Characterization of a novel protein kinase D: Caenorhabditis elegans DKF-1 is activated by translocation-phosphorylation and regulates movement and growth in vivo

Protein kinase D (PKD) isoforms are protein kinase C (PKC) effectors in diacylglycerol (DAG)-regulated signaling pathways. Key physiological processes are placed under DAG control by the distinctive substrate specificity and intracellular distribution of PKDs. Comprehension of the roles of PKDs in homeostasis and signal transduction requires further knowledge of regulatory interplay among PKD and PKC isoforms, analysis of PKC-independent PKD activation, and characterization of functions controlled by PKDs in vivo. Caenorhabditis elegans and mammals share conserved signaling mechanisms, molecules, and pathways Thus, characterization of the C. elegans PKDs could yield insights into regulation and functions that apply to all eukaryotic PKDs. C. elegans DKF-1 (D kinase family-1) contains tandem DAG binding (C1) modules, a PH (pleckstrin homology) domain, and a Ser/Thr protein kinase segment, which are homologous with domains in classical PKDs. DKF-1 and PKDs have similar substrate specificities. Phorbol 12-myristate 13-acetate (PMA) switches on DKF-1 catalytic activity in situ by promoting phosphorylation of a single amino acid Thr(588) in the activation loop. DKF-1 phosphorylation and activation are unaffected when PKC activity is eliminated by inhibitors. Both phosphorylation and kinase activity of DKF-1 are extinguished by substituting Ala for Thr(588) or Gln for Lys(455) ("kinase dead") or incubating with protein phosphatase 2C. Thus, DKF-1 is a PMA-activated, PKC-independent D kinase. In vivo, dkf-1 gene promoter activity is evident in neurons. Both dkf-1 gene disruption (null phenotype) and RNA interference-mediated depletion of DKF-1 protein cause lower body paralysis. Targeted DKF-1 expression corrected this locomotory defect in dkf-1 null animals. Supraphysiological expression of DKF-1 limited C. elegans growth to approximately 60% of normal length.     

Conserved domains subserve novel mechanisms and functions in DKF-1, a Caenorhabditis elegans protein kinase D

Protein kinase D (PKD) isoforms are effectors in signaling pathways controlled by diacylglycerol. PKDs contain conserved diacylglycerol binding (C1a, C1b), pleckstrin homology (PH), and Ser/Thr kinase domains. However, the properties of conserved domains may vary within the context of distinct PKD polypeptides. Such functional/structural malleability (plasticity) was explored by studying Caenorhabditis elegans D kinase family-1 (DKF-1), a PKD that governs locomotion in vivo. Phorbol ester binding with C1b alone activates classical PKDs by relieving C1-mediated inhibition. In contrast, C1a avidly ligated phorbol 12-myristate 13-acetate (PMA) and anchored DKF-1 at the plasma membrane. C1b bound PMA (moderate affinity) and cooperated with C1a in targeting DKF-1 to membranes. Mutations at a "Pro(11)" position in C1 domains were inactivating; kinase activity was minimal at PMA concentrations that stimulated wild type DKF-1 approximately 10-fold. DKF-1 mutants exhibited unchanged, maximum kinase activity after cells were incubated with high PMA concentrations. Titration in situ revealed that translocation and activation of wild type and mutant DKF-1 were tightly and quantitatively linked at all PMA concentrations. Thus, C1 domains positively regulated phosphotransferase activity by docking DKF-1 with pools of activating lipid. A PH domain inhibits kinase activity in classical PKDs. The DKF-1 PH module neither inhibited catalytic activity nor bound phosphoinositides. Consequently, the PH module is an obligatory, positive regulator of DKF-1 activity that is compromised by mutation of Lys(298) or Trp(396). Phosphorylation of Thr(588) switched on DKF-1 kinase activity. Persistent phosphorylation of Thr(588) (activation loop) promoted ubiquitinylation and proteasome-mediated degradation of DKF-1. Each DKF-1 domain displayed novel properties indicative of functional malleability (plasticity).     

Role of the regulatory domain of protein kinase D2 in phorbol ester binding, catalytic activity, and nucleocytoplasmic shuttling

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.     

