Protein Kinase D Activation by Mutations within Its Pleckstrin Homology Domain

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) 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 C (PKC) 1The abbreviations used are: PKC, protein kinase C; DMEM, Dulbecco's modified Eagle's medium; GF, I GF 1092030X; PBS, phosphate-buffered saline; PCR, polymerase chain reaction; PDB, phorbol-12,13-dibutyrate; PH, pleckstrin homology; PKD, protein kinase D; PAGE, polyacrylamide gel electrophoresis. 1The abbreviations used are: PKC, protein kinase C; DMEM, Dulbecco's modified Eagle's medium; GF, I GF 1092030X; PBS, phosphate-buffered saline; PCR, polymerase chain reaction; PDB, phorbol-12,13-dibutyrate; PH, pleckstrin homology; PKD, protein kinase D; PAGE, polyacrylamide gel electrophoresis.has been implicated in the signal transduction of a wide range of biological responses including changes in cell morphology, differentiation, and proliferation (1Kikkawa U. Nishizuka Y. Annu. Rev. Cell Biol. 1986; 2: 149-154Crossref PubMed Scopus (442) Google Scholar, 2Rozengurt E. Science. 1986; 234: 161-166Crossref PubMed Scopus (845) Google Scholar, 3Nishizuka Y. Science. 1992; 258: 607-614Crossref PubMed Scopus (4215) Google Scholar, 4Weinstein I.B. Cancer Res. 1988; 48: 4135-4143PubMed Google Scholar, 5Herschmann H.R. Annu. Rev. Biochem. 1991; 60: 281-319Crossref PubMed Scopus (943) Google Scholar). Molecular cloning has demonstrated the presence of multiple related PKC isoforms (3Nishizuka Y. Science. 1992; 258: 607-614Crossref PubMed Scopus (4215) Google Scholar, 6Dekker L.V. Parker P.J. Trends Biochem. Sci. 1994; 19: 73-77Abstract Full Text PDF PubMed Scopus (918) Google Scholar, 7Hug H. Sarre T.F. Biochem. J. 1993; 291: 329-343Crossref PubMed Scopus (1217) Google Scholar),i.e. classic PKCs (α, β1, β2, and γ), novel PKCs (δ, ε, η, and θ), and atypical PKCs (ζ and λ) 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 (8Valverde A.M. Sinnett-Smith J. Van Lint J. Rozengurt E. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 8572-8576Crossref PubMed Scopus (357) Google Scholar). The catalytic domain of PKD is distantly related to Ca2+-regulated kinases and shows little similarity to the highly conserved regions of the kinase subdomains of the PKC family (9Rozengurt E. Sinnett-Smith J. Lint J.V. Valverde A.M. Mutat. Res. 1995; 333: 153-160Crossref PubMed Scopus (48) Google Scholar). 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 (8Valverde A.M. Sinnett-Smith J. Van Lint J. Rozengurt E. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 8572-8576Crossref PubMed Scopus (357) Google Scholar, 10Van Lint J. Sinnett-Smith J. Rozengurt E. J. Biol. Chem. 1995; 270: 1455-1461Abstract Full Text Full Text PDF PubMed Scopus (154) Google Scholar). 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 (8Valverde A.M. Sinnett-Smith J. Van Lint J. Rozengurt E. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 8572-8576Crossref PubMed Scopus (357) Google Scholar,10Van Lint J. Sinnett-Smith J. Rozengurt E. J. Biol. Chem. 1995; 270: 1455-1461Abstract Full Text Full Text PDF PubMed Scopus (154) Google Scholar). Immunopurified PKD is markedly stimulated by PDB or diacylglycerol in the presence of phosphatidylserine (10Van Lint J. Sinnett-Smith J. Rozengurt E. J. Biol. Chem. 1995; 270: 1455-1461Abstract Full Text Full Text PDF PubMed Scopus (154) Google Scholar). The human protein kinase PKCμ (11Johannes F.J. Prestle J. Eis S. Oberhagemann P. Pfizenmaier K. J. Biol. Chem. 1994; 269: 6140-6148Abstract Full Text PDF PubMed Google Scholar, 12Dieterich S. Herget T. Link G. Bottinger H. Pfizenmaier K. Johannes F.J. FEBS Lett. 1996; 381: 183-187Crossref PubMed Scopus Google with homology to