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 Ser 744 and Ser 748 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 32 P-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 Ser 744 and Ser 748 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 C (PKC), 1 a major target for the tumor promoting phorbol esters, has been implicated in the signal transduction of a wide range of biological responses, including changes in cell morphology, differentiation, and proliferation (1-5). Molecular cloning has demonstrated the presence of multiple related PKC isoforms (5-8) i.e. classic PKCs (␣, ␤1, ␤2, and ␥), novel PKCs (␦, ⑀, , and ) and atypical PKCs ( and ) all of which possess a highly conserved catalytic domain. Despite intense investigation, the events occurring downstream of specific isoforms of PKC remain poorly defined.The newly identified PKD is a mouse serine/threonine protein kinase with distinct structural and enzymological properties (9). The catalytic domain of PKD is distantly related to Ca 2ϩ -regulated kinases and shows little similarity to the highly conserved regions of the kinase subdomains of the PKC family (10). Consistent with this, PKD does not phosphorylate a variety of substrates utilized by PKCs, indicating that PKD is a protein kinase with distinct substrate specificity (9, 11). In contrast to all known PKCs, including mammalian, Drosophila, and yeast isoforms (12), the NH 2 -terminal region of PKD contains a pleckstrin homology domain that regulates enzyme activity (13) and lacks a sequence with homology to a typical PKC autoinhibitory pseudosubstrate motif (9). However, the amino-terminal region of PKD contains a tandem repeat of cysteine-rich, zinc finger-like motifs that binds phorbol esters with high affinity (9). Immunopurified PKD is markedly stimulated by either biologically active...

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 coimmunoprecipitates 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.Stimulation of several plasma membrane receptors by hormones, growth factors, and cytokines causes the rapid hydrolysis of phosphoinositides, which results in an increase of the lipid-derived second messenger diacylglycerol (DAG) 1 (1). DAG causes the activation of protein kinase C (PKC) (2, 3), which in turn affects several cellular processes including cell growth and differentiation, changes in cell morphology, neuronal development, and endocrine and exocrine secretion (2, 4 -6). PKC isoforms are divided into three major groups on the basis of their primary structure and biochemical properties (7-9). The conventional PKCs (␣, ␤1, ␤2, and ␥) contain a regulatory cysteine-rich domain that binds DAG and phorbol esters and a calcium binding domain responsible for the calcium-dependent modulation of their kinase activity. The novel PKCs (␦, ⑀, , and ) are sensitive to DAG or phorbol esters but are calciumindependent. The third group is known as the atypical PKCs ( and /), which are unresponsive to calcium, DAG, or phorbol esters.Protein kinase D (PKD, also known as PKC) is a serine/ threonine kinase distantly related to the PKC family. It contains a conserved DAG/phorbol ester-binding cysteine-rich domain like the classical and novel PKC isoforms but has singular enzymological and structural properties (10 -13). Unlike all oth...

The importance of activation loop phosphorylation in the regulation of protein kinase D (PKD/protein kinase C (PKC) ) activity has become controversial. In order to clarify the mechanism(s) of PKD activation, we developed a novel phosphospecific antibody recognizing phosphorylated Ser 748 in PKD (pS748

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 (ϳ16-fold) and exogenous syntide-2 peptide substrate phosphorylation assays (ϳ12-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 C (PKC)1 has been implicated in the signal transduction of a wide range of biological responses including changes in cell morphology, differentiation, and proliferation (1-5). Molecular cloning has demonstrated the presence of multiple related PKC isoforms (3, 6, 7), i.e. classic PKCs (␣, ␤1, ␤2, and ␥), novel PKCs (␦, ⑀, , and ), and atypical PKCs ( and ) all of which possess a highly conserved catalytic domain.The newly identified PKD is a mouse serine/threonine protein kinase with distinct structural and enzymological properties (8). The catalytic domain of PKD is distantly related to Ca 2ϩ -regulated kinases and shows little similarity to the highly conserved regions of the kinase subdomains of the PKC family (9). Consistent with this, PKD does not phosphorylate a variety of substrates utilized by PKCs indicating that PKD is a protein kinase with distinct substrate specificity (8, 10). However, the amino-terminal region of PKD contains a tandem repeat of cysteine-rich, zinc finger-like motifs that bind phorbol esters with high affinity (8, 10). Immunopurified PKD is markedly stimulated by PDB or diacylglycerol in the presence of phosphatidylserine (10). The human protein kinase PKC (11, 12) with 92% homology to PKD (extending to 98% homology in the catalytic domain) is also stimulated by phorbol esters and phospholipids (12). These in vitro results indicate that PKD/ PKC is a novel phorbol ester/diacylglycerol-stimulated protein kinase. Recently a new mechanism of PKD activation has been identified (13). Specifically, exposure of intact cells to biologically active phorbol esters and membrane-permeant diacylglycerol induces phosphorylation-dependent PKD activation via a PKC-dependent pathway (13). Thus, PKD can functio...

The results presented here demonstrate that protein kinase D (PKD) and PKC transiently coexpressed in COS-7 cells form complexes that can be immunoprecipitated from cell lysates using specific antisera to PKD or PKC. The presence of PKC 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 PKC 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 PKC⑀, and did not bind PKC. When PKC was coexpressed with PKD mutants containing either complete or partial deletions of the PH domain, both PKC 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 PKC. Furthermore, a glutathione S-transferase-PKDPH fusion protein bound preferentially to PKC. These results indicate that the PKD PH domain can discriminate between closely related structures of a single enzyme family, e.g. novel PKCs ⑀ and , thereby revealing a previously undetected degree of specificity among protein-protein interactions mediated by PH domains.