Protein kinase C phosphorylates protein kinase D activation loop Ser744 and Ser748 and releases autoinhibition by the pleckstrin homology domain

Persistent activation of protein kinase D (PKD) via protein kinase C (PKC)-mediated signal transduction is accompanied by phosphorylation at Ser(744) and Ser(748) located in the catalytic domain activation loop, but whether PKC isoforms directly phosphorylate these residues, induce PKD autophosphorylation, or recruit intermediate upstream kinase(s) is unclear. Here, we explore the mechanism whereby PKC activates PKD in response to cellular stimuli. We first assessed in vitro PKC-PKD transphosphorylation and PKD activation. A PKD738-753 activation loop peptide was well phosphorylated by immunoprecipitated PKC isoforms, consistent with similarities between the loop and their known substrate specificities. A similar peptide with glutamic acid replacing Ser(748) was preferentially phosphorylated by PKCepsilon, suggesting that PKD containing phosphate at Ser(748) is rapidly targeted by this isoform at Ser(744). When incubated in the presence of phosphatidylserine, phorbol 12,13-dibutyrate and ATP, intact PKD slowly autophosphorylated in the activation loop but only at Ser(748). In contrast, addition of purified PKCepsilon to the incubation mixture induced rapid Ser(744) and Ser(748) phosphorylation, concomitant with persistent 2-3-fold increases in PKD activity, measured using reimmunoprecipitated PKD to phosphorylate an exogenous peptide, syntide-2. We also further examined pleckstrin homology domain-mediated PKD regulation to determine its relationship with activation loop phosphorylation. The high constitutive activity of the pleckstrin homology (PH) domain deletion mutant PKD-deltaPH was not abrogated by mutation of Ser(744) and Ser(748) to alanines, suggesting that one function of activation loop phosphorylation in the PKD activation mechanism is to relieve autoinhibition by the PH domain. These studies provide evidence of a direct PKCepsilon-PKD phosphorylation cascade and provide additional insight into the activation mechanism.     

Individual C1 domains of PKD3 in phorbol ester-induced plasma membrane translocation of PKD3 in intact cells

Protein kinase D3 is a novel member of the serine/threonine kinase family PKD. The regulatory region of PKD contains a tandem repeat of C1 domains designated C1a and C1b that bind diacylglycerol and phorbol esters, and are important membrane targeting modules. Here, we investigate the activities of individual C1 domains of PKD3 and their roles in phorbol ester-induced plasma membrane translocation of PKD3. Truncated C1a of PKD3 binds [(3)H]phorbol 12, 13-dibutyrate with high affinity, but no binding activity is detected for C1b. Meanwhile, mutations in C1a of truncated C1ab of PKD3 lead to the loss of binding affinity, while these mutations in C1b have little impact, indicating that C1a is responsible for most of the phorbol ester-binding activities of PKD3. C1a and C1b of the GFP-tagged full length PKD3 are then mutated to assess their roles in phorbol ester-induced plasma membrane translocation in intact cells. At low concentration of phorbol 12-myristate 13-acetate (PMA), the plasma membrane translocations of the C1a and C1ab mutants are significantly impaired, reflecting an important role of C1a in this process. However, at higher PMA concentrations, all C1 mutants exhibit increased rates of translocation as compared to that of wild-type PKD3, which parallel their enhanced activation by PMA, implying that PKD3 kinase activity affects membrane targeting. In line with this, a constitutive active PKD3-GFP translocates similarly as wild-type PKD3, while a kinase-inactive PKD3 shows little translocation up to 2 muM PMA. In addition, RO 31-8220, a potent PKC inhibitor that blocks PMA-induced PKD3 activation in vivo, significantly attenuates the plasma membrane translocation of wild-type PKD3 at different doses of PMA. Taken together, our results indicate that both C1a and the kinase activity of PKD3 are necessary for the phorbol ester-induced plasma membrane translocation of PKD3. PKC, by directly activating PKD3, regulates its plasma membrane localization in intact cells.     

