Inhibition Of Protein Kinase D1 Research Articles

Protein Kinase D1 Regulates Cardiac Hypertrophy, Potassium Channel Remodeling, and Arrhythmias in Heart Failure.

Background Structural and electrophysiological remodeling characterize heart failure (HF) enhancing arrhythmias. PKD1 (protein kinase D1) is upregulated in HF and mediates pathological hypertrophic signaling, but its role in K+ channel remodeling and arrhythmogenesis in HF is unknown. Methods and Results We performed echocardiography, electrophysiology, and expression analysis in wild-type and PKD1 cardiomyocyte-specific knockout (cKO) mice following transverse aortic constriction (TAC). PKD1-cKO mice exhibited significantly less cardiac hypertrophy post-TAC and were protected from early decline in cardiac contractile function (3 weeks post-TAC) but not the progression to HF at 7 weeks post-TAC. Wild-type mice exhibited ventricular action potential duration prolongation at 8 weeks post-TAC, which was attenuated in PKD1-cKO, consistent with larger K+ currents via the transient outward current, sustained current, inward rectifier K+ current, and rapid delayed rectifier K+ current and increased expression of corresponding K+ channels. Conversely, reduction of slowly inactivating K+ current was independent of PKD1 in HF. Acute PKD inhibition slightly increased transient outward current in TAC and sham wild-type myocytes but did not alter other K+ currents. Sham PKD1-cKO versus wild-type also exhibited larger transient outward current and faster early action potential repolarization. Tachypacing-induced action potential duration alternans in TAC animals was increased and independent of PKD1, but diastolic arrhythmogenic activities were reduced in PKD1-cKO. Conclusions Our data indicate an important role for PKD1 in the HF-related hypertrophic response and K+ channel downregulation. Therefore, PKD1 inhibition may represent a therapeutic strategy to reduce hypertrophy and arrhythmias; however, PKD1 inhibition may not prevent disease progression and reduced contractility in HF.

Protein kinase D1 induced epithelial–mesenchymal transition and invasion in salivary adenoid cystic carcinoma via E‐cadherin/Snail regulation

Salivary adenoid cystic carcinoma (SACC) is a malignant tumor, which is characterized by a higher incidence of distant metastasis. The aim of this study was to investigate the role and mechanism of protein kinase D1 (PKD1) in regulating the epithelial-mesenchymal transition (EMT) and promotes the metastasis in SACC. We analyzed the expression of PKD1 in 40 SACC patients and different metastatic potential cell lines. Then, we investigated whether the migration and growth of SACC were regulated by PKD1 using shRNA interference or inhibition of kinase active in vitro cell. Moreover, the mechanism by which PKD1 regulates the stability of Snail protein was determined. Finally, nude mice were used to testify the function of PKD1 via tail vein injection. PKD1 was correlated with metastasis and poor prognosis of SACC patients. PKD1 inhibition attenuated proliferation, migration, invasion, and EMT of SACC cells. Conversely, kinase active PKD1 could induce EMT and promoted cell migration in human HSG cell. Furthermore, downregulation of PKD1 regulated Snail via phosphorylation at Ser-11 on Snail protein and promotion of proteasome-mediated degradation, and reduced lung metastasis in vivo. Our results suggest that PKD1 induces the EMT and promotes the metastasis, which illustrate that PKD1 may be a potential prognostic biomarker and serve as a potential therapeutic target for SACC patients.

A role for PKD1 in insulin secretion downstream of P2Y1 receptor activation in mouse and human islets.

Along with insulin, β‐cells co‐secrete the neurotransmitter ATP which acts as a positive autocrine signal via P2Y1 receptors to activate phospholipase C and increase the production of diacylglycerol (DAG). However, the downstream signaling that couples P2Y1 activation to insulin secretion remains to be fully elucidated. Since DAG activates protein kinase D1 (PKD1) to potentiate glucose‐stimulated insulin release, we hypothesized that autocrine ATP signaling activates downstream PKD1 to regulate insulin secretion. Indeed, we find that the P2Y1 receptor agonists, MRS2365 and ATP induce, PKD1 phosphorylation at serine 916 in mouse islets. Similarly, direct depolarization of islets by KCl caused PKD1 activation, which is reduced upon P2Y1 antagonism. Potentiation of insulin secretion by P2Y1 activation was lost from PKD1−/− mouse islets, and knockdown of PKD1 reduced the ability of P2Y1 activation to facilitate exocytosis in single mouse β‐cells. Finally, qPCR analysis confirmed PKD1 transcript (PRKD1) expression in human islets, and insulin secretion assays showed that inhibition of either P2Y1 or PKD1 signaling impaired glucose‐stimulated insulin secretion. Human islets showed donor‐to‐donor variation in their responses to both P2Y1 and PKD1 inhibition, however, and we find that the P2Y1‐PKD1 pathway contributes a substantially greater proportion of insulin secretion from islets of overweight and obese donors. Thus, PKD1 promotes increased insulin secretion, likely mediating an autocrine ATP effect via P2Y1 receptor activation which may be more important in islets of donors who are overweight or obese.

Unveiling the Structural Insights into the Selective Inhibition of Protein Kinase D1.