PKD to homology in the catalytic is also stimulated by phorbol esters and S. Herget T. Link G. Bottinger H. Pfizenmaier K. Johannes F.J. FEBS Lett. 1996; 381: 183-187Crossref PubMed Scopus Google Scholar). These in results that is a novel phorbol protein a of PKD has been identified Sinnett-Smith J. Lint J.V. Rozengurt E. J. 1996; PubMed Scopus Google Scholar). of intact to active phorbol esters and diacylglycerol PKD a Sinnett-Smith J. Lint J.V. Rozengurt E. J. 1996; PubMed Scopus Google Scholar). PKD function in to or of PKCs in signal amino-terminal region of PKD also contains a PH domain which is not in any of the PKCs. PH of amino acids with in sequence S. Science. 1993; PubMed Scopus Google Scholar, A.M. 1988; 333: PubMed Scopus Google Scholar, 1993; PubMed Scopus Google Scholar, 1993; Full Text PDF PubMed Scopus Google Scholar, A. Trends Biochem. Sci. 1994; 19: Full Text PDF PubMed Scopus Google Scholar, J. 1996; Full Text Full Text PDF PubMed Scopus Google Scholar). These been identified in a of and proteins A. Trends Biochem. Sci. 1994; 19: Full Text PDF PubMed Scopus Google Scholar, J. 1996; Full Text Full Text PDF PubMed Scopus Google Scholar, A. T. P. J. Trends Biochem. Sci. 1993; Full Text PDF PubMed Scopus Google Scholar, G. 1995; Scopus Google Scholar). has been that PH homology domain and function and PH been to bind and or proteins as the of proteins J. 1996; Full Text Full Text PDF PubMed Scopus Google Scholar, P.J. 1994; PubMed Scopus Google Scholar, K. T.F. A. Biol. 1995; PubMed Scopus Google Scholar, H. J. 1995; PubMed Scopus Google Scholar, K. J. Biol. Chem. 1995; 270: Full Text Full Text PDF PubMed Scopus Google Scholar). The of the PH domain is for the and of PH including kinase S. Science. 1993; PubMed Scopus Google Scholar, P. Science. 1993; PubMed Scopus Google Scholar, T. S. A. H. K. 1995; 2: Full Text PDF PubMed Scopus Google Scholar, 1995; PubMed Scopus Google kinase K. J. Biol. Chem. 1995; 270: Full Text Full Text PDF PubMed Scopus Google and the serine/threonine kinase by the T.F. K. A. 1995; Full Text PDF PubMed Scopus Google Scholar). These to the function of the PH domain in the regulation of PKD the present we that a PKD the entire PH domain high basal kinase activity. active of PKD phorbol esters as as the protein is only slightly further by treatment with PDB in PKD mutants of the PH domain or with single amino acid substitutions within the PH domain also high basal kinase activity. Our results that the PH domain of PKD plays a negative role in the regulation of PKD kinase the of several PH to has been the function of domain A. Trends Biochem. Sci. 1994; 19: Full Text PDF PubMed Scopus Google Scholar, J. 1996; Full Text Full Text PDF PubMed Scopus Google Scholar, A. T. P. J. Trends Biochem. Sci. 1993; Full Text PDF PubMed Scopus Google Scholar, G. 1995; Scopus Google Scholar). The PH of a variety of been or in to with K. T.F. A. Biol. 1995; PubMed Scopus Google Scholar, T.F. K. A. 1995; Full Text PDF PubMed Scopus Google Scholar, T.F. A. Science. PubMed Scopus Google kinase S. J. J. Biol. Chem. 1996; Full Text Full Text PDF PubMed Scopus Google kinase S. Science. 1993; PubMed Scopus Google Scholar, P. Science. 1993; PubMed Scopus Google Scholar, T. S. A. H. K. 1995; 2: Full Text PDF PubMed Scopus Google Scholar, 1995; PubMed Scopus Google kinase K. J. Biol. Chem. 1995; 270: Full Text Full Text PDF PubMed Scopus Google the Y. A. J. Biol. Chem. 1996; Full Text Full Text PDF PubMed Scopus Google Scholar, A. U. Science. 