Regulation of protein kinase D by multisite phosphorylation. Identification of phosphorylation sites by mass spectrometry and characterization by site-directed mutagenesis

Activation of the serine/threonine kinase, protein kinase D (PKD/PKC mu) via a phorbol ester/PKC-dependent pathway involves phosphorylation events. The present study identifies five in vivo phosphorylation sites by mass spectrometry, and the role of four of them was investigated by site-directed mutagenesis. Four sites are autophosphorylation sites, the first of which (Ser(916)) is located in the C terminus; its phosphorylation modifies the conformation of the kinase and influences duration of kinase activation but is not required for phorbol ester-mediated activation of PKD. The second autophosphorylation site (Ser(203)) lies in that region of the regulatory domain, which in PKC mu interacts with 14-3-3tau. The last two autophosphorylation sites (Ser(744) and Ser(748)) are located in the activation loop but are only phosphorylated in the isolated PKD-catalytic domain and not in the full-length PKD; they may affect enzyme catalysis but are not involved in the activation of wild-type PKD by phorbol ester. We also present evidence for proteolytic activation of PKD. The fifth site (Ser(255)) is transphosphorylated downstream of a PKC-dependent pathway after in vivo stimulation with phorbol ester. In vivo phorbol ester stimulation of an S255E mutant no longer requires PKC-mediated events. In conclusion, our results show that PKD is a multisite phosphorylated enzyme and suggest that its phosphorylation may be an intricate process that regulates its biological functions in very distinct ways.     

C1 domains exposed: from diacylglycerol binding to protein-protein interactions

C1 domains, cysteine-rich modules originally identified in protein kinase C (PKC) isozymes, are present in multiple signaling families, including PKDs, chimaerins, RasGRPs, diacylglycerol kinases (DGKs) and others. Typical C1 domains bind the lipid second messenger diacylglycerol (DAG) and DAG-mimetics such as phorbol esters, and are critical for governing association to membranes. On the contrary, atypical C1 domains possess structural determinants that impede phorbol ester/DAG binding. C1 domains are generally expressed as twin modules (C1A and C1B) or single domains. Biochemical and cellular studies in PKC and PKD isozymes revealed that C1A and C1B domains are non-equivalent as lipid-binding motifs or translocation modules. It has been recently determined that individual C1 domains have unique patterns of ligand recognition, driven in some cases by subtle structural differences. Insights from recent 3-D studies on beta2-chimaerin and Munc13-1 revealed that their single C1 domains are sterically blocked by intramolecular interactions, suggesting that major conformational changes would be required for exposing the site of DAG interaction. Thus, it is clear that the protein context plays a major role in determining whether binding of DAG to the C1 domain would lead to enzyme activation or merely serves as an anchoring mechanism.     

The Protein Scaffold NHERF-1 Controls the Amplitude and Duration of Localized Protein Kinase D Activity

