H. Lee Moffitt Cancer Center and Research Institute , University of South Florida , Tampa 33612 , USA .
Mol Cell Biol 16 : 4327-36 ( 1996)
Abstract
We have investigated the regulation of p27kip1 , a cyclin-dependent kinase inhibitor , in BALB/c 3T3 cells during growth factor-stimulated transition from quiescence ( G0 ) to a proliferative ( G1 ) state .
The level of p27kip1 protein falls dramatically after mitogenic stimulation and is accompanied by a decrease in cyclin E associated p27kip1 , as well as a transient increase in cyclin D1-associated p27kip1 that later declines concomitantly with the loss of total p27kip1 .
Analysis of metabolically labelled cells revealed that cyclin D2 , cyclin D3 , and cdk4 were also partnered with p27kip1 in quiescent BALB/c 3T3 cells and that this association decreased after platelet-derived growth factor ( PDGF ) treatment .
Furthermore , the decline in p27kip1 and reduced association with cyclin D3 , initiated by the addition of PDGF but not plasma-derived factors , suggested that these changes are involved in competence , the first step in the exit from G0 .
Synthesis of p27kip1 as determined by incorporation of [ 35S]methionine was repressed upon mitogenic stimulation , and PDGF was sufficient to elicit this repression within 2 to 3 h . Pulse-chase experiments demonstrated the reduced rate of synthesis was not the result of an increased rate of degradation .
Full repression of p27kip1 synthesis required the continued presence of PDGF and failed to occur in the presence of the RNA polymerase inhibitor , 6-dichlorobenzimidazole riboside .
These characteristics demonstrate that repression was a late effect of PDGF and was consistent with our finding that conditional expression of activated H-ras did not affect synthesis of p27kip1 .
Northern ( RNA ) analysis of p27kip1 mRNA revealed that the repression was not accompanied by a corresponding decrease in p27kip1 mRNA , suggesting that the PDGF-regulated decrease in p27kip1 expression occurred through a translational mechanism .