1995; PubMed Scopus Google Scholar, U. J. Biol. Chem. 1995; 270: Full Text Full Text PDF PubMed Scopus Google Scholar, S. Biol. 1996; PubMed Scopus Google and K. J. Biol. Chem. Full Text Full Text PDF Scopus Google demonstrated that the PH domain plays role in the regulation of the enzyme activity. that the of the PH domain is for enzyme newly identified PKD contains a PH domain inserted between the cysteine-rich and the kinase catalytic domain (9Rozengurt E. Sinnett-Smith J. Lint J.V. Valverde A.M. Mutat. Res. 1995; 333: 153-160Crossref PubMed Scopus (48) Google Scholar). The presence of the PH domain PKD from all of the PKC the present we or the PH domain of PKD and the regulatory properties of the PKD to that PH for enzyme results that partial or deletions of the PH domain of PKD in PKD mutants that a high of basal kinase activity. amino acid substitutions (e.g. R447C and W538A) within the PH domain also of The R447C a in the PH domain of kinase is for the of in and in S. Science. 1993; PubMed Scopus Google Scholar, P. Science. 1993; PubMed Scopus Google Scholar, T. S. A. H. K. 1995; 2: Full Text PDF PubMed Scopus Google and a in the serine/threonine kinase of enzyme by T.F. K. A. 1995; Full Text PDF PubMed Scopus Google Scholar, T.F. A. Science. PubMed Scopus Google Scholar). The the only amino acid in the and is conserved in all PH A. Trends Biochem. Sci. 1994; 19: Full Text PDF PubMed Scopus Google Scholar, A. T. P. J. Trends Biochem. Sci. 1993; Full Text PDF PubMed Scopus Google Scholar). the as as partial or deletions of the PH domain in active of results demonstrated that treatment of intact with PDB, or PKD that cell and Sinnett-Smith J. Lint J.V. Rozengurt E. J. 1996; PubMed Scopus Google Scholar, Rozengurt E. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). of including the of PKC and of PKD with mutants of and Sinnett-Smith J. Lint J.V. Rozengurt E. J. 1996; PubMed Scopus Google that PKD by phosphorylation in intact a signal transduction Our results that PKD deletions of the PH domain or the single amino acid within the of the PH domain were only slightly stimulated further by treatment of the with PDB, that PKD active by PH domain is that the of the PH domain is for PKD in a of low catalytic kinase low basal activity of protein kinases is by the between domain within the enzyme with catalytic the of substrates J. Biol. Chem. Full Text PDF PubMed Google Scholar). all of the PKC family from to human PKCs possess that is of the first cysteine-rich domain T. J. 1993; PubMed Scopus Google Scholar). PKD does not a region in a (9Rozengurt E. Sinnett-Smith J. Lint J.V. Valverde A.M. Mutat. Res. 1995; 333: 153-160Crossref PubMed Scopus (48) Google Scholar). is that PKD is also by as protein the region has not been identified of is to that the PH domain of PKD has function by as a novel role of PH in enzyme the of partial or deletions of the PH domain of PKD or single amino acid substitutions within domain active of PKD in is by the results the that the PH domain of PKD bind that is by or by by treatment of intact with However, PH domain as and of proteins and enzyme of enzyme activity A. Trends Biochem. Sci. 1994; 19: Full Text PDF PubMed Scopus Google Scholar, J. 1996; Full Text Full Text PDF PubMed Scopus Google Scholar, A. T. P. J. Trends Biochem. Sci. 1993; Full Text PDF PubMed Scopus Google Scholar, G. 1995; Scopus Google Scholar). to between of the results demonstrate, for the first time, that the PH domain of PKD plays a negative role in the regulation of PKD kinase activity. Protein kinase C (PKC) 1The abbreviations used are: PKC, protein kinase C; DMEM, Dulbecco's modified Eagle's medium; GF, I GF 1092030X; PBS, phosphate-buffered saline; PCR, polymerase chain reaction; PDB, phorbol-12,13-dibutyrate; PH, pleckstrin homology; PKD, protein kinase D; PAGE, polyacrylamide gel electrophoresis. 