Protein kinase D orchestrates the activation of DRAK2 in response to TCR-induced Ca2+ influx and mitochondrial reactive oxygen generation

DRAK2 is a serine/threonine kinase highly enriched in lymphocytes that raises the threshold for T cell activation and maintains T cell survival following productive activation. T cells lacking DRAK2 are prone to activation under suboptimal conditions and exhibit enhanced calcium responses to AgR stimulation. Despite this, mice lacking DRAK2 are resistant to organ-specific autoimmune diseases due to defective autoreactive T cell survival. DRAK2 kinase activity is induced by AgR signaling, and in this study we show that the induction of DRAK2 activity requires Ca(2+) influx through the Ca(2+) release-activated Ca(2+) channel formed from Orai1 subunits. Blockade of DRAK2 activity with the protein kinase D (PKD) inhibitor G?6976 or expression of a kinase-dead PKD mutant prevented activation of DRAK2, whereas a constitutively active PKD mutant promoted DRAK2 function. Knockdown of PKD in T cells strongly blocked endogenous DRAK2 activation following TCR ligation, implicating PKD as an essential intermediate in the activation of DRAK2 by Ca(2+) influx. Furthermore, we identify DRAK2 as a novel substrate of PKD, and demonstrate that DRAK2 and PKD physically interact under conditions that activate PKD. Mitochondrial generation of reactive oxygen intermediates was necessary and sufficient for DRAK2 activation in response to Ca(2+) influx. Taken together, DRAK2 and PKD form a novel signaling module that controls calcium homeostasis following T cell activation.     

KCl activates mitogen-activated protein kinase in rabbit bailar artery

The objective of the present study was to investigate if MAPK can be activated by a non-receptor agonist KCl, which depolarizes membrane to increase intracellular Ca(2+) and contracts cerebral arteries. Rabbit basilar arteries were used in isometric tension and western blot analysis studies. KCl produced a concentration-dependent contraction and an elevation of phospho-MAPK, which can be abolished by nicardipine, a voltage-dependent Ca(2+) channel blocker, and by PD98059 or U0126, MAPK kinase inhibitors. Thus, MAPK can be activated by the elevation of intracellular Ca(2+), independent of the activation of either G-protein coupled receptors or receptor tyrosine kinase. KCl which not only depolarizes membrane potentials, opens voltage-dependent Ca(2+), and increases intracellular Ca(2+), but also, probably by elevation of intracellular Ca(2+), triggers the activation of MAPK which seems responsible for a predominant part of the contraction of KCl in the rabbit basilar arteries.     

G-PKDrep-live, a genetically encoded FRET reporter to measure PKD activity at the trans-Golgi-network

The serine/threonine protein kinase D (PKD) is recruited to the trans-Golgi-network (TGN) by interaction with diacylglycerol (DAG) and Arf1 and promotes the fission of vesicles containing cargo destined for the plasma membrane. PKD activation is mediated by PKC(-induced phosphorylation. However, signaling pathways that activate PKD specifically at the TGN are only poorly characterized. Recently we created G-PKDrep, a genetically encoded fluorescent reporter for PKD activity at the TGN in fixed cells. To establish a reporter useful for monitoring Golgi-specific PKD activity in living cells we now refined G-PKDrep to generate G-PKDrep-live. Specifically, phosphorylation of G-PKDrep-live expressed in mammalian cells results in changes of fluorescence resonance energy transfer (FRET), and allows for indirect imaging of PKD activity. In a proof-of-principle experiment using phorbolester treatment, we demonstrate the reporter's capability to track rapid activation of PKD at the TGN. Furthermore, activation-induced FRET changes are reversed by treatment with PKD-specific pharmacological inhibitors. Thus, the newly developed reporter G-PKDrep-live is a suitable tool to visualize dynamic changes in PKD activity at the TGN in living cells. See accompanying commentary by Gautam DOI: 10.1002/biot.201100424.     

