Characterization of EVL-I as a protein kinase D substrate.

EVL-I is a splice variant of EVL (Ena/VASP like protein), whose in vivo function and regulation are still poorly understood. We found that Protein Kinase D (PKD) interacts in vitro and in vivo with EVL-I and phosphorylates EVL-I in a 21 amino acid alternately-included insert in the EVH2 domain. Following knockdown of the capping protein CPbeta and spreading on laminin, phosphorylated EVL-I can support filopodia formation and the phosphorylated EVL-I is localized at filopodial tips. Furthermore, we found that the lamellipodial localization of EVL-I is unaffected by phosphorylation, but that impairment of EVL-I phosphorylation is associated with ruffling of lamellipodia upon PDBu stimulation. Besides the lamellipodial and filopodial localization of phosphorylated EVL-I in fibroblasts, we determined that EVL-I is hyperphosphorylated and localized in the cell-cell contacts of certain breast cancer cells and mouse embryo keratinocytes. Taken together, our results show that phosphorylated EVL-I is present in lamellipodia, filopodia and cell-cell contacts and suggest the existence of signaling pathways that may affect EVL-I via phosphorylation of its EVH2 domain.

Characterization of cortactin as an in vivo protein kinase D substrate: interdependence of sites and potentiation by Src.

Protein Kinase D (PKD) has been implicated in the regulation of actin turnover at the leading edge, invasion and migration. In particular, a complex between cortactin, paxillin and PKD in the invadopodia of invasive breast cancer cells has been described earlier, but so far this complex remained ill defined. Here we have investigated the possible role of PKD as a cortactin kinase. Using a mass spectrometric approach, we found that PKD phosphorylates cortactin on Ser 298 in the 6th cortactin repeat region and on Ser 348, right before the helical-proline rich domain of cortactin. We developed phosphospecific antibodies against these phosphorylated sequences, and used them as tools to follow the in vivo phosphorylation of cortactin by PKD. Examination of cortactin phosphorylation kinetics revealed that Ser 298 serves as a priming site for subsequent phosphorylation of Ser 348. Src, a well-known cortactin kinase, strongly potentiated the in vivo PKD mediated cortactin phosphorylation. This Src effect is neither mediated by pre-phosphorylation of cortactin nor by activation of PKD by Src. Phosphorylation of cortactin by PKD does not affect its subcellular localization, nor does it affect its translocation to podosomes or membrane ruffles. Moreover, there was no effect of PKD mediated cortactin phosphorylation on EGF receptor degradation and LPA induced migration. Taken together, these data establish cortactin as a novel PKD substrate and reveal a novel connection between Src and PKD.

An enzyme-linked immunosorbent assay for protein kinase D activity using phosphorylation site-specific antibodies.

The protein kinase D (PKD) family is a novel group of kinases that are involved in the regulation of cell proliferation and apoptosis, and several other physiological processes. Hence, these enzymes are attractive targets for pharmacological intervention, but no specific PKD inhibitors are known. With this in mind, we have developed a high-throughput, non-radioactive enzyme-linked immunosorbent assay (ELISA) method to monitor the PKD activity with myelin basic protein (MBP) as substrate. We determined that MBP is phosphorylated by PKD on Ser-160 and that this phosphorylation can be quantified in ELISAs, by the use of phosphorylation site-specific antibodies. Antibodies were developed that are highly specific for the MBP peptide sequence surrounding the phosphorylated Ser-160. We show that our high-throughput kinase assay is useful not only for determining the cellular PKD activity but also to screen for PKD-inhibitory compounds. Our ELISA has advantages over the current radioisotope kinase assay in terms of simplicity and environmental safety.

Protein kinase D induces transcription through direct phosphorylation of the cAMP-response element-binding protein.

Protein kinase D (PKD), a family of serine/threonine kinases, can be activated by a multitude of stimuli in a protein kinase C-dependent or -independent manner. PKD is involved in signal transduction pathways controlling cell proliferation, apoptosis, motility, and protein trafficking. Despite its versatile functions, few genuine in vivo substrates for PKD have been identified. In this study we demonstrate that the transcription factor cAMP-response element-binding protein (CREB) is a direct substrate for PKD. PKD1 and CREB interact in cells, and activated PKD1 provokes CREB phosphorylation at Ser-133 both in vitro and in vivo. A constitutive active mutant of PKD1 stimulates GAL4-CREB-mediated transcription in a Ser-133-dependent manner, activates CRE-responsive promoters, and increases the expression of CREB target genes. PKD1 also enhances transcription mediated by two other members of the CREB family, ATF-1 and CREM. Our results describe a novel mechanism for PKD-induced signaling through activation of the transcription factor CREB and suggest that stimulus-induced phosphorylation of CREB, reported to be mediated by protein kinase C, may involve downstream activated PKD.

