Protein kinase D (PKD) is activated by phosphorylation in intact cells stimulated by phorbol esters, cell permeant diacylglycerols, bryostatin, neuropeptides, and growth factors, but the critical activating residues in PKD have not been identified. Here, we show that substitution of Ser⁷⁴⁴ and Ser⁷⁴⁸ with alanine (PKD-S744A/S748A) completely blocked PKD activation induced by phorbol-12,13-dibutyrate (PDB) treatment of intact cells as assessed by autophosphorylation and exogenous syntide-2 peptide substrate phosphorylation assays. Conversely, replacement of both serine residues with glutamic acid (PKD-S744E/S748E) markedly increased basal activity (7.5-fold increase compared with wild type PKD). PKD-S744E/S748E mutant was only slightly further stimulated by PDB treatment in vivo, suggesting that phosphorylation of these two sites induces maximal PKD activation. Two-dimensional tryptic phosphopeptide analysis obtained from PKD mutants immunoprecipitated from ³²P-labeled transfected COS-7 cells showed that two major spots present in the PDB-stimulated wild type PKD or the kinase-dead PKD-D733A phosphopeptide maps completely disappeared in the kinase-deficient triple mutant PKD-D733A/S744E/S748E. Our results indicate that PKD is activated by phosphorylation of residues Ser⁷⁴⁴ and Ser⁷⁴⁸ and thus provide the first example of a non-RD kinase that is up-regulated by phosphorylation of serine/threonine residues within the activation loop.

Protein kinase D (PKD) is a serine/threonine kinase regulated by diacylglycerol signaling pathways with unique domain composition and enzymatic properties, still awaiting identification of its specific substrate(s). Here we have isolated, cloned, and characterized a novel protein from PC12 cells, termed Kidins220 (kinase D-interacting substrate of 220 kDa), as the first identified PKD physiological substrate. Kidins220 contains 11 ankyrin repeats and four transmembrane domains within the N-terminal region. We have shown that Kidins220 is an integral membrane protein selectively expressed in brain and neuroendocrine cells, where it concentrates at the tip of neurites. In PC12 cells, PKD co-immunoprecipitates and phosphorylates endogenous Kidins220. This phosphorylation is increased after stimulating PKD activity in vivo by phorbol-12, 13-dibutyrate treatment. A constitutively active PKD mutant (PKD-S744E/S748E) phosphorylates recombinant Kindins220-VSVG in vitro in the absence of phorbol-12,13-dibutyrate. Conversely, Kidins220-VSVG phosphorylation is abolished when a dominant negative mutant of PKD (PKD-D733A) is used. Moreover, a peptide within the Kidins220 sequence, containing serine 919 in a consensus motif for PKD-specific phosphorylation, behaved as the best peptide substrate to date. Substitution of serine 919 to alanine abrogated peptide phosphorylation. Furthermore, by generating an antibody recognizing Kidins220 phosphorylated on serine 919, we show that phorbol ester treatment causes the specific phosphorylation of this residue in PC12 cells in vivo. Our results provide the first physiological substrate for PKD and indicate that Kidins220 is phosphorylated by PKD at serine 919 in vivo.

The importance of activation loop phosphorylation in the regulation of protein kinase D (PKD/protein kinase C (PKC) μ) activity has become controversial. In order to clarify the mechanism(s) of PKD activation, we developed a novel phosphospecific antibody recognizing phosphorylated Ser⁷⁴⁸ in PKD (pS748). Western blot analysis with the pS748 antibody, carried out with a variety of PKD forms and in a variety of cell types including full-length PKD transfected in COS-7 and HEK 293 cells, a green fluorescent protein-PKD fusion protein transfected in either Swiss 3T3 fibroblasts or Madin-Darby canine kidney epithelial cells, and endogenous PKD expressed in A20 lymphocytes and Rat-1 fibroblasts, indicated that Ser⁷⁴⁸ phosphorylation was absent from unstimulated cells. In contrast, dramatic increases in Ser⁷⁴⁸ phosphorylation were induced by phorbol esters, bombesin, or cross-linking of B lymphocyte antigen receptors or by cotransfection with active PKCε or PKCη. Western analysis using a second phosphospecific antibody, which primarily recognizes PKD phosphorylated at Ser⁷⁴⁴, revealed that Ser⁷⁴⁴phosphorylation accompanies Ser⁷⁴⁸ phosphorylation during PKD activation in vivo. Ser⁷⁴⁴/Ser⁷⁴⁸ phosphorylation requires PKC but not PKD activity, indicative of transphosphorylation. Our results provide new experimental evidence indicating that activation loop phosphorylation at Ser⁷⁴⁴ and Ser⁷⁴⁸ occurs during PKD activation in vivo and support the notion of a PKC-PKD phosphorylation cascade.

