Protein kinase D (PKD) activation in intact cells through a protein kinase C‐dependent signal transduction pathway.

Protein kinase D (PKD) is a serine/threonine protein kinase that is directly stimulated in vitro by phorbol esters and diacylglycerol in the presence of phospholipids. Here, we examine the regulation of PKD in living cells. Our results demonstrate that tumour‐promoting phorbol esters, membrane‐permeant diacylglycerol and serum growth factors rapidly induced PKD activation in immortalized cell lines (e.g. Swiss 3T3 and Rat‐1 cells), in secondary cultures of mouse embryo fibroblasts and in COS‐7 cells transiently transfected with a PKD expression construct. PKD activation was maintained during cell disruption and immunopurification and was associated with an electrophoretic mobility shift and enhanced 32P incorporation into the enzyme, but was reversed by treatment with alkaline phosphatase. PKD was activated, deactivated and reactivated in response to consecutive cycles of addition and removal of PDB. PKD activation was completely abrogated by exposure of the cells to the protein kinase C inhibitors GF I and Ro 31–8220. In contrast, these compounds did not inhibit PKD activity when added directly in vitro. Co‐transfection of PKD with constitutively activated mutants of PKCs showed that PKCepsilon and eta but not PKCzeta strongly induced PKD activation in COS‐7 cells. Thus, our results indicate that PKD is activated in living cells through a PKC‐dependent signal transduction pathway.

[1]  C. Proud.  p70 S6 kinase: an enigma with variations. , 1996, Trends in biochemical sciences.

[2]  K. Pfizenmaier,et al.  In vitro activation and substrates of recombinant, baculovirus expressed human protein kinase Cμ , 1996, FEBS letters.

[3]  Á. Valverde,et al.  Protein kinase D (PKD): a novel target for diacylglycerol and phorbol esters. , 1995, Mutation research.

[4]  G. Milne,et al.  Residues in the Second Cysteine-rich Region of Protein Kinase C δ Relevant to Phorbol Ester Binding as Revealed by Site-directed Mutagenesis (*) , 1995, The Journal of Biological Chemistry.

[5]  Jonathan A. Cooper,et al.  Rho family members: Activators of MAP kinase cascades , 1995, Cell.

[6]  J. Hurley,et al.  Crystal structure of the Cys2 activator-binding domain of protein kinase Cδ in complex with phorbol ester , 1995, Cell.

[7]  T. Hunter,et al.  The eukaryotic protein kinase superfamily: kinase (catalytic) domain structure and classification 1 , 1995, FASEB journal : official publication of the Federation of American Societies for Experimental Biology.

[8]  E. Génot,et al.  Analysis of the Role of Protein Kinase C-α, -ϵ, and -ζ in T Cell Activation (*) , 1995, The Journal of Biological Chemistry.

[9]  J. Exton,et al.  Stimulation of Phospholipase D by Epidermal Growth Factor Requires Protein Kinase C Activation in Swiss 3T3 Cells (*) , 1995, The Journal of Biological Chemistry.

[10]  E. Rozengurt,et al.  Expression and Characterization of PKD, a Phorbol Ester and Diacylglycerol-stimulated Serine Protein Kinase (*) , 1995, The Journal of Biological Chemistry.

[11]  A. Toker,et al.  Activation of protein kinase C family members by the novel polyphosphoinositides PtdIns-3,4-P2 and PtdIns-3,4,5-P3. , 1994, The Journal of biological chemistry.

[12]  Á. Valverde,et al.  Molecular cloning and characterization of protein kinase D: a target for diacylglycerol and phorbol esters with a distinctive catalytic domain. , 1994, Proceedings of the National Academy of Sciences of the United States of America.

[13]  M. Kazanietz,et al.  Close similarity of baculovirus-expressed n-chimaerin and protein kinase C alpha as phorbol ester receptors. , 1994, The Journal of biological chemistry.

[14]  A. Wittinghofer,et al.  Direct stimulation of Vav guanine nucleotide exchange activity for Ras by phorbol esters and diglycerides , 1994, Molecular and cellular biology.

[15]  J. Prestle,et al.  PKCu is a novel, atypical member of the protein kinase C family. , 1994, The Journal of biological chemistry.