Apoptosis signal regulating kinase-1 connects reactive oxygen species to p38 MAPK-induced mitochondrial apoptosis in UVB-irradiated human keratinocytes.

The p38 MAPK pathway controls critical premitochondrial events culminating in apoptosis of UVB-irradiated human keratinocytes, but the upstream mediators of this stress signal are not completely defined. This study shows that in human keratinocytes exposed to UVB the generation of reactive oxygen species (ROS) acts as a mediator of apoptosis signal regulating kinase-1 (Ask-1), a redox-sensitive mitogen-activated protein kinase kinase kinase (MAP3K) regulating p38 MAPK and JNK cascades. The NADPH oxidase antagonist diphenylene iodonium chloride and the EGFR inhibitor AG1487 prevent UVB-mediated ROS generation, the activation of the Ask-1-p38 MAPK stress response pathway, and apoptosis, evidencing the link existing between the early plasma membrane-generated ROS and the activation of a lethal cascade initiated by Ask-1. Consistent with this, Ask-1 overexpression considerably sensitizes keratinocytes to UVB-induced mitochondrial apoptosis. Although the JNK pathway is also stimulated after UVB, the killing effect of Ask-1 overexpression is reverted by p38 MAPK inhibition, suggesting that Ask-1 exerts its lethal effects mainly through the p38 MAPK pathway. Moreover, p38alpha(-/-) murine embryonic fibroblasts are protected from UVB-induced apoptosis even if JNK activation is fully preserved. These results argue for an important role of the UVB-generated ROS as mediators of the Ask-1-p38 MAPK pathway that, by culminating in apoptosis, restrains the propagation of potentially mutagenic keratinocytes.

Role of endoplasmic reticulum depletion and multidomain proapoptotic BAX and BAK proteins in shaping cell death after hypericin-mediated photodynamic therapy.

Both the commitment event and the modality of cell death in photodynamic therapy (PDT) remain poorly defined. We report that PDT with endoplasmic reticulum (ER)-associating hypericin leads to an immediate loss of SERCA2 protein levels, causing disruption of Ca2+ homeostasis and cell death. Protection of SERCA2 protein rescues ER-Ca2+ levels and prevents cell death, suggesting that SERCA2 photodestruction with consequent incapability of the ER to maintain intracellular Ca2+ homeostasis is causal to cell killing. Apoptosis is rapidly initiated after ER-Ca2+ depletion and strictly requires the BAX/BAK gateway at the mitochondria. Bax-/-Bak-/- double-knockout (DKO) cells are protected from apoptosis but undergo autophagy-associated cell death as revealed by electron microscopy and biochemical analysis. Autophagy inhibitors, but not caspase antagonists, significantly reduce death of DKO cells, suggesting that sustained autophagy is lethal. Thus, following ER photodamage and consequent disruption of Ca2+ homeostasis, BAX and BAK proteins model PDT-mediated cell killing, which is executed through apoptosis in their presence or via an autophagic pathway in their absence.

Targeted inhibition of p38alpha MAPK suppresses tumor-associated endothelial cell migration in response to hypericin-based photodynamic therapy.

Photodynamic therapy (PDT) is an established anticancer modality and hypericin is a promising photosensitizer for the treatment of bladder tumors. We show that exposure of bladder cancer cells to hypericin PDT leads to a rapid rise in the cytosolic calcium concentration which is followed by the generation of arachidonic acid by phospholipase A2 (PLA2). PLA2 inhibition significantly protects cells from the PDT-induced intrinsic apoptosis and attenuates the activation of p38 MAPK, a survival signal mediating the up-regulation of cyclooxygenase-2 that converts arachidonic acid into prostanoids. Importantly, inhibition of p38alpha MAPK blocks the release of vascular endothelial growth factor and suppresses tumor-promoted endothelial cell migration, a key step in angiogenesis. Hence, targeted inhibition of p38alpha MAPK could be therapeutically beneficial to PDT, since it would prevent COX-2 expression, the inducible release of growth and angiogenic factors by the cancer cells, and cause an increase in the levels of free arachidonic acid, which promotes apoptosis.

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.

Phosphorylation of histone deacetylase 7 by protein kinase D mediates T cell receptor-induced Nur77 expression and apoptosis.

