NGF REGULATES THE PC12 CELL-CYCLE MACHINERY THROUGH SPECIFIC-INHIBITION OF THE CDK KINASES AND INDUCTION OF CYCLIN D1

We have examined the effects of NGF on components of the PC12 cell cyc le machinery. We show that NGF represses over 6-8 d the levels of spec ific cdk kinase proteins and the G2-M phase specific cyclin B1 and the S phase marker PCNA as well as the level of phosphorylation of the re tinoblastoma (Rb) protein. All of these changes may provide a basis fo r a NGF block to cell cycling. Unexpectedly, the G1 phase-specific cyc lin D1 was dramatically increased by inducers of differentiation (NGF and FGF), but not by inducers of proliferation (EGF and insulin). Alth ough the levels of cyclin D1/cdk2 and cyclin D1/cdk4 complexes increas ed following NGF treatment, as did cyclin D1/Rb complexes, the associa ted kinase activities declined, indicating that NGF also induces an in hibitor of cdk kinase activity. In agreement, NGF induced the cdk inhi bitory protein, p21, which was found in cyclin D1/cdk kinase complexes after NGF treatment. We show that vector over expression of cyclin D1 in PC12 is sufficient on its own to arrest the cells in G1 phase and inhibit expression of PCNA. These results indicate that NGF induction of cyclin D1 and inactivation of cdk kinases, the latter possibly by i ncrease of p21, play a central role in the NGF block of PC12 cell cycl ing.