Figure 6.

PKN inhibits Cdc25C activity in vitro. Cdc25C tyrosine phosphatase activity was assayed by using the Cdc2 (N133A)/cyclin B complex purified from Sf9 cells as the phosphorylated substrate. Affinity-purified GST/Cdc25C was incubated with GST/PKN () or GST/PKN ()-K644E; then Cdc2 dephosphorylation was initiated by addition of the Cdc2 (N133A)/cyclin B complex. Dephosphorylation of Tyr-15 of Cdc2 was analyzed by Western blotting with antiphospho-Tyr-15 antibody (Upper). The same blot was reprobed with anti-Cdc2 antibody (Lower). GST/Cdc25C, GST/PKN (), and GST/PKN ()-K644E are indicated as Cdc25C, PKN, and PKN(KN), respectively.