Figure 4

In T98G cells, inhibition of JNKs by SP600125 or JNK-IN-8 decreases DNA synthesis (a), Rb-kinase activity (b), and phosphorylation of CDK4 (c) and p21 (d). (a) T98G cells were stimulated with FBS for 18 h in the presence or absence of SP600125 or JNK-IN-8. BrdU was present during the last 30 min. The fraction of nuclei having incorporated BrdU was determined (mean+range of duplicate dishes). (b) In the same cell cultures, extracts of cells treated for the indicated times were Ip with anti-cyclin D1, anti-cyclin D3 or anti-p21 antibodies, assayed for Rb-kinase activity, separated by SDS–PAGE and immunoblotted. Rb fragment phosphorylated in vitro at T826 (Rb-kinase activity), CDK4, cyclin D1, cyclin D3, p21 and p27 were detected using specific antibodies. After densitometry quantitation, the Rb-kinase activity was normalized to the amount of co-Ip CDK4 and expressed as % of the FBS-stimulated condition without JNK inhibitor (Rb Kin./CDK4; ND, values could not be determined because of insufficient CDK4 signal). (c, d) The same immunoprecipitates analyzed in b were separated by 2D-gel electrophoresis followed by immunodetection of CDK4 (c) and p21 (d). The percentage of the T172-phosphorylated form of CDK4 (black arrows) versus the sum of Ip CDK4 forms is determined by densitometry (% p-CDK4) (c). Arrows in d indicate the phosphorylated form of p21. Results in a–c were reproduced at least five times with SP600125 and three times with JNK-IN-8 in independent experiments, with similar observations. (e) Cell lysates from serum-starved quiescent T98G cells were Ip with p27 antibody (Ip p27), incubated with ATP with or without recombinant JNK1, and separated by 2D-gel electrophoresis followed by CDK4 immunodetection.