Fig. 4.

p160^ROCK phosphorylates Cdc25A. (A) In vitro kinase activity (K) of immunoprecipitated p160^ROCK from TGF-β treated NMuMG cells with either wild-type Cdc25A or an S123A mutant of Cdc25A as substrate. Western blot for Cdc25A (W) also was done from the same cell lysate as a control. Cdc25A* indicates immunodetection of both S123A and wild-type (wt) isoforms. (B) In vivo kinase activity of NMuMG cells treated with TGF-β and/or Y23637 was metabolically labeled with [³²P]orthophosphate, and Cdc25A was immunoprecipitated from equal amounts of extracts. (C) In vivo kinase activity in NIH 3T3 cells expressing GST-tagged wild-type Cdc25A in addition to Ost or control cDNA was determined. After [³²P]orthophosphate labeling and TGF-β and/or Y23637 treatment, GST-Cdc25A was pulled down with glutathione beads and separated by electrophoresis. The result is representative of at least two independent experiments.