Molecular cloning and characterization of the human protein kinase D2 - A novel member of the protein kinase D family of serine threonine kinases

We have isolated the full-length cDNA of a novel human serine threonine pro tein kinase gene. The deduced protein sequence contains two cysteine-rich m otifs at the N terminus, a pleckstrin homology domain, and a catalytic doma in containing all the characteristic sequence motifs of serine protein kina ses. It exhibits the strongest homology to the serine threonine protein kin ases PKD/PKC mu, and PKC nu, particularly in the duplex zinc finger-like cy steine-rich motif, in the pleckstrin homology domain and in the protein kin ase domain, In contrast, it shows only a low degree of sequence similarity to other members of the PKC family, Therefore, the new protein has been ter med protein kinase D2 (PKD2). The mRNA of PKD2 is widely expressed in human and murine tissues. It encodes a protein with a molecular mass of 105 kDa in SDS-polyacrylamide gel electrophoresis, which is expressed in various hu man cell lines, including HL60 cells, which do not express PKC mu. lit vivo phorbol ester binding studies demonstrated a concentration-dependent bindi ng of [H-3]phorbol 12,13-dibutyrate to PKD2. The addition of phorbol 12,13- dibutyrate in the presence of dioleoylphosphatidylserine stimulated the aut ophosphorylation of PKD2 in a synergistic fashion. Phorbol esters also stim ulated autophosphorylation of PKD2 in intact cells. PKD2 activated by phorb ol esters efficiently phosphorylated the exogenous substrate histone H1. In addition, we could identify the C-terminal Ser(876) residue as an in vivo phosphorylation site within PKD2. Phosphorylation of Ser(876) of PKD2 corre lated with the activation status of the kinase. Finally, gastrin was found to be a physiological activator of PKD2 in human AGS-B cells stably transfe cted with the CCKB/gastrin receptor. Thus, PKD2 is a novel phorbol ester- a nd growth factor-stimulated protein kinase.