IDENTIFICATION OF IN-VIVO PHOSPHORYLATION SITES REQUIRED FOR PROTEIN-KINASE-D ACTIVATION

Protein kinase D (PKD) is activated by phosphorylation in intact cells stimulated by phorbol esters, cell permeant diacylglycerols, bryostat in, neuropeptides, and growth factors, but the critical activating res idues in PKD have not been identified. Here, we show that substitution of Ser(744) and Ser(748) with alanine (PKD-S744A/S748A) completely bl ocked PKD activation induced by phorbol-12,13-dibutyrate (PDB) treatme nt of intact cells as assessed by autophosphorylation and exogenous sy ntide-2 peptide substrate phosphorylation assays. Conversely, replacem ent of both serine residues with glutamic acid (PKD-S744E/S748E) marke dly increased basal activity (7.5-fold increase compared with wild typ e PKD), PKD-S744E/S748E mutant was only slightly further stimulated by PDB treatment in vivo, suggesting that phosphorylation of these two s ites induces maximal PKD activation. Two-dimensional tryptic phosphope ptide analysis obtained from PKD mutants immunoprecipitated from P-32- labeled transfected COS-7 cells showed that two major spots present in the PDB-stimulated wild type PKD or the kinase-dead PKD-D733A phospho peptide maps completely disappeared in the kinase-deficient triple mut ant PKD-D733A/S744E/S748E. Our results indicate that PKD is activated by phosphorylation of residues Ser(744) and Ser(748) and thus provide the first example of a non-PH kinase that is up-regulated by phosphory lation of serine/threonine residues within the activation loop.