REPRESSION OF P27(KIP1) SYNTHESIS BY PLATELET-DERIVED GROWTH-FACTOR IN BALB C 3T3 CELLS/

We have investigated the regulation of p27(kip1), a cyclin-dependent k inase inhibitor, in BALB/c 3T3 cells during growth factor-stimulated t ransition from quiescence (G(0)) to a proliferative (G(1)) state. The level of p27(kip1) protein falls dramatically after mitogenic stimulat ion and is accompanied by a decrease in cyclin E associated p27(kip1), aS well as a transient increase in cyclin D1-associated p27(kip1) tha t later declines concomitantly with the loss of total p27(kip1). Analy sis of metabolically labelled cells revealed that cyclin D2, cyclin D3 , and cdk4 were also partnered with p27(kip1) in quiescent BALB/c 3T3 cells and that this association decreased after platelet-derived growt h factor (PDGF) treatment. Furthermore, the decline in p27(kip1) and r educed association with cyclin D3, initiated by the addition of PDGF b ut not plasma-derived factors, suggested that these changes are involv ed in competence, the first step in the exit from G,. Synthesis of p27 (kip1) aS determined by incorporation of [S-35]methionine was represse d upon mitogenic Stimulation, and PDGF was sufficient to elicit this r epression within 2 to 3 h. Pulse-chase experiments demonstrated the re duced rate of synthesis was not the result of an increased rate of deg radation. Full repression of p27(kip1) synthesis required the continue d presence of PDGF and frilled to occur in the presence of the RNA pol ymerase inhibitor 5,6-dichlorobenzimidazole riboside. These characteri stics demonstrate that repression was a late effect of PDGF and was co nsistent with our finding that conditional expression of activated H-r as did not affect synthesis of p27(kip1). Northern (RNA) analysis of p 27(kip1) mRNA revealed that the repression was not accompanied by a co rresponding decrease in p27(kip1) mRNA, suggesting that the PDGF-regul ated decrease in p27(kip1) expression occurred through a translational mechanism.