P27Kip1 and p21Cip1 are not required for the formation of active D cyclin-cdk4 complexes

Abstract

Our studies address questions pertaining to the regulation of D cyclin-cdk4 activity, and the following results were obtained. Conditions that increased the abundance of the D cyclins also increased the abundance of enzymatically active D cyclin-cdk4 complexes in mouse embryo fibroblasts (MEFs) lacking both p27(Kip1) and p21(Cip1) (p27/p21(-/-)). Such conditions included ectopic expression of cyclin D1 and inhibition of D cyclin degradation by the proteasome inhibitor MG132. However, as determined by treatment of wild-type MEFs with MG132, maximal accumulation of D cyclin-cdk4 complexes required p27(Kip1) and p21(Cip1) and coincided with the formation of inactive D cyclin-cdk4-p27(Kip1) or -p21(Cip1) complexes. p27(Kip1) or p21(Cip1) also increased the abundance of D cyclin-cdk4 complexes and reduced amounts of cdk4 activity when ectopically expressed in p27/p21(-/-) MEFs. Lastly, increases in the stability of the D cyclins accounted for their greater abundance in wild-type MEFs than in p27/p21(-/-) MEFs. We conclude that (i) D cyclin-cdk4 complexes are formed and become active in the absence of p27(Kip1) and p21(Cip1) and (ii) p27(Kip1) and p21(Cip1) maximize the accumulation but inhibit the activity of D cyclin-cdk4 complexes. We suggest that D cyclin-cdk4 complexes are more stable when bound to p27(Kip1) or p21(Cip1) and that formation of ternary complexes also stabilizes the D cyclins.

FIG. 1.

Effect of cyclin D1 overexpression on cyclin D1-cdk4 abundance and activity in p27/p21^−/− MEFs. Exponentially growing p27/p21^−/− MEFs were transfected with 15 μg of plasmid alone (vector) or with plasmid encoding flag-tagged cyclin D1. Cells transfected with plasmid alone were harvested 24 h after transfection. Cells transfected with plasmid encoding cyclin D1 were harvested 24 or 32 h after transfection. Amounts of cyclin D1 (both ectopic and endogenous) were determined by Western blotting of cell extracts with antibody to cyclin D1. The association of cdk4 with cyclin D1 was determined by Western blotting of cyclin D1 immune complexes with antibody to cdk4. Amounts of cdk4 activity were determined by in vitro kinase assays of cyclin D1 immunoprecipitates with GST-Rb as substrate. IP, immunoprecipitation; pRb, phosphorylated Rb.

FIG. 2.

Effect of MG132 on the abundance and activity of D cyclin-cdk4 complexes in wild-type and p27/p21^−/− MEFs. Logarithmically growing MEFs received 20 μM MG132 for the indicated times. Amounts of cyclin D1, cyclin D3, cdk4, p27^Kip1, p21^Cip1, and β-actin (loading control) were determined by Western blotting of cell extracts. Amounts of cyclin D1-associated cdk4 were determined by Western blotting of cyclin D1 immunoprecipitates with antibody to cdk4. Amounts of cdk4-associated cyclin D3, p27^Kip1, and p21^Cip1 were determined by Western blotting of cdk4 immunoprecipitates with antibody to cyclin D3. Amounts of cdk4 activity were determined by in vitro kinase assays of cdk4 immunoprecipitates (IP). pRb, phosphorylated Rb.

FIG. 3.

Amounts and associated activities of cyclins D1 and D3 in p27/p21^−/− MEFs infected with Ad-p27^Kip1. Asynchronously growing p21/p27^−/− MEFs were infected with Ad-p27^Kip1 at the indicated MOIs. Cells were harvested 25 h after infection. Amounts of p27^Kip1, cyclin D1, cyclin D3, and cdk4 were determined by Western blotting of cell extracts. Amounts of cyclin D1-associated cdk4, cdk4-associated cyclin D3, and cdk4-associated p27^Kip1 were determined by immunoprecipitation (IP) and immunoblot analysis. Kinase activity was assayed in cyclin D1 and cdk4 immunoprecipitates with GST-Rb as substrate. pRb, phosphorylated Rb.

FIG. 4.

Amounts and associated activities of cyclins D1 and D3 in p27/p21^−/− MEFs infected with Ad-p21^Cip1. Experiments were performed as described in the legend to Fig. 3, except that p27/p21^−/− MEFs were infected with Ad-p21^Cip1 rather than Ad-p27^Kip1.

FIG. 5.

Lack of effect of p27^Kip1 on cyclin D1 mRNA expression. Exponentially growing p27/p21^−/− MEFs were infected with adenovirus without insert (Ad) or adenovirus encoding p27^Kip1 (Ad-p27^Kip1) at an MOI of 500. Cells were harvested 30 or 40 h after infection. Amounts of cyclin D1 protein and mRNA were determined by Western blotting and Northern blotting, respectively. Amounts of 28S and 18S rRNA are shown as loading controls. The asterisk denotes a nonspecific band.

FIG. 6.

Effect of p27^Kip1 and p21^Cip1 on the stability of cyclin D1 and cyclin D3. Growing p27/p21^−/− MEFs were infected with Ad-p27^Kip1 or Ad-p21^Cip1 at an MOI of 500. Twenty-five hours later infected and uninfected cultures received 10 μg of cycloheximide/ml. Cells were harvested at the indicated times, and amounts of cyclin D1 and cyclin D3 were determined by Western blotting. Different exposures of the Western blots were chosen to obtain roughly equivalent amounts of cyclin D1 or cyclin D3 at time zero.

FIG. 7.

Model showing the effects of p27^Kip1 and p21^Cip1 on the abundance and activity of D cyclin-cdk4 complexes in wild-type cells. The D cyclins in three different pools are shown, and the activity and stability of binary and ternary complexes are indicated. See the text for details.