FIG. 8.

Direct phosphorylation of PKD by PKCδ stimulates PKD. (A) An in vitro kinase reaction was performed with purified recombinant PKD and PKCδ (250 ng of PKD, 100 ng of PKCδ, 100 μM unlabeled ATP). PKD then was analyzed for activation loop phosphorylation (anti-pS738/742 antibody). The blot was stripped and reprobed against PKD. (B) PKD, PKCδ, or Abl was added alone or in combination, as indicated, to an unlabeled in vitro kinase assay mixture (200 ng of PKD, 100 ng of PKCδ and/or 20 ng of Abl, 150 μM unlabeled ATP). PKD was immunoprecipitated, and after extensive washing in kinase buffer, a PKD substrate peptide assay was performed as described in Materials and Methods. All results are typical of two independent experiments.