[16]  R. Bell,et al.  A phorbol ester binding domain of protein kinase C gamma. High affinity binding to a glutathione-S-transferase/Cys2 fusion protein. , 1994, The Journal of biological chemistry.

[17]  D. Fesquet,et al.  Calmodulin-dependent protein kinase II mediates inactivation of MPF and CSF upon fertilization of Xenopus eggs , 1993, Nature.

[18]  L. Dekker,et al.  Mutagenesis of the regulatory domain of rat protein kinase C-eta. A molecular basis for restricted histone kinase activity. , 1993, The Journal of biological chemistry.

[19]  T. Toda,et al.  Two novel protein kinase C‐related genes of fission yeast are essential for cell viability and implicated in cell shape control. , 1993, The EMBO journal.

[20]  H. Hidaka,et al.  Purification and characterization of Ca2+/calmodulin-dependent protein kinase V from rat cerebrum. , 1993, The Journal of biological chemistry.

[21]  H. Hug,et al.  Protein kinase C isoenzymes: divergence in signal transduction? , 1993, The Biochemical journal.

[22]  P. Parker,et al.  Characterization of protein kinase C isotype expression in adult rat heart. Protein kinase C-epsilon is a major isotype present, and it is activated by phorbol esters, epinephrine, and endothelin. , 1993, Circulation research.

[23]  M. Berridge.  Inositol trisphosphate and calcium signalling , 1993, Nature.

[24]  Y. Nishizuka.  Intracellular signaling by hydrolysis of phospholipids and activation of protein kinase C. , 1992, Science.

[25]  E. Rozengurt,et al.  Bombesin, vasopressin, and endothelin stimulation of tyrosine phosphorylation in Swiss 3T3 cells. Identification of a novel tyrosine kinase as a major substrate. , 1992, The Journal of biological chemistry.

[26]  J. Exton,et al.  Purification and characterization of the zeta isoform of protein kinase C from bovine kidney. , 1992, The Journal of biological chemistry.

[27]  Y. Nishizuka,et al.  Isolation and characterization of the epsilon subspecies of protein kinase C from rat brain. , 1992, Proceedings of the National Academy of Sciences of the United States of America.

[28]  W. R. Bishop,et al.  Identification and characterization of zinc binding sites in protein kinase C. , 1991, Science.

[29]  D. Burns,et al.  Protein kinase C contains two phorbol ester binding domains. , 1991, The Journal of biological chemistry.

[30]  J. Zheng,et al.  Crystal structure of the catalytic subunit of cyclic adenosine monophosphate-dependent protein kinase. , 1991, Science.

[31]  S. Brenner,et al.  A phorbol ester/diacylglycerol-binding protein encoded by the unc-13 gene of Caenorhabditis elegans. , 1991, Proceedings of the National Academy of Sciences of the United States of America.

[32]  J. Exton.  Signaling through phosphatidylcholine breakdown. , 1990, The Journal of biological chemistry.

[33]  Y Nishizuka,et al.  Alfred P. Sloan Jr. prize. Studies and prospectives of the protein kinase C family for cellular regulation , 1989, Cancer.

[34]  I. Weinstein,et al.  The origins of human cancer: molecular mechanisms of carcinogenesis and their implications for cancer prevention and treatment--twenty-seventh G.H.A. Clowes memorial award lecture. , 1988, Cancer research.

[35]  E. Rozengurt.  Early signals in the mitogenic response. , 1986, Science.

[36]  N. Divecha,et al.  Phospholipid signaling , 1995, Cell.

[37]  S. Hanks,et al.  Protein kinase catalytic domain sequence database: identification of conserved features of primary structure and classification of family members. , 1991, Methods in enzymology.

[38]  H. Herschman.  Primary response genes induced by growth factors and tumor promoters. , 1991, Annual review of biochemistry.

[39]  B. Sefton,et al.  Acid and base hydrolysis of phosphoproteins bound to immobilon facilitates analysis of phosphoamino acids in gel-fractionated proteins. , 1989, Analytical biochemistry.

[40]  Y. Ono.  Protein kinase ζ subspecies from rat brain ; Its structure, expression and properties , 1989 .

[41]  Y. Nishizuka,et al.  The role of protein kinase C in transmembrane signalling. , 1986, Annual review of cell biology.