Diacylglycerol mediates the T-cell receptor-driven Ca(2+) influx in T cells by a novel mechanism independent of protein kinase C activation

The mechanism of Ca(2+) influx in nonexcitable cells is not known yet. According to the capacitative hypothesis, Ca(2+) influx is triggered by IP(3)-mediated Ca(2+) release from the intracellular Ca(2+) stores. Conversely, many workers have reported a lack of association between release and influx. In this work, the role of diacylglycerol (DAG) as the mediator of T-cell receptor (TCR)-driven Ca(2+) influx in T cells was investigated. Stimulation of mouse splenic T cells with naturally occurring DAG caused Ca(2+) entry in a dose- and time-dependent manner. Such stimulation was blocked by Ni(2+), a divalent cation known to block Ca(2+) channels. Inhibition of protein kinase C (PKC) by calphostin C did not inhibit, but slightly enhanced, the DAG-stimulated Ca(2+) entry. However, inhibition of DAG metabolism by DAG kinase and lipase inhibitors enhanced the DAG-stimulated Ca(2+) entry. DAG lipase and kinase inhibitors also enhanced the Ca(2+) entry in T cells stimulated through TCR/CD3 complex with anti-CD3 antibody. Calphostin C did not affect the anti-CD3-stimulated Ca(2+) entry. These results showed that TCR-driven Ca(2+) influx in T cells is mediated by DAG through a novel mechanism(s) independent of PKC activation.     

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.     

Phospholipase C, protein kinase C, Ca(2+)/calmodulin-dependent protein kinase II, and tyrosine phosphorylation are involved in carbachol-induced phospholipase D activation in Chinese hamster ovary cells expressing muscarinic acetylcholine receptor of Caenorhabditis elegans

Recently, we have isolated a cDNA encoding a muscarinic acetylcholine receptor (mAChR) from Caenorhabditis elegans. To investigate the regulation of phospholipase D (PLD) signaling via a muscarinic receptor, we generated stable transfected Chinese hamster ovary (CHO) cells that overexpress the mAChR of C. elegans (CHO-GAR-3). Carbachol (CCh) induced inositol phosphate formation and a significantly higher Ca(2+) elevation and stimulated PLD activity through the mAChR; this was insensitive to pertussis toxin, but its activity was abolished by the phospholipase C (PLC) inhibitor U73122. Western blot analysis revealed several apparent tyrosine-phosphorylated protein bands after CCh treatment. The CCh-induced PLD activation and tyrosine phosphorylation were significantly reduced by the protein kinase C (PKC) inhibitor calphostin C and down-regulation of PKC and the tyrosine kinase inhibitor genistein. Moreover, the Ca(2+)-calmodulin-dependent protein kinase II (CaM kinase II) inhibitor KN62, in addition to chelation of extracellular or intracellular Ca(2+) by EGTA and BAPTA/AM, abolished CCh-induced PLD activation and protein tyrosine phosphorylation. Taken together, these results suggest that the PLC/PKC-PLD pathway and the CaM kinase II/tyrosine kinase-PLD pathway are involved in the activation of PLD through mAChRs of C. elegans.     

Targeting protein kinase C activity reporter to discrete intracellular regions reveals spatiotemporal differences in agonist-dependent signaling

Protein kinase C (PKC) family members transduce an abundance of diverse intracellular signals. Here we address the role of spatial and temporal segregation in signal specificity by measuring the activity of endogenous PKC at defined intracellular locations in real time in live cells. We targeted a genetically encoded fluorescence resonance energy transfer-based reporter for PKC activity, C kinase activity reporter (CKAR) (Violin, J. D., Zhang, J., Tsien, R. Y., and Newton, A. C. (2003) J. Cell Biol. 161, 899-909), to the plasma membrane, Golgi, cytosol, mitochondria, or nucleus by fusing appropriate targeting sequences to the NH2 or COOH terminus of CKAR. Measuring the phosphorylation of the reporter in the presence of PKC inhibitors, activators, and/or phosphatase inhibitors shows that activity at each region is under differential control by phosphatase activity; nuclear activity is completely suppressed by phosphatases, whereas membrane-associated activity is the least suppressed by phosphatases. UTP stimulation of endogenous P2Y receptors in COS 7 cells reveals spatiotemporally divergent PKC responses. Imaging the second messengers Ca2+ and diacylglycerol (DAG) reveal that PKC activity at each location is driven by an initial spike in Ca2+, followed by location-specific diacylglycerol generation. In response to UTP, phosphorylation of GolgiCKAR was sustained the longest, driven by the persistence of DAG, whereas phosphorylation of CytoCKAR was of the shortest duration, driven by high phosphatase activity. Our data reveal that the magnitude and duration of PKC signaling is location-specific and controlled by the level of phosphatase activity and persistence of DAG at each location.     