Protein kinase D (PKD) transduces an abundance of signals downstream of diacylglycerol production. The mammalian PKD family consists of three isoforms, PKD1, PKD2, and PKD3; of these PKD1 and PKD2 contain PDZ-binding motifs at their carboxyl termini. Here we show that membrane-localized NHERF scaffold proteins provide a nexus for tightly controlled PKD signaling via a PDZ domain interaction. Using a proteomic array containing 96 purified PDZ domains, we have identified the first PDZ domain of NHERF-1 as an interaction partner for the PDZ-binding motifs of both PKD1 and PKD2. A fluorescence resonance energy transfer-based translocation assay reveals a transient association of PKD1 and PKD2 with NHERF-1 in live cells that is triggered by phorbol ester stimulation and, importantly, differs strikingly from the sustained translocation to plasma membrane. Targeting a fluorescence resonance energy transfer-based kinase activity reporter for PKD to NHERF scaffolds reveals a unique signature of PKD activation at the scaffold that is distinct from that of general cytosolic or plasma membrane activity. Specifically, agonist-evoked activation of PKD at the scaffold is rapid and sustained but blunted in magnitude when compared with cytosolic PKD. Thus, live cell imaging of PKD activity demonstrates ultrasensitive control of kinase signaling at the scaffold compared with bulk activity in the cytosol or at the plasma membrane.Protein kinase D (PKD)² plays a role in numerous processes including cell proliferation, cell survival, immune cell signaling, gene expression, vesicle trafficking, and neuronal development (1). The PKD family consists of three members belonging to the Ca²⁺/calmodulin-dependent kinase group of serine/threonine protein kinases. Each isoform contains a conserved catalytic core and an amino-terminal regulatory moiety. This regulatory region contains two cysteine-rich (C1) domains and a pleckstrin homology domain that autoinhibits the kinase (2). The C1 domains are membrane-targeting modules that bind diacylglycerol (DAG) and its functional analogues, phorbol esters, thus recruiting PKD to membranes (3). The PKD1 and PKD2 isoforms additionally contain PDZ-binding motifs at their carboxyl termini that can target the kinases to distinct subcellular scaffolds through interactions with PDZ domain-containing proteins (4).PKD transduces signals downstream of the second messenger DAG. In addition to membrane recruitment by DAG, activation of PKD requires phosphorylation by novel protein kinase C (PKC) family members at two sites within its catalytic core (5, 6). The novel PKCs themselves contain C1 domains and are allosterically activated by DAG-mediated membrane binding; thus, DAG production leads to PKD activation through coincident activation of the novel PKCs and localization of PKD near its upstream kinases. Hence, activation of phospholipase C (PLC)-coupled receptors (such as certain G protein-coupled receptors (GPCRs) or receptor tyrosine kinases) results in the production of second messengers including DAG, and this leads to recruitment and activation of the novel PKCs and thus also PKD.PDZ (PSD-95, Discs large, ZO-1) domains are compact, globular structures of ∼90 residues, occurring in one or multiple copies within a protein, that mediate protein-protein interactions (7). These interactions occur via binding to other PDZ domains or, more commonly, by recognition of short amino acid motifs in the carboxyl termini of target proteins commonly terminating in a hydrophobic residue (8). In the case of PKD1 and PKD2, the last four amino acids are VSIL and ISVL, respectively. Here we identify Na⁺/H⁺ exchanger regulatory factor 1 (NHERF-1) as a PDZ domain-containing protein that interacts with the PDZ-binding motif of both PKD1 and PKD2.NHERF-1 was originally cloned as a critical protein component for the inhibition of Na⁺/H⁺ exchanger 3 by protein kinase A (9). NHERF-1 is 52% identical to NHERF-2, a family member with which it shares the conserved domain structure of two PDZ domains followed by an ezrin-radixin-moesin (ERM)-binding region (10). Parallel studies demonstrating its ability to strongly interact with ezrin independently identified NHERF-1 as ERM-binding phosphoprotein 50 (11). Via this ERM-binding region, NHERF-1 and NHERF-2 are predominantly localized near the actin cytoskeleton, thus poising them near the plasma membrane where they function as scaffolds. Since these original cloning reports, numerous studies have identified over 30 binding partners of these scaffold proteins including GPCRs, tyrosine kinase receptors, other adaptor proteins, signaling enzymes, and ion channels (12, 13).Here we identify PKD1 and PKD2 as NHERF-1-interacting proteins. Using a fluorescence resonance energy transfer (FRET)-based assay to assess molecular proximity, both PKD1 and PKD2 are shown to transiently associate with NHERF-1 following PKD activation. Furthermore, through use of genetically encoded reporters for PKD activity, we show a unique signature of PKD activation at the NHERF scaffold. Specifically, signaling is more tightly regulated at the scaffold than in the cytosol or bulk plasma membrane. Phosphatase activity is higher at NHERF than at the plasma membrane, resulting in a more rapidly reversible PKD response at the scaffold, and following an agonist-evoked response, PKD signaling is prolonged compared with the length of response in the cytosol. Our data identify NHERF-1 as a novel nexus of PKD signaling and raise the possibility that PKD may act as a novel regulator of proteins at the NHERF scaffold.     

