Protein Kinase Cδ Mediates Neurogenic but Not Mitogenic Activation of Mitogen-Activated Protein Kinase in Neuronal Cells

Although activation of mitogen-activated protein (MAP) kinases (extracellular signal-related kinase 1 [ERK1] and -2) by growth factors can lead to a variety of cellular fates, including growth and differentiation, the mechanism by which specificity is determined is not known. One explanation for the observed differences involves the duration of ERK activation (32). For example, epidermal growth factor (EGF) induces transient activation of ERKs and stimulates proliferation of pheochromocytoma (PC12) cells, while fibroblast-derived growth factor (FGF) and nerve growth factor (NGF) stimulate prolonged ERK activation and induce cellular differentiation (46). Another potential explanation is that there are differences in the intracellular signals that couple growth factors to ERKs. Several molecules exclusive of the linear Ras, c-Raf, MAP kinase kinase (MEK), and MAP kinase pathways have been shown to mediate ERK activation (4, 13, 41, 47) and among these are members of the protein kinase C (PKC) family. To date, 11 members of the PKC superfamily have been identified (reviewed in references 9, 22, and 36). The PKCs have been classified into three groups based upon their ability to be activated by Ca2+ and diacylglycerol (DAG). The classical PKCs (cPKCs) are activated by both Ca2+ and DAG and include the α, βI, βII, and γ isoforms. The Ca2+-independent but DAG-dependent isoforms (θ, η, δ, and ɛ) comprise the novel PKCs (nPKCs). Finally, the atypical PKCs (aPKCs), ζ, ι/λ, and μ, are both Ca2+ and DAG independent. Various PKCs have been shown to mediate or modulate the activation of ERKs by growth factors, hormones, and phorbol esters (3, 31, 41, 45). Many of these PKCs act as potentiators of cell cycle progression. Interestingly, PKCδ differs from other closely related PKCs, such as PKCɛ. PKCδ has a distinct subcellular localization (15) and mediates tetradecanoyl phorbol acetate-induced differentiation of murine myeloid progenitor cells into macrophages (34) as well as the secretory response of antigen-stimulated rat basophilic RBL-2H3 cells (34, 44). Overexpression of PKCδ leads to growth arrest in vascular smooth muscle, capillary endothelial, NIH 3T3, and CHO cells (14, 20, 33, 48), a function often associated with differentiation. Furthermore, N-myc-induced transformation in a rat neuroblastoma cell line results in a decrease in PKCδ expression (2). Consistent with these studies, PKCδ blocks cellular transformation by Src and has been postulated to be a tumor suppressor (29). Finally, PKCδ, but not PKCɛ, is translocated to the membrane in response to NGF in PC12 cells (38). Thus, PKCδ appears to have a growth-inhibiting and differentiating function in a variety of cell types. In the present study, we have investigated the role of PKCδ in the activation of MAP kinase and the induction of neurite outgrowth in PC12 and H19-7 cells. The conditionally immortalized H19-7 cell line was generated by transducing rat E17 hippocampal cells with a retroviral vector expressing a temperature-sensitive simian virus 40 large T antigen (10). At the nonpermissive temperature, when T is inactivated, H19-7 cells differentiate upon stimulation by FGF, but not upon exposure to EGF (10, 25, 26). In contrast to PC12 cells, H19-7 cells lack the Trk receptor and therefore are nonresponsive to NGF (11). We now show that PKCδ is required for ERK activation by FGF and other differentiating factors in both H19-7 and PC12 cells. Preincubation of cells with rottlerin, a PKC inhibitor with specificity for the δ isoform (17), blocks activation of MEK but not Raf, suggesting that PKCδ acts either downstream or in parallel with Raf. Finally, rottlerin inhibits neurite outgrowth in response to activated Raf in H19-7 cells and FGF or NGF in PC12 cells. The results presented here suggest that PKCδ is required for the activation of MEK by FGF or NGF in neuronal cells, and this pathway may account for some of the selective effects of differentiating versus growth-promoting factors.