CDK2 and PKA participate in p19 sequential phosphorylation.

<p>(<b>A</b>) CDK and PKA involvement in endogenous p19 phosphorylation. WI-38 fibroblasts were incubated with roscovitine (RSC, 10 µM), or with H-89 (1 µM) for 1 hour before the damaging treatments (4 mJ/cm² UV light, 10 µM cisplatin or 20 µM ß-amyloid peptide). p19 phosphorylation was analyzed by autoradiography. (<b>B, C</b>) Effect of CDK and PKA inhibition on the phosphorylation of T141 mutants. WI-38 cells were transfected with the indicated p19 constructs expression plasmids, incubated with roscovitine or H-89 for 1 hour and then treated with UV light (4 mJ/cm²) or β-amyloid peptide (20 µM) for 2 hours. p19wt or the mutants were immunoprecipitated with anti-V5 antibody and the immunocomplexes were analyzed by autorradiography and immunoblotting. (<b>D</b>) Measurement of CDK1 and CDK2 activities in the phosphorylation process of endogenous p19. WI-38 fibroblasts were incubated for 24 hours with specific CDK1 or CDK2 antisense oligonucleotides before treatment with UV radiation (4 mJ/cm²). After 2 hours, p19 was immunoprecipitated and phosphorylation observed by autoradiography as mentioned before (upper panel). Northern blot results show the efficiency of the antisense oligonucleotides (lower panel).