Regulated nucleocytoplasmic transport of protein kinase D in response to G protein-coupled receptor activation

Protein kinase D (PKD)/protein kinase C mu is a serine/threonine protein kinase activated by growth factors, antigen-receptor engagement, and G protein-coupled receptor (GPCR) agonists via a phosphorylation-dependent mechanism that requires protein kinase C (PKC) activity. In order to investigate the dynamic mechanisms associated with GPCR signaling, the intracellular distribution of PKD was analyzed in live cells by imaging fluorescent protein-tagged PKD and in fixed cells by immunocytochemistry. We found that PKD shuttled between the cytoplasm and the nucleus in both fibroblasts and epithelial cells. Cell stimulation with mitogenic GPCR agonists that activate PKD induced a transient nuclear accumulation of PKD that was prevented by inhibiting PKC activity. The nuclear import of PKD requires its cys2 domain in conjunction with a nuclear import receptor, while its nuclear export requires its pleckstrin homology domain and a competent Crm1-dependent nuclear export pathway. This study thus characterizes the regulated nuclear transport of a signaling molecule in response to mitogenic GPCR agonists and positions PKD as a serine kinase whose kinase activity and intracellular localization is coordinated by PKC.     

Expression of the protein kinase D (PKD) family during mouse embryogenesis

The serine/threonine protein kinase D (PKD) family comprises of three members, PKD1 (PKCmu), PKD2 and PKD3 (PKCnu). Like the related C-type protein kinases (PKCs), PKDs are activated by diacylglycerol (DAG). PKDs have been implicated in numerous intracellular signaling pathways including vesicular transport, cell proliferation, survival, migration and immune responses. While experimental data on this recently discovered kinase family are starting to accumulate family member specific information is still sparse and only small effort has been taken to functionally differentiate the three PKDs. To address this issue we followed the expression patterns of PKD1, 2 and 3 during the development of the mouse embryo. Using specific probe sets for RT-PCR and in situ hybridization, we demonstrate shared and differential expression domains for the three PKD family members in both neuronal and non-neuronal tissues.     

Integrin signaling regulates blastocyst adhesion to fibronectin at implantation: intracellular calcium transients and vesicle trafficking in primary trophoblast cells

Accumulating evidence indicates that the endometrial extracellular matrix (ECM) modulates trophoblast adhesion during mouse blastocyst implantation. In previous studies of adhesion-competent mouse blastocysts, we have demonstrated that integrin-mediated fibronectin (FN)-binding activity on the apical surface of trophoblast cells is initially low, but becomes strengthened after embryos are exposed to FN. In the present study, we have examined whether the ligand-induced upregulation of trophoblast adhesion to FN is mediated by integrin signaling. The strengthening of adhesion to FN required integrin ligation, which rapidly elevated cytoplasmic-free Ca(2+). Chelation of intracellular Ca(2+) using BAPTA-AM, or inhibition of the Ca(2+)-dependent proteins, protein kinase C or calmodulin, significantly attenuated the effect of FN on binding activity. Furthermore, direct elevation of cytoplasmic Ca(2+) levels with ionomycin upregulated FN-binding activity, demonstrating that Ca(2+) signaling is required and sufficient for strong adhesion to FN. Ca(2+) signaling may induce protein trafficking, a known requirement for ligand-induced upregulation of FN-binding activity. Indeed, intracellular vesicles accumulated in adhesion-competent blastocysts, but were absent after exposure to either FN or ionomycin. These findings suggest that, during implantation, contact between peri-implantation blastocysts and FN elevates intracellular Ca(2+), which strengthens trophoblast adhesion to ECM through protein redistribution.     

Activation of nuclear Ca(2+)/calmodulin-dependent protein kinase II and brain-derived neurotrophic factor gene expression by stimulation of dopamine D2 receptor in transfected NG108-15 cells

We identified the isoforms of Ca(2+) /calmodulin-dependent protein kinase II (CaM kinase II) subunits in rat striatum. All four subunits of CaM kinase II alpha, beta, gamma and delta were detected including the isoforms of alphaB, gammaA, gammaA', gammaA.B, delta3 and delta7 with nuclear localization signal. We established NG108-15 cells with the stably expressed dopamine D2L receptor (D2LR, long form), which is an alternative splicing variant. The cells were termed NGD2L. Immunostaining demonstrated that D2LR was localized in plasma membranes. Calcium imaging with fluo-3 AM revealed that quinpirole, a D2R agonist, increased the intracellular Ca(2+), which was blocked by treatment with sulpiride and pertussis toxin in NGD2L cells, but not in mock cells. Furthermore, stimulation of D2LR with quinpirole in NGD2L cells activated the nuclear isoform of CaM kinase II. Stimulation of D2LR increased the expression of exon III- and IV-BDNF mRNA. Overexpression of CaM kinase II delta3 increased exon IV- but not exon III-BDNF mRNA. These results suggest that D2R is involved in the activation of the nuclear isoform of CaM kinase II and thereby in stimulation of gene expression through Ca(2+) signaling.     

