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We have isolated the full-length cDNA of a novel human serine threonine protein kinase gene. The deduced protein sequence contains two cysteine-rich motifs at the N terminus, a pleckstrin homology domain, and a catalytic domain containing all the characteristic sequence motifs of serine protein kinases. It exhibits the strongest homology to the serine threonine protein kinases PKD/PKCmicro and PKCnu, particularly in the duplex zinc finger-like cysteine-rich motif, in the pleckstrin homology domain and in the protein kinase 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 termed 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 human cell lines, including HL60 cells, which do not express PKCmicro. In vivo phorbol ester binding studies demonstrated a concentration-dependent binding of [(3)H]phorbol 12,13-dibutyrate to PKD2. The addition of phorbol 12,13-dibutyrate in the presence of dioleoylphosphatidylserine stimulated the autophosphorylation of PKD2 in a synergistic fashion. Phorbol esters also stimulated autophosphorylation of PKD2 in intact cells. PKD2 activated by phorbol 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 correlated with the activation status of the kinase. Finally, gastrin was found to be a physiological activator of PKD2 in human AGS-B cells stably transfected with the CCK(B)/gastrin receptor. Thus, PKD2 is a novel phorbol ester- and growth factor-stimulated protein kinase.

Recently, we cloned a novel serine/threonine kinase termed protein kinase D2 (PKD2). PKD2 can be activated by phorbol esters both in vivo and in vitro but also by gastrin via the cholecystokinin/CCK B receptor in human gastric cancer cells stably transfected with the CCK B /gastrin receptor (AGS-B cells). Here we identify the mechanisms of gastrin-induced PKD2 activation in AGS-B cells. PKD2 phosphorylation in response to gastrin was rapid, reaching a maximum after 10 min of incubation. Our data demonstrate that gastrin-stimulated PKD2 activation involves a heterotrimeric G␣ q protein as well as the activation of phospholipase C. Furthermore, we show that PKD2 can be activated by classical and novel members of the protein kinase C (PKC) family such as PKC␣, PKC⑀, and PKC. These PKCs are activated by gastrin in AGS-B cells. Thus, PKD2 is likely to be a novel downstream target of specific PKCs upon the stimulation of AGS-B cells with gastrin. Our data suggest a two-step mechanism of activation of PKD2 via endogenously produced diacylglycerol and the activation of PKCs.We recently described the cloning of a novel human serine/ threonine kinase termed PKD2 1 (1). This kinase exhibits a high homology to PKD/PKC and PKC but not to the classical, novel, and atypical members of the protein kinase C family. PKD2 has two cysteine-rich domains at its N terminus that bind phorbol esters and diacylglycerol and is potently activated by phorbol esters in vitro and in vivo. In addition, gastrin activates PKD2 via the CCK B /gastrin receptor (also termed the CCK2 receptor). However, the precise mechanism leading to the activation of PKD2 in response to gastrin has not been characterized.Gastrin is implicated in a wide range of fundamental biological responses such as secretion, growth, and neoplastic transformation (2). Therefore, there is considerable interest in the elucidation of signaling pathways mediated by the CCK B /gastrin receptor in nontransformed and transformed cells. Gastrin, CCK, and CCK-related peptides exert their effects by binding to specific G protein-coupled receptor subtypes. The CCK B /gastrin receptor binds gastrin and CCK with similar affinity, whereas the CCK A (also called CCK1) receptor exhibits a 500-fold higher affinity for CCK than for gastrin (3). Upon binding to the CCK B /gastrin receptor, gastrin stimulates multiple signaling pathways. One of the earliest signal transduction pathways activated in response to gastrin is the stimulation of ␤-isoforms of PLC, leading to IP 3 -mediated Ca 2ϩ -mobilization from internal stores and DAG-induced stimulation of classical and novel isoforms of the protein kinase C family.In this manuscript we identify the precise signaling mechanism leading to activation of PKD2 in response to gastrin. We show that the activation of PKD2 by gastrin in AGS-B cells involves a G␣ q heterotrimeric G protein, PLC, and possibly various PKC isoforms, making PKD2 a novel downstream target of members of the PKC family. EXPERIMENTAL PROCEDURES Materials-[␥-32 P]ATP (5000 Ci/mmol; ...

Protein kinase D2 (PKD2) belongs to the PKD family of serine/threonine kinases that is activated by phorbol esters and G protein-coupled receptors (GPCRs). Its C-terminal regulatory domain comprises two cysteine-rich domains (C1a/C1b) followed by a pleckstrin homology (PH) domain. Here, we examined the role of the regulatory domain in PKD2 phorbol ester binding, catalytic activity, and subcellular localization: The PH domain is a negative regulator of kinase activity. C1a/C1b, in particular C1b, is required for phorbol ester binding and gastrin-stimulated PKD2 activation, but it has no inhibitory effect on the catalytic activity. Gastrin triggers nuclear accumulation of PKD2 in living AGS-B cancer cells. C1a/C1b, not the PH domain, plays a complex role in the regulation of nucleocytoplasmic shuttling: We identified a nuclear localization sequence in the linker region between C1a and C1b and a nuclear export signal in the C1a domain. In conclusion, our results define the critical components of the PKD2 regulatory domain controlling phorbol ester binding, catalytic activity, and nucleocytoplasmic shuttling and reveal marked differences to the regulatory properties of this domain in PKD1. These findings could explain functional differences between PKD isoforms and point to a functional role of PKD2 in the nucleus upon activation by GPCRs.

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