The molecular basis of thymocyte negative selection, a crucial mechanism in establishing central tolerance, is not yet resolved. Histone deacetylases (HDACs) have emerged as key transcriptional regulators in several major developmental programs. Recently, we showed that the class IIa member, HDAC7, regulates negative selection by repressing expression of Nur77, an orphan nuclear receptor involved in antigen-induced apoptosis of thymocytes. Engagement of the T cell receptor (TCR) alleviates this repression through phosphorylation-dependent nuclear exclusion of HDAC7. However, the identity of the TCR-activated kinase that phosphorylates and inactivates HDAC7 was still unknown. Here, we demonstrate that TCR-induced nuclear export of HDAC7 and Nur77 expression is mediated by activation of protein kinase D (PKD). Indeed, active PKD stimulates HDAC7 nuclear export and Nur77 expression. In contrast, inhibition of PKD prevents TCR-mediated nuclear exclusion of HDAC7 and associated Nur77 activation. Furthermore, we show that HDAC7 is an interaction partner and a substrate for PKD. We identify four serine residues in the NH(2) terminus of HDAC7 as targets for PKD. More importantly, a mutant of HDAC7 specifically deficient in phosphorylation by PKD, inhibits TCR-mediated apoptosis of T cell hybridomas. These findings indicate that PKD is likely to play a key role in the signaling pathways controlling negative selection.

PKCepsilon-PKD1 signaling complex at Z-discs plays a pivotal role in the cardiac hypertrophy induced by G-protein coupling receptor agonists.

Cardiac hypertrophy is triggered in response to mechanical stress and various neurohumoral factors, such as G-protein coupling receptor (GPCR) and gp130 cytokine receptor agonists. Recent studies have suggested cardiac Z-disc plays a pivotal role to regulate these cellular responses. Here, we demonstrate stimulations with GPCR agonists (norepinephrine, angiotensin II, and endothelin 1) and phorbol ester activated and translocated protein kinase D1 (PKD1) to the Z-discs in neonatal rat cardiomyocytes in a protein kinase C (PKC)-dependent manner, whereas gp130 agonist did not. Especially, upon the alpha-adrenergic receptor agonist stimulations, following the PKCepsilon-PKD1 complex formation, PKCepsilon-dependent activation of PKD1 was essential to induce hypertrophic responses. Constitutively active mutant of either PKD1 or PKCepsilon also induced cardiac hypertrophy ex vivo. Taken together, the PKCepsilon-PKD1 complex at Z-discs could play a pivotal role in the cardiac hypertrophy induced by GPCR agonists, at least alpha-adrenergic receptor agonist.

Activation of p38 MAPK is required for Bax translocation to mitochondria, cytochrome c release and apoptosis induced by UVB irradiation in human keratinocytes.

This study establishes that activation of p38 MAPK by UVB represents a crucial signal required for the conformational change and translocation of Bax to the mitochondria in human keratinocytes. UVB-induced Bax translocation and mitochondrial cytochrome c release, which precede caspase activation and other endpoints of the apoptotic program such as chromatin fragmentation and loss of mitochondrial transmembrane potential, are blocked by genetic or pharmacological inhibition of the p38alpha MAPK. Inhibition of p38 MAPK strongly reduces the UVB-induced formation of sunburn cells and blocks Bax conformational change both in cultured human keratinocytes and in human skin, providing clear evidence for the physiological role of the p38 MAPK-Bax pathway in the removal of precancerous, UVB-damaged keratinocytes. Furthermore, we show that Bcl-2 overexpression, but not the pan-caspase inhibitor zVAD-fmk, blocks Bax conformational change and its subsequent translocation downstream of p38 MAPK. These data indicate that the activation of p38 MAPK by UVB engages a caspase-independent death signal leading to mitochondrial membrane permeabilization and apoptosis in human keratinocytes and suggest that p38 MAPK might have a preventive role in the process of photocarcinogenesis.

Doxorubicin-induced activation of protein kinase D1 through caspase-mediated proteolytic cleavage: identification of two cleavage sites by microsequencing.