1The abbreviations used are: PKC, protein kinase C; DMEM, Dulbecco's modified Eagle's medium; GF, I GF 1092030X; PBS, phosphate-buffered saline; PCR, polymerase chain reaction; PDB, phorbol-12,13-dibutyrate; PH, pleckstrin homology; PKD, protein kinase D; PAGE, polyacrylamide gel electrophoresis.has been implicated in the signal transduction of a wide range of biological responses including changes in cell morphology, differentiation, and proliferation (1Kikkawa U. Nishizuka Y. Annu. Rev. Cell Biol. 1986; 2: 149-154Crossref PubMed Scopus (442) Google Scholar, 2Rozengurt E. Science. 1986; 234: 161-166Crossref PubMed Scopus (845) Google Scholar, 3Nishizuka Y. Science. 1992; 258: 607-614Crossref PubMed Scopus (4215) Google Scholar, 4Weinstein I.B. Cancer Res. 1988; 48: 4135-4143PubMed Google Scholar, 5Herschmann H.R. Annu. Rev. Biochem. 1991; 60: 281-319Crossref PubMed Scopus (943) Google Scholar). Molecular cloning has demonstrated the presence of multiple related PKC isoforms (3Nishizuka Y. Science. 1992; 258: 607-614Crossref PubMed Scopus (4215) Google Scholar, 6Dekker L.V. Parker P.J. Trends Biochem. Sci. 1994; 19: 73-77Abstract Full Text PDF PubMed Scopus (918) Google Scholar, 7Hug H. Sarre T.F. Biochem. J. 1993; 291: 329-343Crossref PubMed Scopus (1217) Google Scholar),i.e. classic PKCs (α, β1, β2, and γ), novel PKCs (δ, ε, η, and θ), and atypical PKCs (ζ and λ) all of which possess a highly conserved catalytic The newly identified PKD is a mouse serine/threonine protein kinase with distinct structural and enzymological properties (8Valverde A.M. Sinnett-Smith J. Van Lint J. Rozengurt E. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 8572-8576Crossref PubMed Scopus (357) Google Scholar). The catalytic domain of PKD is distantly related to Ca2+-regulated kinases and shows little similarity to the highly conserved regions of the kinase subdomains of the PKC family (9Rozengurt E. Sinnett-Smith J. Lint J.V. Valverde A.M. Mutat. Res. 1995; 333: 153-160Crossref PubMed Scopus (48) Google Scholar). 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 (8Valverde A.M. Sinnett-Smith J. Van Lint J. Rozengurt E. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 8572-8576Crossref PubMed Scopus (357) Google Scholar, 10Van Lint J. Sinnett-Smith J. Rozengurt E. J. Biol. Chem. 1995; 270: 1455-1461Abstract Full Text Full Text PDF PubMed Scopus (154) Google Scholar). 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 (8Valverde A.M. Sinnett-Smith J. Van Lint J. Rozengurt E. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 8572-8576Crossref PubMed Scopus (357) Google Scholar,10Van Lint J. Sinnett-Smith J. Rozengurt E. J. Biol. Chem. 1995; 270: 1455-1461Abstract Full Text Full Text PDF PubMed Scopus (154) Google Scholar). Immunopurified PKD is markedly stimulated by PDB or diacylglycerol in the presence of phosphatidylserine (10Van Lint J. Sinnett-Smith J. Rozengurt E. J. Biol. Chem. 1995; 270: 1455-1461Abstract Full Text Full Text PDF PubMed Scopus (154) Google Scholar). The human protein kinase PKCμ (11Johannes F.J. Prestle J. Eis S. Oberhagemann P. Pfizenmaier K. J. Biol. Chem. 1994; 269: 6140-6148Abstract Full Text PDF PubMed Google Scholar, 12Dieterich S. Herget T. Link G. Bottinger H. Pfizenmaier K. Johannes F.J. FEBS Lett. 1996; 381: 183-187Crossref PubMed Scopus Google with homology to PKD to homology in the catalytic is also stimulated by phorbol esters and S. Herget T. Link G. Bottinger H. Pfizenmaier K. Johannes F.J. FEBS Lett. 1996; 381: 183-187Crossref PubMed Scopus Google Scholar). These in results that is a novel phorbol protein a of PKD has been identified Sinnett-Smith J. Lint J.V. Rozengurt E. J. 1996; PubMed Scopus Google Scholar). of intact to active phorbol esters and diacylglycerol PKD a Sinnett-Smith J. Lint J.V. Rozengurt E. J. 1996; PubMed Scopus Google Scholar). PKD function in to or of PKCs in signal The amino-terminal region of PKD also contains a PH domain which is not in any of the PKCs. PH of amino acids with in sequence S. Science. 