Figure 5
From: JNKs function as CDK4-activating kinases by phosphorylating CDK4 and p21
In transfected CHO cells, JNK inhibition and CDK2/CDK7 inhibition by R-roscovitine decrease the activity of p21-bound cyclin D1-CDK4 and phosphorylation of both p21 and CDK4. CHO cells were transfected with plasmids encoding cyclin D1 and CDK4 together with p21 either at high (p21) or low (p21 1/10) expression level, in the absence or presence of JNK-IN-8 and/or R-roscovitine (Rosco). As previously shown in these experimental conditions,23 a low expression of p21 permits the activity and phosphorylation of cyclin D-CDK4 and the S130 phosphorylation of p21, whereas higher expression of p21 inhibits phosphorylations of both p21 and CDK4. (a) Effect of the inhibitors on c-Jun S63 phosphorylation (P-Jun) analyzed by immunoblotting from cell lysates (WCE). (b) These cell lysates were Ip with anti-p21 antibody, assayed for their Rb-kinase activity, separated by SDS–PAGE and immunoblotted with the indicated antibodies. (c) The same cell lysates were Ip with anti-cyclin D1 or anti-p21 antibodies and separated by 2D-gel electrophoresis followed by immunodetection of CDK4 and p21. Black arrows, T172-phosphorylated CDK4; green arrows, S130-phosphorylated form of p21 as previously identified.23 In a–c chemiluminescence images of blots were captured with a Solo7S camera (Vilber-Lourmat, Marne-la-Vallée, France) and quantified using the Bio1D software (Vilber-Lourmat). The Rb-kinase activity was normalized to the amount of co-Ip CDK4 and expressed as % of the condition without inhibitor (Rb kin./CDK4 in b). The percentage of the T172-phosphorylated form of CDK4 versus the sum of Ip CDK4 forms is also determined (% p-CDK4 in c).