Recent studies have demonstrated the importance of protein kinase D (PKD) in cell proliferation and apoptosis. Here, we report that in vitro cleavage of recombinant PKD1 by caspase-3 generates two alternative active PKD fragments. N-terminal sequencing of these fragments revealed two distinct caspase-3 cleavage sites located between the acidic and pleckstrin homology (PH) domains of PKD1. Moreover, we present experimental evidence that PKD1 is an in vitro substrate for both initiator and effector caspases. During doxorubicin-induced apoptosis, a zVAD-sensitive caspase induces cleavage of PKD1 at two sites, generating fragments with the same molecular masses as those determined in vitro. The in vivo caspase-dependent generation of the PKD1 fragments correlates with PKD1 kinase activation. Our results indicate that doxorubicin-mediated apoptosis induces activation of PKD1 through a novel mechanism involving the caspase-mediated proteolysis.

Up-regulation of cyclooxygenase-2 and apoptosis resistance by p38 MAPK in hypericin-mediated photodynamic therapy of human cancer cells.

Photodynamic Therapy (PDT) is an approved anticancer therapy that kills cancer cells by the photochemical generation of reactive oxygen species following absorption of visible light by a photosensitizer, which selectively accumulates in tumors. We report that hypericin-mediated PDT of human cancer cells leads to up-regulation of the inducible cyclooxygenase-2 (COX-2) enzyme and the subsequent release of PGE2. Dissection of the signaling pathways involved revealed that the selective activation of p38 MAPK alpha and beta mediate COX-2 up-regulation at the protein and messenger levels. The p38 MAPK inhibitor, PD169316, abrogated COX-2 expression in PDT-treated cells, whereas overexpression of the drug-resistant PD169316-insensitive p38 MAPK alpha and beta isoforms restored COX-2 levels in the presence of the kinase inhibitor. Transcriptional regulation by nuclear factor-kappaB was not involved in COX-2 up-regulation by PDT. The half-life of the COX-2 messenger was drastically shortened by p38 MAPK inhibition in transcriptionally arrested cells, suggesting that p38 MAPK mainly acts by stabilizing the COX-2 transcript. Overexpression of WT-p38 MAPK increased cellular resistance to PDT-induced apoptosis, and inhibiting this pathway exacerbated cell death and prevented PGE2 secretion. Hence, the combination of PDT with pyridinyl imidazole inhibitors of p38 MAPK may improve the therapeutic efficacy of PDT by blocking COX-2 up-regulation, which contributes to tumor growth by the release of growth- and pro-angiogenic factors, as well as by sensitizing cancer cells to apoptosis.

In vitro polarized transport of L-phenylalanine in human nasal epithelium and partial characterization of the amino acid transporters involved.

PURPOSE: The purpose of this study was to provide functional and molecular evidence to support the existence of large neutral amino acid transporters in human nasal epithelium using nasal primary cell culture model. METHODS: L-Phenylalanine was used as a model substrate to characterize carrier-mediated permeation of amino acids across human nasal epithelium. The influence of temperature, concentration, other amino acids, metabolic/transport inhibitors, and polarity/stereo-selectivity on transport of the model compound was investigated. Reverse transcriptase polymerase chain reaction was used for molecular characterization of the existence of the transporters. RESULTS: The transport of L-phenylalanine across the human nasal epithelium was polarized (apical --> basolateral >> basolateral --> apical), saturable (Km = 1.23 mM; Vmax = 805.1 nmol/mg protein/min) and stereo-selective (permeation of L-phenylalanine >> D-Phenylalanine). Its permeation was significantly (< 0.05) reduced by cationic, small and large neutral amino acids, oubain, amiloride, sodium-free medium, and temperature lowering. Reverse transcriptase polymerase chain reaction revealed the presence of the broad-scope cationic-dependent amino acid transporter gene (y+LAT-2) in the human nasal epithelium. CONCLUSIONS: Based on the results of this study, one may postulate that the human nasal epithelium expresses L-amino acid transporters. More studies are necessary for detailed characterization of the transporters.

Protein kinase D: a family affair.

The protein kinase D family of enzymes consists of three isoforms: PKD1/PKCmu PKD2 and PKD3/PKCnu. They all share a similar architecture with regulatory sub-domains that play specific roles in the activation, translocation and function of the enzymes. The PKD enzymes have recently been implicated in very diverse cellular functions, including Golgi organization and plasma membrane directed transport, metastasis, immune responses, apoptosis and cell proliferation.

Ultraviolet B radiation-induced apoptosis in human keratinocytes: cytosolic activation of procaspase-8 and the role of Bcl-2.

