Subcellular Fractions
  • Time Factors
  • Tyrosine / chemistry
  • Tyrosine / metabolism*
  • src-Family Kinases / metabolism*

Substances

  • NF-kappa B
  • Tyrosine
  • Nerve Growth Factor
  • Proline
  • src-Family Kinases
  • Protein Kinase C