Mapping of Atypical Protein Kinase C within the Nerve Growth Factor Signaling Cascade: Relationship to Differentiation and Survival of PC12 Cells

Abstract

The pathway by which atypical protein kinase C (aPKC) contributes to nerve growth factor (NGF) signaling is poorly understood. We previously reported that in PC12 cells NGF-induced activation of mitogen-activated protein kinase (MAPK) occurs independently of classical and nonclassical PKC isoforms, whereas aPKC isoforms were shown to be required for NGF-induced differentiation. NGF-induced activation of PKC-ι was observed to be dependent on phosphatidylinositol 3-kinase (PI3K) and led to coassociation of PKC-ι with Ras and Src. Expression of dominant negative mutants of either Src (DN2) or Ras (Asn-17) impaired activation of PKC-ι by NGF. At the level of Raf-1, neither PKC-ι nor PI3 kinase was required for activation; however, PKC-ι could weakly activate MEK. Inhibitors of PKC-ι activity and PI3K had no effect on NGF-induced MAPK or p38 activation but reduced NGF-stimulated c-Jun N-terminal kinase activity. Src, PI3K, and PKC-ι were likewise required for NGF-induced NF-κB activation and cell survival, whereas Ras was not required for either survival or NF-κB activation but was required for differentiation. IKK existed as a complex with PKC-ι, Src and IκB. Consistent with a role for Src in regulating NF-κB activation, an absence of Src activity impaired recruitment of PKC-ι into an IKK complex and markedly impaired NGF-induced translocation of p65/NF-κB to the nucleus. These findings reveal that in PC12 cells, aPKCs comprise a molecular switch to regulate differentiation and survival responses coupled downstream to NF-κB. On the basis of these findings, Src emerges as a critical upstream regulator of both PKC-ι and the NF-κB pathway.

ACKNOWLEDGMENTS

We thank numerous investigators who provided cDNAs, reagents, and cell lines for this study. We thank members of our laboratory for fruitful discussion.

This research was funded by NINDS grant RO2-NS33661 and the Auburn University Biogrants Program.