1993; PubMed Scopus Google Scholar, A.M. 1988; 333: PubMed Scopus Google Scholar, 1993; PubMed Scopus Google Scholar, 1993; Full Text PDF PubMed Scopus Google Scholar, A. Trends Biochem. Sci. 1994; 19: Full Text PDF PubMed Scopus Google Scholar, J. 1996; Full Text Full Text PDF PubMed Scopus Google Scholar). These been identified in a of and proteins A. Trends Biochem. Sci. 1994; 19: Full Text PDF PubMed Scopus Google Scholar, J. 1996; Full Text Full Text PDF PubMed Scopus Google Scholar, A. T. P. J. Trends Biochem. Sci. 1993; Full Text PDF PubMed Scopus Google Scholar, G. 1995; Scopus Google Scholar). has been that PH homology domain and function and PH been to bind and or proteins as the of proteins J. 1996; Full Text Full Text PDF PubMed Scopus Google Scholar, P.J. 1994; PubMed Scopus Google Scholar, K. T.F. A. Biol. 1995; PubMed Scopus Google Scholar, H. J. 1995; PubMed Scopus Google Scholar, K. J. Biol. Chem. 1995; 270: Full Text Full Text PDF PubMed Scopus Google Scholar). The of the PH domain is for the and of PH including kinase S. Science. 1993; PubMed Scopus Google Scholar, P. Science. 1993; PubMed Scopus Google Scholar, T. S. A. H. K. 1995; 2: Full Text PDF PubMed Scopus Google Scholar, 1995; PubMed Scopus Google kinase K. J. Biol. Chem. 1995; 270: Full Text Full Text PDF PubMed Scopus Google and the serine/threonine kinase by the T.F. K. A. 1995; Full Text PDF PubMed Scopus Google Scholar). These to the function of the PH domain in the regulation of PKD activity. the present we that a PKD the entire PH domain high basal kinase activity. active of PKD phorbol esters as as the protein is only slightly further by treatment with PDB in PKD mutants of the PH domain or with single amino acid substitutions within the PH domain also high basal kinase activity. Our results that the PH domain of PKD plays a negative role in the regulation of PKD kinase activity. the of several PH to has been the function of domain A. Trends Biochem. Sci. 1994; 19: Full Text PDF PubMed Scopus Google Scholar, J. 1996; Full Text Full Text PDF PubMed Scopus Google Scholar, A. T. P. J. Trends Biochem. Sci. 1993; Full Text PDF PubMed Scopus Google Scholar, G. 1995; Scopus Google Scholar). The PH of a variety of been or in to with K. T.F. A. Biol. 1995; PubMed Scopus Google Scholar, T.F. K. A. 1995; Full Text PDF PubMed Scopus Google Scholar, T.F. A. Science. PubMed Scopus Google kinase S. J. J. Biol. Chem. 1996; Full Text Full Text PDF PubMed Scopus Google kinase S. Science. 1993; PubMed Scopus Google Scholar, P. Science. 1993; PubMed Scopus Google Scholar, T. S. A. H. K. 1995; 2: Full Text PDF PubMed Scopus Google Scholar, 1995; PubMed Scopus Google kinase K. J. Biol. Chem. 1995; 270: Full Text Full Text PDF PubMed Scopus Google the Y. A. J. Biol. Chem. 1996; Full Text Full Text PDF PubMed Scopus Google Scholar, A. U. Science. 1995; PubMed Scopus Google Scholar, U. J. Biol. Chem. 1995; 270: Full Text Full Text PDF PubMed Scopus Google Scholar, S. Biol. 1996; PubMed Scopus Google and K. J. Biol. Chem. Full Text Full Text PDF Scopus Google demonstrated that the PH domain plays role in the regulation of the enzyme activity. that the of the PH domain is for enzyme newly identified PKD contains a PH domain inserted between the cysteine-rich and the kinase catalytic domain (9Rozengurt E. Sinnett-Smith J. Lint J.V. Valverde A.M. Mutat. Res. 1995; 333: 153-160Crossref PubMed Scopus (48) Google Scholar). The presence of the PH domain PKD from all of the PKC the present we or the PH domain of PKD and the regulatory properties of the PKD to that PH for enzyme results that partial or deletions of the PH domain of PKD in PKD mutants that a high of basal kinase activity. amino acid substitutions (e.g. R447C and W538A) within the PH domain also of The R447C a in the PH domain of kinase is for the of in and in S. Science. 