Protein kinase C-mediated bidirectional regulation of DNA synthesis, RB protein phosphorylation, and cyclin-dependent kinases in human vascular endothelial cells

Abstract

In human umbilical vein endothelial cells, activators of protein kinase C (PKC) exert cell cycle-dependent, bidirectional growth regulatory effects. Thus, phorbol 12,13-dibutyrate or 1,2-dioctanoylglycerol potentiates growth factor-induced DNA synthesis up to 3-fold when they act during the early G1 phase, whereas they completely inhibit the initiation of DNA synthesis when they act in the late G1 phase. In addition, the PKC activators induce a rapid inhibition of the ongoing DNA synthesis when they are applied after entry into the S phase. The effects of the PKC activators in both stimulatory and inhibitory directions are abolished in PKC-downregulated cells. The cell cycle-dependent, PKC-mediated bidirectional growth regulation is closely associated with either potentiation or inhibition of RB protein phosphorylation and the histone H1 kinase activity of cyclin-dependent kinases (cdks) cdc2 and cdk2, which normally accumulate along the G1 to the S phase transition. Northern and Western blot analyses of cdc2 and cdk2 have revealed that PKC regulates the cdks at multiple steps in distinct ways. Thus, for cdc2, the levels of mRNA and protein as well as the extent of post-translational modification are all subject to the PKC-mediated regulation. In contrast, the level of mRNA or protein of cdk2 is not affected by PKC stimulation at any phase of the cell cycle. These results demonstrate the existence of a complex array of PKC-cdk signaling pathways, which mediate temporally organized bimodal growth regulation in endothelial cells.