Activation mechanisms of conventional protein kinase C isoforms are determined by the ligand affinity and conformational flexibility of their C1 domains

The regulatory domains of conventional and novel protein kinases C (PKC) have two C1 domains (C1A and C1B) that have been identified as the interaction site for diacylglycerol (DAG) and phorbol ester. It has been reported that C1A and C1B domains of individual PKC isoforms play different roles in their membrane binding and activation; however, DAG affinity of individual C1 domains has not been quantitatively determined. In this study, we measured the affinity of isolated C1A and C1B domains of two conventional PKCs, PKCalpha and PKCgamma, for soluble and membrane-incorporated DAG and phorbol ester by isothermal calorimetry and surface plasmon resonance. The C1A and C1B domains of PKCalpha have opposite affinities for DAG and phorbol ester; i.e. the C1A domain with high affinity for DAG and the C1B domain with high affinity for phorbol ester. In contrast, the C1A and C1b domains of PKCgamma have comparably high affinities for both DAG and phorbol ester. Consistent with these results, mutational studies of full-length proteins showed that the C1A domain is critical for the DAG-induced activation of PKCalpha, whereas both C1A and C1B domains are involved in the DAG-induced activation of PKCgamma. Further mutational studies in conjunction with in vitro activity assay and monolayer penetration analysis indicated that, unlike the C1A domain of PKCalpha, neither the C1A nor the C1B domain of PKCgamma is conformationally restricted. Cell studies with enhanced green fluorescent protein-tagged PKCs showed that PKCalpha did not translocate to the plasma membrane in response to DAG at a basal intracellular calcium concentration due to the inaccessibility of its C1A domain, whereas PKCgamma rapidly translocated to the plasma membrane under the same conditions. These data suggest that differential activation mechanisms of PKC isoforms are determined by the DAG affinity and conformational flexibility of their C1 domains.     

Dual phospholipase C/diacylglycerol requirement for protein kinase D1 activation in lymphocytes

The serine/threonine kinase protein kinase D1 (PKD1) is a protein kinase C (PKC) substrate that mediates antigen receptor signal transduction in lymphocytes. PKC phosphorylates serines 744/748 within the PKD1 catalytic domain, and this is proposed to be necessary and sufficient for enzyme activation. Hence, a PKD1 mutant with alanine substituted at positions 744 and 748 (PKD-S744A/S748A) is catalytically inactive. Conversely, a PKD1 mutant with glutamic residues substituted at positions 744 and 748 as phospho-mimics (PKD-S744E/S748E) is constitutively active when expressed in Cos7 or HeLa cells. The present study reveals that Ser-744/Ser-748 phosphorylation is required for PKD1 activation in lymphocytes. However, PKD-S744E/S748E is not constitutively active but, like the wild type enzyme, requires antigen receptor triggering or phorbol ester stimulation. Antigen receptor activation of wild type PKD is dependent on phospholipase C (PLC)/diacylglycerol (DAG) and PKC, whereas PKD-S744E/S748E is only dependent on PLC/DAG but no longer requires PKC. Hence, substitution of serines 744 and 748 with glutamic residues as phospho-mimics bypasses the PKC requirement for PKD1 activation but does not bypass the need for antigen receptors, PLC, or DAG. In lymphocytes, PKD1 is, thus, not regulated by PLC and PKC in a linear pathway; rather, PKD1 activation has more stringent requirements for integration of dual PLC signals, one mediated by PKCs and one that is PKC-independent.     

Oxidative stress induces protein kinase D activation in intact cells. Involvement of Src and dependence on protein kinase C