1993; PubMed Scopus Google Scholar, P. Science. 1993; PubMed Scopus Google Scholar, T. S. A. H. K. 1995; 2: Full Text PDF PubMed Scopus Google and a in the serine/threonine kinase of enzyme by T.F. K. A. 1995; Full Text PDF PubMed Scopus Google Scholar, T.F. A. Science. PubMed Scopus Google Scholar). The the only amino acid in the and is conserved in all PH A. Trends Biochem. Sci. 1994; 19: Full Text PDF PubMed Scopus Google Scholar, A. T. P. J. Trends Biochem. Sci. 1993; Full Text PDF PubMed Scopus Google Scholar). the as as partial or deletions of the PH domain in active of results demonstrated that treatment of intact with PDB, or PKD that cell and Sinnett-Smith J. Lint J.V. Rozengurt E. J. 1996; PubMed Scopus Google Scholar, Rozengurt E. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). of including the of PKC and of PKD with mutants of and Sinnett-Smith J. Lint J.V. Rozengurt E. J. 1996; PubMed Scopus Google that PKD by phosphorylation in intact a signal transduction Our results that PKD deletions of the PH domain or the single amino acid within the of the PH domain were only slightly stimulated further by treatment of the with PDB, that PKD active by PH domain is that the of the PH domain is for PKD in a of low catalytic kinase low basal activity of protein kinases is by the between domain within the enzyme with catalytic the of substrates J. Biol. Chem. Full Text PDF PubMed Google Scholar). all of the PKC family from to human PKCs possess that is of the first cysteine-rich domain T. J. 1993; PubMed Scopus Google Scholar). PKD does not a region in a (9Rozengurt E. Sinnett-Smith J. Lint J.V. Valverde A.M. Mutat. Res. 1995; 333: 153-160Crossref PubMed Scopus (48) Google Scholar). is that PKD is also by as protein the region has not been identified of is to that the PH domain of PKD has function by as a novel role of PH in enzyme the of partial or deletions of the PH domain of PKD or single amino acid substitutions within domain active of PKD in is by the results the that the PH domain of PKD bind that is by or by by treatment of intact with However, PH domain as and of proteins and enzyme of enzyme activity A. Trends Biochem. Sci. 1994; 19: Full Text PDF PubMed Scopus Google Scholar, J. 1996; Full Text Full Text PDF PubMed Scopus Google Scholar, A. T. P. J. Trends Biochem. Sci. 1993; Full Text PDF PubMed Scopus Google Scholar, G. 1995; Scopus Google Scholar). to between of the results demonstrate, for the first time, that the PH domain of PKD plays a negative role in the regulation of PKD kinase activity. the of several PH to has been the function of domain A. Trends Biochem. Sci. 1994; 19: Full Text PDF PubMed Scopus Google Scholar, J. 1996; Full Text Full Text PDF PubMed Scopus Google Scholar, A. T. P. J. Trends Biochem. Sci. 1993; Full Text PDF PubMed Scopus Google Scholar, G. 1995; Scopus Google Scholar). The PH of a variety of been or in to with K. T.F. A. Biol. 1995; PubMed Scopus Google Scholar, T.F. K. A. 1995; Full Text PDF PubMed Scopus Google Scholar, T.F. A. Science. PubMed Scopus Google kinase S. J. J. Biol. Chem. 1996; Full Text Full Text PDF PubMed Scopus Google kinase S. Science. 1993; PubMed Scopus Google Scholar, P. Science. 1993; PubMed Scopus Google Scholar, T. S. A. H. K. 1995; 2: Full Text PDF PubMed Scopus Google Scholar, 1995; PubMed Scopus Google kinase K. J. Biol. Chem. 1995; 270: Full Text Full Text PDF PubMed Scopus Google the Y. A. J. Biol. Chem. 1996; Full Text Full Text PDF PubMed Scopus Google Scholar, A. U. Science. 