Protein kinase D (PKD) is a serine/threonine protein kinase that contains a cysteine-rich repeat sequence homologous to that seen in the regulatory domain of protein kinase C (PKC) and a catalytic domain with only a low degree of sequence similarity to PKCs. PKD also contains a pleckstrin homology (PH) domain inserted between the cysteine-rich motifs and the catalytic domain that is not present in any of the PKCs. To investigate the function of the PH domain in the regulation of PKD activity, we determined the kinase activity of several PKD PH domain mutants immunoprecipitated from lysates of transiently transfected COS-7 cells. Deletion of the entire PH domain (amino acids 429–557) markedly increased the basal activity of the enzyme as assessed by autophosphorylation (∼16-fold) and exogenous syntide-2 peptide substrate phosphorylation assays (∼12-fold). Mutant PKD proteins with partial deletions or single amino acid substitutions within the PH domain (e.g. R447C and W538A) also exhibited increased basal kinase activity. These constitutive active mutants of PKD were only slightly further stimulated by phorbol-12,13-dibutyrate treatment of intact cells. Our results demonstrate, for the first time, that the PKD PH domain plays a negative role in the regulation of enzyme activity.

Protein kinase D (PKD; also known as PKCμ) is a serine/threonine kinase activated by diacylglycerol signalling pathways in a variety of cells. PKD has been described previously as Golgi‐localized, but herein we show that it is present within the cytosol of quiescent B cells and mast cells and moves rapidly to the plasma membrane after antigen receptor triggering. The membrane redistribution of PKD requires the diacylglycerol‐binding domain of the enzyme, but is independent of its catalytic activity and does not require the integrity of the pleckstrin homology domain. Antigen receptor signalling initiates in glycosphingolipid‐enriched microdomains, but membrane‐associated PKD does not co‐localize with these specialized structures. Membrane targeting of PKD is transient, the enzyme returns to the cytosol within 10 min of antigen receptor engagement. Strikingly, the membrane‐recycled PKD remains active in the cytosol for several hours. The present work thus characterizes a sustained antigen receptor‐induced signal transduction pathway and establishes PKD as a serine kinase that temporally and spatially disseminates antigen receptor signals away from the plasma membrane into the cytosol.

The results presented here demonstrate that protein kinase D (PKD) and PKCeta transiently coexpressed in COS-7 cells form complexes that can be immunoprecipitated from cell lysates using specific antisera to PKD or PKCeta. The presence of PKCeta in PKD immune complexes was initially detected by in vitro kinase assays which reveal the presence of an 80-kDa phosphorylated band in addition to the 110-kDa band corresponding to autophosphorylated PKD. The association between PKD and PKCeta was further verified by Western blot analysis and peptide phosphorylation assays that exploited the distinct substrate specificity between PKCs and PKD. By the same criteria, PKD formed complexes only very weakly with PKCepsilon, and did not bind PKCzeta. When PKCeta was coexpressed with PKD mutants containing either complete or partial deletions of the PH domain, both PKCeta immunoreactivity and PKC activity in PKD immunoprecipitates were sharply reduced. In contrast, deletion of an amino-terminal portion of the molecule, either cysteine-rich region, or the entire cysteine-rich domain did not interfere with the association of PKD with PKCeta. Furthermore, a glutathione S-transferase-PKDPH fusion protein bound preferentially to PKCeta. These results indicate that the PKD PH domain can discriminate between closely related structures of a single enzyme family, e.g. novel PKCs epsilon and eta, thereby revealing a previously undetected degree of specificity among protein-protein interactions mediated by PH domains.