FIG. 4.
Ask1 phosphorylates p21Cip1 in vitro and in vivo. (A) Bacterially expressed purified p21Cip1 was incubated with recombinant ASK1 in the presence of [γ-32P]ATP with or without addition of SP600125 (10 μM) or SB203580 (10 μM). In vitro kinase assays were carried out as described in Materials and Methods. (B) 293T cells were transfected with plasmids encoding HA-ASK1 or the epitope-tagged kinase-dead mutant ASK-KM. After 24 h, cell lysates were prepared and immunoprecipitated using anti-ASK1 antibody. Immunoprecipitates were used in vitro to phosphorylate either purified p21 or purified p27Kip1. (C). The JNK inhibitor SP600125 potently inhibits ASK1-mediated in vitro phosphorylation of MKK6 or p21Cip1. Recombinant ASK1 was incubated with purified Mal-E-MKK6 peptide, a known substrate, or purified p21Cip1 in the presence of [γ-32P]ATP and increasing concentrations of SP600125 as described in Materials and Methods. (D) ASK1 can phosphorylate p21çip1 in vivo. 293T cells were transfected with plasmids encoding ΔNLSp21 or its mutant S98E and cotransfected with ASK1 or JNK1 expression plasmids. The JNK inhibitor SP600125 was added 10 min before harvesting of cells. Cell lysates were immunoprecipated using anti-p21Cip1 antibody, and phosphorylated p21Cip1 was detected using a PhosphorImager. The same membrane was blotted using antibodies against p21Cip1. Levels of ASK1 and JNK1 were determined on whole-cell lysates (bottom panel). Relative activities for phosphorylation of ΔNLSp21 were calculated from the ratio of optical densities of the 32P-p21Cip1/p21Cip1 Western blot signals and normalized to that for ΔNLSp21. (E) ASK1 or ASK1-KM expression plasmids were cotransfected together with the ΔNLSp21 plasmid into 293T cells. After 24 h, cells were either not stimulated or stimulated with AN (10 μg/ml), a known activator of JNK1 and -2, in the presence of [32P]orthophosphate. After 3 h, p21Cip1 was immunoprecipitated, and phosphorylated p21 was detected using a PhosphorImager. The membrane was reprobed using anti-p21 antibody. The lower panels show levels of pJNK1, and the level of pJNK2 in whole-cell lysates was determined using anti-phospho-JNK antibody. Relative activities for phosphorylation of ΔNLSp21 were calculated from the ratio of optical densities of the 32P-p21Cip1/p21Cip1 Western blot signals.