1995; PubMed Scopus Google Scholar, U. J. Biol. Chem. 1995; 270: Full Text Full Text PDF PubMed Scopus Google Scholar, S. Biol. 1996; PubMed Scopus Google and K. J. Biol. Chem. Full Text Full Text PDF Scopus Google demonstrated that the PH domain plays role in the regulation of the enzyme activity. that the of the PH domain is for enzyme The newly identified PKD contains a PH domain inserted between the cysteine-rich and the kinase catalytic domain (9Rozengurt E. Sinnett-Smith J. Lint J.V. Valverde A.M. Mutat. Res. 1995; 333: 153-160Crossref PubMed Scopus (48) Google Scholar). The presence of the PH domain PKD from all of the PKC the present we or the PH domain of PKD and the regulatory properties of the PKD to that PH for enzyme results that partial or deletions of the PH domain of PKD in PKD mutants that a high of basal kinase activity. amino acid substitutions (e.g. R447C and W538A) within the PH domain also of The R447C a in the PH domain of kinase is for the of in and in S. Science. 1993; PubMed Scopus Google Scholar, P. Science. 1993; PubMed Scopus Google Scholar, T. S. A. H. K. 1995; 2: Full Text PDF PubMed Scopus Google and a in the serine/threonine kinase of enzyme by T.F. K. A. 1995; Full Text PDF PubMed Scopus Google Scholar, T.F. A. Science. PubMed Scopus Google Scholar). The the only amino acid in the and is conserved in all PH A. Trends Biochem. Sci. 1994; 19: Full Text PDF PubMed Scopus Google Scholar, A. T. P. J. Trends Biochem. Sci. 1993; Full Text PDF PubMed Scopus Google Scholar). the as as partial or deletions of the PH domain in active of results demonstrated that treatment of intact with PDB, or PKD that cell and Sinnett-Smith J. Lint J.V. Rozengurt E. J. 1996; PubMed Scopus Google Scholar, Rozengurt E. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). of including the of PKC and of PKD with mutants of and Sinnett-Smith J. Lint J.V. Rozengurt E. J. 1996; PubMed Scopus Google that PKD by phosphorylation in intact a signal transduction Our results that PKD deletions of the PH domain or the single amino acid within the of the PH domain were only slightly stimulated further by treatment of the with PDB, that PKD active by PH domain is that the of the PH domain is for PKD in a of low catalytic kinase activity. The low basal activity of protein kinases is by the between domain within the enzyme with catalytic the of substrates J. Biol. Chem. Full Text PDF PubMed Google Scholar). all of the PKC family from to human PKCs possess that is of the first cysteine-rich domain T. J. 1993; PubMed Scopus Google Scholar). PKD does not a region in a (9Rozengurt E. Sinnett-Smith J. Lint J.V. Valverde A.M. Mutat. Res. 1995; 333: 153-160Crossref PubMed Scopus (48) Google Scholar). is that PKD is also by as protein the region has not been identified of is to that the PH domain of PKD has function by as a novel role of PH in enzyme the of partial or deletions of the PH domain of PKD or single amino acid substitutions within domain active of PKD in is by the results the that the PH domain of PKD bind that is by or by by treatment of intact with However, PH domain as and of proteins and enzyme of enzyme activity A. Trends Biochem. Sci. 1994; 19: Full Text PDF PubMed Scopus Google Scholar, J. 1996; Full Text Full Text PDF PubMed Scopus Google Scholar, A. T. P. J. Trends Biochem. Sci. 1993; Full Text PDF PubMed Scopus Google Scholar, G. 1995; Scopus Google Scholar). to between of the results demonstrate, for the first time, that the PH domain of PKD plays a negative role in the regulation of PKD kinase activity.