Phosphorylation-dependent protein kinase D activation

The novel mouse serine-threonine kinase protein kinase D (PKD) is activated in intact Swiss 3T3 cells stimulated by phorbol esters, cell permeant diac ylglycerols, bryostatin, neuropeptides and growth factors via a phosphoryla tion-dependent mechanism requiring protein kinase C (PKC) activity. Structu ral comparison of the PKD catalytic domain with other kinases reveals a clo se similarity with MEK family kinases, which are activated upon phosphoryla tion of key serine and threonine residues in a region termed the activation loop. To study the regulation of PKD, we transfected mutant PKD cDNAs in w hich putative activation loop serine residues 744 and 748 were mutated to e ither alanine or glutamic acid into COS-7 cells. Replacement of serines 744 and 748 with alanine prevented activation of the overexpressed PKD form up on phorbol ester treatment of cells, whereas replacement with glutamic acid results in full constitutive activation. Single serine to glutamic acid re placement mutants were partially activated. In vivo P-32-labeling and two-d imensional phosphopeptide mapping of PKD and catalytically inactive PKD mut ants at serine 744, 748 or at both residues revealed that phorbol ester-sen sitive phosphopeptides could be selectively eliminated from patterns observ ed as a result of these mutations. Treatment of cells with the PKC inhibito r GFI also prevented the appearance of phosphopeptide spots occuring in res ponse to phorbol ester stimulation. These results provide direct evidence t hat PKD becomes activated in vivo as a consequence of PKC-mediated phosphor ylation of serines 744 and 748. These results support our view of PKD as an important clement in PKC signal transduction.