Figure 1
From: JNKs function as CDK4-activating kinases by phosphorylating CDK4 and p21
JNKs phosphorylate p21-bound CDK4 in vitro, but ERK1/2 and CAK do not. (a, b) CHO cells were transfected with plasmids encoding p21, cyclin D1 or cyclin D3 and CDK4 (a) or T172A CDK4 (b). Cell lysates were Ip with cyclin D1 or cyclin D3 antibodies. The co-immunoprecipitates were incubated with the indicated recombinant kinases and ATP, separated by 2D-gel electrophoresis, and immunodetected with antibodies against CDK4 or its T172 phosphorylation (p-CDK4(T172)). 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. (c) As a positive control for CAK activity in the experiment shown in a, Ip cyclin D3-CDK4 complexes (Ip cyclin D3) from CHO cells transfected only with plasmids encoding CDK4 and cyclin D3 were incubated with ATP and CAK or JNK1, and analyzed by 2D-gel electrophoresis and CDK4 immunodetection. Because cyclin D3-CDK4 complexes produced in CHO cells in the absence of p21 expression are phosphorylated at T172, they were dephosphorylated by incubation with λ phosphatase (+λ PPase) before incubation with kinases (c). Black arrows indicate the increased abundance of the T172-phosphorylated form of CDK4.