Protein kinase D (PKD) is a protein serine kinase that is directly stimulated in vitro by phorbol esters and diacylglycerol in the presence of phospholipids, and activated by phorbol esters, neuropeptides, and platelet-derived growth factor via protein kinase C (PKC) in intact cells. Recently, oxidative stress was shown to activate transfected PKC isoforms via tyrosine phosphorylation, but PKD activation was not demonstrated. Here, we report that oxidative stress initiated by addition of H(2)O(2) (0.15-10 mm) to quiescent Swiss 3T3 fibroblasts activates PKD in a dose- and time- dependent manner, as measured by autophosphorylation and phosphorylation of an exogenous substrate, syntide-2. Oxidative stress also activated transfected PKD in COS-7 cells but not a kinase-deficient mutant PKD form or a PKD mutant with critical activating serine residues 744 and 748 mutated to alanines. Genistein, or the specific Src inhibitors PP-1 and PP-2 (1-10 micrometer) inhibited H(2)O(2)-mediated PKD activation by 45%, indicating that Src contributes to this signaling pathway. PKD activation by H(2)O(2) was also selectively potentiated by cotransfection of PKD together with an active form of Src (v-Src) in COS-7 cells, as compared with PDB-mediated activation. The specific phospholipase C inhibitor, partly blocked H(2)O(2)-mediated but not PDB-mediated PKD activation. In contrast, PKC inhibitors blocked H(2)O(2) or PDB-mediated PKD activation essentially completely, suggesting that whereas Src mediates part of its effects via phospholipase C activation, PKC acts more proximally as an upstream activator of PKD. Together, these studies reveal that oxidative stress activates PKD by initiating distinct Src-dependent and -independent pathways involving PKC.     

Activation loop Ser744 and Ser748 in protein kinase D are transphosphorylated in vivo.

The importance of activation loop phosphorylation in the regulation of protein kinase D (PKD/protein kinase C (PKC) mu) 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). Western blot analysis with the pS748 antibody, carried out with a variety of PKD forms and in a variety of cell types including full-length PKD transfected in COS-7 and HEK 293 cells, a green fluorescent protein-PKD fusion protein transfected in either Swiss 3T3 fibroblasts or Madin-Darby canine kidney epithelial cells, and endogenous PKD expressed in A20 lymphocytes and Rat-1 fibroblasts, indicated that Ser(748) phosphorylation was absent from unstimulated cells. In contrast, dramatic increases in Ser(748) phosphorylation were induced by phorbol esters, bombesin, or cross-linking of B lymphocyte antigen receptors or by cotransfection with active PKCepsilon or PKCeta. Western analysis using a second phosphospecific antibody, which primarily recognizes PKD phosphorylated at Ser(744), revealed that Ser(744) phosphorylation accompanies Ser(748) phosphorylation during PKD activation in vivo. Ser(744)/Ser(748) phosphorylation requires PKC but not PKD activity, indicative of transphosphorylation. Our results provide new experimental evidence indicating that activation loop phosphorylation at Ser(744) and Ser(748) occurs during PKD activation in vivo and support the notion of a PKC-PKD phosphorylation cascade.     

Activation of protein kinase D by signaling through the alpha subunit of the heterotrimeric G protein G(q)

Protein kinase D (PKD/PKCmu) immunoprecipitated from COS-7 cells transiently transfected with a constitutively active alpha subunit of G(q) (Galpha(q)Q209L) exhibited a marked increase in basal activity, which was not further enhanced by treatment of the cells with phorbol 12,13-dibutyrate. In contrast, transient transfection of COS-7 cells with activated Galpha(12)Q229L or Galpha(13)Q226L neither promoted PKD activation nor interfered with the increase of PKD activity induced by phorbol 12,13-dibutyrate. The addition of aluminum fluoride to cells co-transfected with PKD and wild type Galpha(q) induced a marked increase in PKD activity, which was comparable with that induced by expression of Galpha(q)Q209L. Treatment with the protein kinase C inhibitor GF I or Ro 31-8220 prevented the increase in PKD activity induced by aluminum fluoride. Expression of a COOH-terminal fragment of Galpha(q) that acts in a dominant negative fashion attenuated PKD activation in response to agonist stimulation of bombesin receptor. PKD activation in response to either Galpha(q) or bombesin was completely prevented by mutation of Ser(744) and Ser(748) to Ala in the kinase activation loop of PKD. Our results show that Galpha(q) activation is sufficient to stimulate sustained PKD activation via protein kinase C and indicate that the endogenous Galpha(q) mediates PKD activation in response to acute bombesin receptor stimulation.     

