Figure 2

JNKs, ERK1/2 and p38α phosphorylate p21 in vitro. (a, b) CHO cells were transfected with plasmids encoding CDK4, p21 and cyclin D1 or cyclin D3, or with the p21 plasmid alone (right column in b). Cell lysates were Ip with antibodies against cyclin D1 or cyclin D3 (a, b) or against p21 (b). The immunoprecipitates were incubated with the indicated recombinant kinases and ATP and separated by 2D-gel electrophoresis followed by immunodetection with antibodies against p21 (a, b) or its T57 phosphorylation (p-p21(T57) in b. Colored arrows indicate phosphorylated forms of p21. The experiment in a was reproduced four times using cyclin D1-CDK4-p21 complexes and three times using cyclin D3-CDK4-p21 complexes, with similar results. The experiment in b was reproduced three times with similar results. (c) The T57D phosphomimetic mutation of p21 increases the activity of p21-bound cyclin D1-CDK4 and cyclin D3-CDK4 complexes. CHO cells were transfected with plasmids encoding cyclin D1 or cyclin D3, CDK4 and wt p21 or T57D p21. Cell lysates were Ip with antibodies directed against cyclin D1, cyclin D3 or p21, assayed for their Rb-kinase activity, separated by SDS–PAGE, and immunoblotted with the indicated antibodies. The experiment in c was reproduced three times in cyclin D3-CDK4-p21 complexes and performed twice in cyclin D1-CDK4-p21 complexes, with similar results.