Redistribution of the CDK inhibitor p27 between different cyclin.CDK complexes in the mouse fibroblast cell cycle and in cells arrested with lovastatin or ultraviolet irradiation.

Abstract

The cyclin-dependent kinase (CDK) inhibitor p27 binds and inhibits the kinase activity of several CDKs. Here we report an analysis of the behavior and partners of p27 in Swiss 3T3 mouse fibroblasts during normal mitotic cell cycle progression, as well as in cells arrested at different stages in the cycle by growth factor deprivation, lovastatin treatment, or ultraviolet (UV) irradiation. We found that the level of p27 is elevated in cells arrested in G0 by growth factor deprivation or contact inhibition. In G0, p27 was predominantly monomeric, although some portion was associated with residual cyclin A.Cdk2. During G1, all of p27 was associated with cyclin D1.Cdk4 and was then redistributed to cyclin A.Cdk2 as cells entered S phase. The loss of the monomeric p27 pool as cyclins accumulate in G1 is consistent with the in vivo and in vitro data showing that p27 binds better to cyclin.CDK complexes than to monomeric CDKs. In growing cells, the majority of p27 was associated with cyclin D1 and the level of p27 was significantly lower than the level of cyclin D1. In cells arrested in G1 with lovastatin, cyclin D1 was degraded and p27 was redistributed to cyclin A.Cdk2. In contrast to p21 (which is a p27-related CDK inhibitor and is induced by UV irradiation), the level of p27 was reduced after UV irradiation, but because cyclin D1 was degraded more rapidly than p27, there was a transient increase in binding of p27 to cyclin A.Cdk2. These data suggest that cyclin D1.Cdk4 acts as a reservoir for p27, and p27 is redistributed from cyclin D1.Cdk4 to cyclin A.Cdk2 complexes during S phase, or when cells are arrested by growth factor deprivation, lovastatin treatment, or UV irradiation. It is likely that a similar principle of redistribution of p27 is used by the cell in other instances of cell cycle arrest.

Early G1 growth arrest of hybridoma B cells by DMSO involves cyclin D2 inhibition and p21[CIP1] induction

“…Similar mechanisms of CDK inhibition by p27 [KIP1] were previously reported earlier for growth inhibition mediated by TGFb and lovastatin (Chen et al, 1996;Hall et al, 1995;Peters, 1994;Poon et al, 1995). In these situations p27 [KIP1] redistribution was attributed (i) to the inhibition of cyclin D1 expression in cells arrested by lovastatin (Poon et al, 1995) and (ii) to the synthesis of the p15 [INK4B] inhibitor in the case of TGFb (Hannon and Beach, 1994;Reynisdottir et al, 1995). In 7TD1 cells this last possibility can be ruled out since we demonstrated that p15 [INK4B] and p16 [INK4A] are not stimulated by DMSO (data not shown).…”

“…Indeed, since in 7TD1 cells cyclin D2/CDK4 is principally associated with p27 [KIP1] the DMSO-dependent cyclin D2 inhibition provides free p27 [KIP1] molecules that can associate with cyclin E/CDK2 complexes. Similar mechanisms of CDK inhibition by p27 [KIP1] were previously reported earlier for growth inhibition mediated by TGFb and lovastatin (Chen et al, 1996;Hall et al, 1995;Peters, 1994;Poon et al, 1995). In these situations p27 [KIP1] redistribution was attributed (i) to the inhibition of cyclin D1 expression in cells arrested by lovastatin (Poon et al, 1995) and (ii) to the synthesis of the p15 [INK4B] inhibitor in the case of TGFb (Hannon and Beach, 1994;Reynisdottir et al, 1995).…”

“…To date, the mechanisms underlying these antiproliferative e ects are a matter of great interest. Several lines of evidence have demonstrated that these molecules inhibit cell progression through di erent mechanisms such as alteration of the CDK expression, modi®cation of cyclin levels (Gorospe et al, 1996;L'Allemain et al, 1997;Poon et al, 1995;Tiefenbrun et al, 1996), regulation of CDK activity through phosphorylation/dephosphorylation (Tiefenbrun et al, 1996) and induction or redistribution of CDK inhibitors (CKIs) (Florenes et al, 1996;Hengst et al, 1994;Koyoma et al, 1996;Poon et al, 1995;Reynisdottir et al, 1995).…”

Inhibition of Myc-dependent apoptosis by eukaryotic translation initiation factor 4E requires cyclin D1

“…One possibility is that cyclin D1 functions by activating Cdk4 or Cdk6 or sequestering Cdk inhibitors (Poon et al, 1995), p21 and p27, which have been strongly linked to apoptosis when expressed at the wrong time in the cell cycle (Duttaroy et al, 1997;Katayose et al, 1997;Wang et al, 1997). The most widely recognized function of cyclin D-dependent kinases is phosphorylation of pRb which disrupts association of pRb with E2F family proteins (Sherr and Roberts, 1999).…”

“…The presence of c-Myc in REF/Myc/4E clones was veri®ed to rule out the possibility that any new phenotype observed was due to loss of c-Myc expression. For immunoblot analysis of D cyclins, 40 mg of cytoplasmic extract prepared using a NP-40 lysis bu er (Poon et al, 1995) was subjected to SDS ± PAGE (12%) under reducing conditions, and blotted onto nitrocellulose. The blot was proved using primary antibodies against cyclins D1, D2, and D3 (Santa Cruz Biotechnologies, Inc., Santa Cruz, CA, USA; 1 : 200), appropriate horseradish peroxidase labeled secondary antibodies, and visualized by ECL.…”