Protein kinase D potentiates DNA synthesis and cell proliferation induced by bombesin, vasopressin, or phorbol esters in Swiss 3T3 cells

We examined whether protein kinase D (PKD) overexpression in Swiss 3T3 cells potentiates the proliferative response to either the G protein-coupled receptor agonists bombesin and vasopressin or the biologically active phorbol ester phorbol 12,13-dibutyrate (PDBu). In order to generate Swiss 3T3 cells stably overexpressing PKD, cultures of these cells were infected with retrovirus encoding murine PKD and green fluorescent protein (GFP) expressed as two separate proteins translated from the same mRNA. GFP was used as a marker for selection of PKD-positive cells. PKD overexpressed in Swiss 3T3 cells was dramatically activated by cell treatment with bombesin or PDBu as judged by in vitro kinase autophosphorylation assays and exogenous substrate phosphorylation. Concomitantly, these stimuli induced PKD phosphorylation at Ser(744), Ser(748), and Ser(916). PKD activation and phosphorylation were prevented by exposure of the cells to protein kinase C-specific inhibitors. Addition of bombesin, vasopressin, or PDBu to cultures of Swiss 3T3 cells overexpressing PKD induced a striking increase in DNA synthesis and cell number compared with cultures of Swiss 3T3-GFP cells. In contrast, stimulation of DNA synthesis in response to epidermal growth factor, which acts via protein kinase C/PKD-independent pathways, was not enhanced. Our results demonstrate that overexpression of PKD selectively potentiates mitogenesis induced by bombesin, vasopressin, or PDBu in Swiss 3T3 cells.     

Effects on ligand interaction and membrane translocation of the positively charged arginine residues situated along the C1 domain binding cleft in the atypical protein kinase C isoforms

The C1 domain zinc finger structure is highly conserved among the protein kinase C (PKC) superfamily members. As the interaction site for the second messenger sn-1,2-diacylglycerol (DAG) and for the phorbol esters, the C1 domain has been an important target for developing selective ligands for different PKC isoforms. However, the C1 domains of the atypical PKC members are DAG/phorbol ester-insensitive. Compared with the DAG/phorbol ester-sensitive C1 domains, the rim of the binding cleft of the atypical PKC C1 domains possesses four additional positively charged arginine residues (at positions 7, 10, 11, and 20). In this study, we showed that mutation to arginines of the four corresponding sites in the C1b domain of PKCdelta abolished its high potency for phorbol 12,13-dibutyrate in vitro, with only marginal remaining activity for phorbol 12-myristate 13-acetate in vivo. We also demonstrated both in vitro and in vivo that the loss of potency to ligands was cumulative with the introduction of the arginine residues along the rim of the binding cavity rather than the consequence of loss of a single, specific residue. Computer modeling reveals that these arginine residues reduce access of ligands to the binding cleft and change the electrostatic profile of the C1 domain surface, whereas the basic structure of the binding cleft is still maintained. Finally, mutation of the four arginine residues of the atypical PKC C1 domains to the corresponding residues in the deltaC1b domain conferred response to phorbol ester. We speculate that the arginine residues of the C1 domain of atypical PKCs may provide an opportunity for the design of ligands selective for the atypical PKCs.     

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 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.     

Protein kinase D intracellular localization and activity control kinase D-interacting substrate of 220-kDa traffic through a postsynaptic density-95/discs large/zonula occludens-1-binding motif

Protein kinase D (PKD) controls protein traffic from the trans-Golgi network (TGN) to the plasma membrane of epithelial cells in an isoform-specific manner. However, whether the different PKD isoforms could be selectively regulating the traffic of their specific substrates remains unexplored. We identified the C terminus of the different PKDs that constitutes a postsynaptic density-95/discs large/zonula occludens-1 (PDZ)-binding motif in PKD1 and PKD2, but not in PKD3, to be responsible for the differential control of kinase D-interacting substrate of 220-kDa (Kidins220) surface localization, a neural membrane protein identified as the first substrate of PKD1. A kinase-inactive mutant of PKD3 is only able to alter the localization of Kidins220 at the plasma membrane when its C terminus has been substituted by the PDZ-binding motif of PKD1 or PKD2. This isoform-specific regulation of Kidins220 transport might not be due to differences among kinase activity or substrate selectivity of the PKD isoenzymes but more to the adaptors bound to their unique C terminus. Furthermore, by mutating the autophosphorylation site Ser(916), located at the critical position -2 of the PDZ-binding domain within PKD1, or by phorbol ester stimulation, we demonstrate that the phosphorylation of this residue is crucial for Kidins220-regulated transport. We also discovered that Ser(916) trans-phosphorylation takes place among PKD1 molecules. Finally, we demonstrate that PKD1 association to intracellular membranes is critical to control Kidins220 traffic. Our findings reveal the molecular mechanism by which PKD localization and activity control the traffic of Kidins220, most likely by modulating the recruitment of PDZ proteins in an isoform-specific and phosphorylation-dependent manner.     