Trophoblast adhesion of the peri-implantation mouse blastocyst is regulated by integrin signaling that targets phospholipase C

Integrin signaling modulates trophoblast adhesion to extracellular matrices during blastocyst implantation. Fibronectin (FN)-binding activity on the apical surface of trophoblast cells is strengthened after elevation of intracellular Ca(2+) downstream of integrin ligation by FN. We report here that phosphoinositide-specific phospholipase C (PLC) mediates Ca(2+) signaling in response to FN. Pharmacological agents used to antagonize PLC (U73122) or the inositol phosphate receptor (Xestospongin C) inhibited FN-induced elevation of intracellular Ca(2+) and prevented the upregulation of FN-binding activity. In contrast, inhibitors of Ca(2+) influx through either voltage-gated or non-voltage-gated Ca(2+) channels were without effect. Inhibition of protein tyrosine kinase activity by genistein, but not G-protein inhibition by suramin, blocked FN-induced intracellular Ca(2+) signaling and upregulation of adhesion, consistent with involvement of PLC-gamma. Confocal immunofluorescence imaging of peri-implantation blastocysts demonstrated that PLC-gamma2, but not PLC-gamma1 nor PLC-beta1, accumulated near the outer surface of the embryo. Phosphotyrosine site-directed antibodies revealed phosphorylation of PLC-gamma2, but not PLC-gamma1, upon integrin ligation by FN. These data suggest that integrin-mediated activation of PLC-gamma to initiate phosphoinositide signaling and intracellular Ca(2+) mobilization is required for blastocyst adhesion to FN. Signaling cascades regulating PLC-gamma could, therefore, control a critical feature of trophoblast differentiation during peri-implantation development.     

Vasopressin-induced intracellular redistribution of protein kinase D in intestinal epithelial cells

The spatio-temporal changes of signaling molecules in response to G protein-coupled receptors (GPCR) stimulation is a poorly understood process in intestinal epithelial cells. Here we investigate the dynamic mechanisms associated with GPCR signaling in living rat intestinal epithelial cells by characterizing the intracellular translocation of protein kinase D (PKD), a serine/threonine protein kinase involved in mitogenic signaling in intestinal epithelial cells. Analysis of the intracellular steady-state distribution of green fluorescent protein (GFP)-tagged PKD indicated that in non-stimulated IEC-18 cells, GFP-PKD is predominantly cytoplasmic. However, cell stimulation with the GPCR agonist vasopressin induces a rapid translocation of GFP-PKD from the cytosol to the plasma membrane that is accompanied by its activation via protein kinase C (PKC)-mediated process and posterior plasma membrane dissociation. Subsequently, active PKD is imported into the nuclei where it transiently accumulates before being exported into the cytosol by a mechanism that requires a competent Crm1 nuclear export pathway. These findings provide evidence for a mechanism by which PKC coordinates in intestinal epithelial cells the translocation and activation of PKD in response to vasopressin-induced GPCR activation.     

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.     

Intracellular redistribution of protein kinase D2 in response to G-protein-coupled receptor agonists

The protein kinase D (PKD) family consists of three serine/threonine protein kinases: PKC mu/PKD, PKD2, and PKC nu/PKD3. While PKD has been the focus of most studies to date, no information is available on the intracellular distribution of PKD2. Consequently, we examined the mechanism that regulates its intracellular distribution in human pancreatic carcinoma Panc-1 cells. Analysis of the intracellular steady-state distribution of fluorescent-tagged PKD2 in unstimulated cells indicated that this kinase is predominantly cytoplasmic. Cell stimulation with the G protein-coupled receptor agonist neurotensin induced a rapid and reversible plasma membrane translocation of PKD2 by a mechanism that requires PKC activity. In contrast to the other PKD isoenzymes, PKD2 activation did not induce its redistribution from the cytoplasm to the nucleus. Thus, this study demonstrates that the regulation of the distribution of PKD2 is distinct from other PKD isoenzymes, and suggests that the differential spatio-temporal localization of these signaling molecules regulates their specific signaling properties.