In this study, we show that ultraviolet B radiation (UVB)-induced apoptosis of human keratinocytes involves mainly cytosolic signals with mitochondria playing a central role. Overexpression of Bcl-2 inhibited UVB-induced apoptosis by blocking the early generation of reactive oxygen species, mitochondrial cardiolipin degradation and cytochrome c release, without affecting Fas ligand (FasL)-induced cell death. It also prevented the subsequent activation of procaspase-3 and -8 as well as Bid cleavage in UVB-treated cells. Comparative analysis of UVB and FasL death pathways revealed a differential role and mechanism of caspase activation, with the UVB-induced activation of procaspase-8 only being a bystander cytosolic event rather than a major initiator mechanism, as is the case for the FasL-induced cell death. Our results suggest that Bcl-2 overexpression, by preventing reactive oxygen species production, helps indirectly to maintain the integrity of lysosomal membranes, and therefore inhibits the release of cathepsins, which contribute to the cytosolic activation of procaspase-8 in UVB-irradiated keratinocytes.

Phosphorylation of Bcl-2 in G2/M phase-arrested cells following photodynamic therapy with hypericin involves a CDK1-mediated signal and delays the onset of apoptosis.

The role of Bcl-2 in photodynamic therapy (PDT) is controversial, and some photosensitizers have been shown to induce Bcl-2 degradation with loss of its protective function. Hypericin is a naturally occurring photosensitizer with promising properties for the PDT of cancer. Here we show that, in HeLa cells, photoactivated hypericin does not cause Bcl-2 degradation but induces Bcl-2 phosphorylation in a dose- and time-dependent manner. Bcl-2 phosphorylation is induced by sublethal PDT doses; increasing the photodynamic stress promptly leads to apoptosis, during which Bcl-2 is neither phosphorylated nor degraded. Bcl-2 phosphorylation involves mitochondrial Bcl-2 and correlates with the kinetics of a G(2)/M cell cycle arrest, preceding apoptosis. The co-localization of hypericin with alpha-tubulin and the aberrant mitotic spindles observed following sublethal PDT doses suggest that photodamage to the microtubule network provokes the G(2)/M phase arrest. PDT-induced Bcl-2 phosphorylation is not altered by either the overexpression or inhibition of p38 mitogen-activated protein kinase (p38 MAPK) and c-Jun NH(2)-terminal protein kinase 1 (JNK1) nor by inhibiting the extracellular signal-regulated kinases (ERKs) or protein kinase C. By contrast, Bcl-2 phosphorylation is selectively suppressed by the cyclin-dependent protein kinase (CDK)-inhibitor roscovitine, completely blocked by the protein synthesis inhibitor cycloheximide and enhanced by the overexpression of CDK1, suggesting a role for this pathway. However, in an in vitro kinase assay, active CDK1/cyclin B1 complex failed to phosphorylate immunoprecipitated Bcl-2, suggesting that this protein kinase may not directly modify Bcl-2. Mutation of serine-70 to alanine in Bcl-2 abolishes PDT-induced phosphorylation and restores the caspase-3 activation to the same levels of the vector-transfected cells, indicating that Bcl-2 phosphorylation may be a signal to delay apoptosis in G(2)/M phase-arrested cells.

Mechanism of activation of protein kinase D2(PKD2) by the CCK(B)/gastrin receptor.

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 alpha(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 alpha, PKC epsilon, and PKC eta. 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.

Protein kinase D: an intracellular traffic regulator on the move.

Recent research has identified protein kinase D (PKD, also called PKCmu) as a serine/threonine kinase with potentially important roles in growth factor signaling as well as in stress-induced signaling. Moreover, PKD has emerged as an important regulator of plasma membrane enzymes and receptors, in some cases mediating cross-talk between different signaling systems. The recent discovery of two additional kinases belonging to the PKD family and the plethora of proteins that interact with PKD point to a multifaceted regulation and a multifunctional role for these enzymes, with functions in processes as diverse as cell proliferation, apoptosis, immune cell regulation, tumor cell invasion and regulation of Golgi vesicle fission.

Getting to know protein kinase D.

The protein kinase D (PKD) enzymes represent a new family of second messenger stimulated kinases, with diacylglycerol as a prime, but not the sole, mediator of activation. Their molecular architecture features a catalytic domain, unrelated to that of all PKC family members, and a large inhibitory, regulatory domain, comprised of two Zinc fingers, and a pleckstrin homology domain. These different sub-domains play distinctive roles in the activation, translocation and biological functions of the kinase. The enzymes have been implicated in signalling mechanisms controlling cell proliferation and programmed cell death and in metastasis, immune responses, and Golgi restructuring and function. A variety of proteins specifically interact with the different sub-domains of the enzymes and direct their wide range of cellular functions.