PKD1 Inhibits AMPKα2 through Phosphorylation of Serine 491 and Impairs Insulin Signaling in Skeletal Muscle Cells

AMP-activated protein kinase (AMPK) is an energy-sensing enzyme whose activity is inhibited in settings of insulin resistance. Exposure to a high glucose concentration has recently been shown to increase phosphorylation of AMPK at Ser^485/491 of its α1/α2 subunit; however, the mechanism by which it does so is not known. Diacylglycerol (DAG), which is also increased in muscle exposed to high glucose, activates a number of signaling molecules including protein kinase (PK)C and PKD1. We sought to determine whether PKC or PKD1 is involved in inhibition of AMPK by causing Ser^485/491 phosphorylation in skeletal muscle cells. C2C12 myotubes were treated with the PKC/D1 activator phorbol 12-myristate 13-acetate (PMA), which acts as a DAG mimetic. This caused dose- and time-dependent increases in AMPK Ser^485/491 phosphorylation, which was associated with a ∼60% decrease in AMPKα2 activity. Expression of a phosphodefective AMPKα2 mutant (S491A) prevented the PMA-induced reduction in AMPK activity. Serine phosphorylation and inhibition of AMPK activity were partially prevented by the broad PKC inhibitor Gö6983 and fully prevented by the specific PKD1 inhibitor CRT0066101. Genetic knockdown of PKD1 also prevented Ser^485/491 phosphorylation of AMPK. Inhibition of previously identified kinases that phosphorylate AMPK at this site (Akt, S6K, and ERK) did not prevent these events. PMA treatment also caused impairments in insulin-signaling through Akt, which were prevented by PKD1 inhibition. Finally, recombinant PKD1 phosphorylated AMPKα2 at Ser⁴⁹¹ in cell-free conditions. These results identify PKD1 as a novel upstream kinase of AMPKα2 Ser⁴⁹¹ that plays a negative role in insulin signaling in muscle cells.     

Protein kinase D-mediated phosphorylation and nuclear export of sphingosine kinase 2

Sphingosine kinase (SPHK) is a key enzyme producing important messenger sphingosine 1-phosphate and is implicated in cell proliferation and suppression of apoptosis. Because the extent of agonist-induced activation of SPHK is modest, signaling via SPHK may be regulated through its localization at specific intracellular sites. Although the SPHK1 isoform has been extensively studied and characterized, the regulation of expression and function of the other isoform, SPHK2, remain largely unexplored. Here we describe an important post-translational modification, namely, phosphorylation of SPHK2 catalyzed by protein kinase D (PKD), which regulates its localization. Upon stimulation of HeLa cells by tumor promoter phorbol 12-myristate 13-acetate, a serine residue in a novel and putative nuclear export signal, identified for the first time, in SPHK2 was phosphorylated followed by SPHK2 export from the nucleus. Constitutively active PKD phosphorylated this serine residue in the nuclear export signal both in vivo and in vitro. Moreover, down-regulation of PKDs through RNA interference resulted in the attenuation of both basal and phorbol 12-myristate 13-acetate-induced phosphorylation, which was followed by the accumulation of SPHK2 in the nucleus in a manner rescued by PKD over-expression. These results indicate that PKD is a physiologically relevant enzyme for SPHK2 phosphorylation, which leads to its nuclear export for subsequent cellular signaling.