Repression of p27^kip1 Synthesis by Platelet-Derived Growth Factor in BALB/c 3T3 Cells

Abstract

We have investigated the regulation of p27 kip1 , a cyclin-dependent kinase inhibitor, in BALB/c 3T3 cells during growth factor-stimulated transition from quiescence (G 0 ) to a proliferative (G 1 ) state. The level of p27 kip1 protein falls dramatically after mitogenic stimulation and is accompanied by a decrease in cyclin E associated p27 kip1, as well as a transient increase in cyclin D1-associated p27 kip1 that later declines concomitantly with the loss of total p27 kip1 . Analysis 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 growth factor (PDGF) treatment. Furthermore, the decline in p27 kip1 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 G 0 . Synthesis of p27 kip1 as determined by incorporation of [ 35 S]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 p27 kip1 synthesis required the continued presence of PDGF and failed to occur in the presence of the RNA polymerase inhibitor 5,6-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 p27 kip1. Northern (RNA) analysis of p27 kip1 mRNA revealed that the repression was not accompanied by a corresponding decrease in p27 kip1 mRNA, suggesting that the PDGF-regulated decrease in p27 kip1 expression occurred through a translational mechanism.Under normal circumstances, proliferation of mammalian cells is a highly controlled process. This control is achieved, in part, by growth factors and cytokines that exert either mitogenic and/or antiproliferative effects in a cell-specific manner (1). Binding of growth-regulatory ligands results in the activation of signaling pathways that trigger direct alteration of specific metabolic pathways as well as immediate and delayed changes in gene expression (65). Mitogenic stimulation initiates a program of sequential synthesis of proteins, termed cyclins, that complex with and activate cyclin-dependent kinases (cdks) (9, 29-31, 39, 52, 54, 64, 67), enzymes that modulate key regulatory events leading to progression through and transitions between different stages of the cell cycle (7,13,20,21,47,55,64). The order of cyclin synthesis during cell cycle traverse has been examined in a variety of cells, including T cells, macrophages, epithelial cells, and fibroblasts (16,30,31,44,45,50,52). The D-type cyclins, which complex with cdk4 and cdk6, are the first cyclins synthesized during the cell cycle. They are detected in mid-G 1 and are believed to function as Rb ...

Evidence for a p23 caspase-cleaved form of p27[KIP1] involved in G1 growth arrest

“…The abundance of p27 is thought to be regulated by translational and post-translational pathways, and less commonly at the level of transcription (Agrawal et al, 1996;Hengst and Reed, 1996;Roberts et al, 1994;Vlach et al, 1997). Our own observation suggests that the in vivo proteolysis of p27 by a CPP32-like caspase during early G 1 arrest could be an additional posttranslational regulation pathway of p27.…”

“…It is generally up-regulated in cells arrested either by cell contact, serum deprivation or after incubation with transforming growth factor-b (TGF-b) Polyak et al, 1994a;Slingerland et al, 1994). In contrast, p27 usually declines in the presence of mitogenic agents like platelet-derived growth factor, macrophage stimulating factor or interleukin-2 (Agrawal et al, 1996;Kato et al, 1994;Nourse et al, 1994). Nevertheless, recent data obtained with p27 nullizygote mice have shown that cell cycle arrest induced by TGF-b, rapamycin or contact inhibition remains una ected (Nakayama et al, 1996), suggesting that p27 is not the only CKI involved in cell growth arrest under these conditions.…”

Primary keratinocytes have an adhesion dependent S phase checkpoint that is absent in immortalized cell lines

“…Autoradiographic ®lm was purchased from Kodak (Rochester, NY, USA). Antibodies utilized in this study were anti-cyclin A, a kind gift from Ed Leof (Mayo Clinic), and anti-Kip (Agrawal et al, 1996).…”

“…Cyclin B binds and activates cdc2, forming a complex that is essential for triggering the onset and passage through mitosis. The regulation of the cyclin proteins appears, for the most part, to be periodic, and is con®ned to the interval in which the corresponding activities are required, although there are exceptions such as cyclin E and cyclin D3 (Agrawal et al, 1995(Agrawal et al, , 1996, which are present even in quiescent cells. In addition to the regulatory roles of cyclins on cdk activity, another class of proteins, cyclin dependent kinase inhibitors (CKI), have been described to provide negative control under conditions where the proliferative response must be attenuated, such as density arrest and in response to DNA damaging agents.…”

Involvement of p21 and p27 in the regulation of CDK activity and cell cycle progression in the regenerating liver

“…p27 is constitutively expressed in many cells, and the level of this protein is elevated in G0 phase and declines as cells in culture enter the cell cycle (Poon et al, 1995;Agrawal et al, 1996;Polyak et al, 1994). p27 may play an important role in governing the growth factor restriction point by ensuring that CDK activity is suppressed during G0 and early G1 phase (Coats et al, 1996;Soos et al, 1996).…”