Although protein kinase D1 (PKD1) has been proved to be an efficient target for anticancer drug development, lack of structural details and substrate binding mechanisms are the main obstacles for the development of selective inhibitors with therapeutic benefits. The present study described the in silico dynamics behaviors of PKD1 in binding with selective and non-selective inhibitors and revealed the critical binding site residues for the selective kinase inhibition. Here, the three dimensional model of PKD1 was initially constructed by homology modeling along with binding site characterization to explore the non-conserved residues. Subsequently, two known inhibitors were docked to the catalytic site and the detailed ligand binding mechanisms and post binding dyanmics were investigated by molecular dynamics simulation and binding free energy calculations. According to the binding site analysis, PKD1 serves several non-conserved residues in the G-loop, hinge and catalytic subunits. Among them, the residues including Leu662, His663, and Asp665 from hinge region made polar interactions with selective PKD1 inhibitor in docking simulation, which were further validated by the molecular dynamics simulation. Both inhibitors strongly influenced the structural dynamics of PKD1 and their computed binding free energies were in accordance with experimental bioactivity data. The identified non-conserved residues likely to play critical role on molecular reorganization and inhibitor selectivity. Taken together, this study explained the molecular basis of PKD1 specific inhibition, which may help to design new selective inhibitors for better therapies to overcome cancer and PKD1 dysregulated disorders.

Structure Based Design of Potent Selective Inhibitors of Protein Kinase D1 (PKD1).

We previously disclosed a series of type I 1/2 inhibitors of NF-κB inducing kinase (NIK). Inhibition of NIK by these compounds was found to be strongly dependent on the inclusion and absolute stereochemistry of a propargyl tertiary alcohol as it forms critical hydrogen bonds (H-bonds) with NIK. We report that inhibition of protein kinase D1 (PKD1) by this class of compounds is not dependent on H-bond interactions of this tertiary alcohol. This feature was leveraged in the design of highly selective inhibitors of PKD1 that no longer inhibit NIK. A structure-based hypothesis based on the position and flexibility of the α-C-helix of PKD1 vs NIK is presented.

Abstract 1289: AICAR inhibits protein kinase D1 activity leading to epigenetic downregulation of immediate early genes via the NF-kB pathway in acute lymphoblastic leukemia

Abstract Acute lymphoblastic leukemia (ALL) is the leading cause of cancer related death in children, and cure rates for relapsed/refractory ALL remain dismal, highlighting the need for novel targeted therapies. We previously uncovered that ALL are vulnerable to metabolic/energy stress and ER-stress via AMP-activated protein kinase (AMPK) activation. In order to identify genome wide metabolic stress and AMPK transcriptionally regulated genes, we used RNA-Seq and compared mRNA profiles in ALL cells treated with the adenosine analog AICAR, an activator of AMPK. RNA-Seq data indicated that high dose AICAR (15 mM/45 min) induced a robust downregulation of a cluster of genes known as the immediate early genes (IEGs), which are critical for cell survival, proliferation and adaptation. AICAR-induced downregulation on IEG expression was dose- and time-dependent, and observed in other cell types (HEK293T, Hela, MEF), indicating this mechanism is conserved in mammalian cells. Using MEF AMPKα2 and AMPKα1/α2 knockout cell lines, we found that these alterations were AMPK-independent. Characterization of AICAR’s mechanism of action identified the protein kinase D1 (PKD1) as responsible for these effects. PKD1 is a Ser/Thr protein kinase involved in many cellular processes important for cancer development and progression, including proliferation, survival, apoptosis, invasion, cell adhesion and angiogenesis. We uncovered that high dose AICAR significantly inhibited PKD1 activation (Ser-910) at the plasma membrane which prevented its nuclear translocation. When PKD1 activity was pharmacologically inhibited by CRT0066101 or downregulated by shRNA, we observed similar IEGs’ downregulation in ALL cells. Conversely, the effect of AICAR on IEGs’ expression was abrogated by PMA, a direct activator of PKD1. In addition, when PKD1 was overexpressed in HEK293T cells, AICAR-induced IEG’s downregulation was partially restored. Using a kinase assay, we found that AICAR, but not ZMP, directly inhibited PKD1 kinase activity. Further, we determined that AICAR suppressed phosphorylation and nuclear export of PKD1-targeted histone deacetylases HDAC4/5, which led to decreased histone H3 acetylation at the IEGs’ promoter region. Finally, ChIP-qPCR indicated that AICAR-induced PKD1 inhibition prevented NF-κB recruitment to IEGs’ promoters. Inhibition of PKD1 activity led to decreased cell proliferation and promoted apoptosis in ALL cells. To confirm the in vivo relevance of our data, single agent and combination experiments using our NSG ALL mouse model are underway. Taken together, we have identified a novel AMPK-independent mechanism leading to AICAR’s inhibition of PKD1-mediated ALL survival. Consequently, co-targeting PDK1 and other pro-survival stress response pathways in ALL cells offers novel strategies to overcome therapeutic resistance. Citation Format: Anna Shvab, Guangyan Sun, Bin Li, Felipe Beckedorff, Guy J. Leclerc, Ramin Shiekhattar, Julio C. Barredo. AICAR inhibits protein kinase D1 activity leading to epigenetic downregulation of immediate early genes via the NF-kB pathway in acute lymphoblastic leukemia [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1289.

Protein kinase D1 inhibition interferes with mitosis progression

Protein kinase D1 (PKD1) plays a vital role in signal transduction, cell proliferation, membrane trafficking, and cancer; however, the majority of the studies up to date had centered primarily on PKD1 functions in interphase, very little is known about its role during cell division. We previously demonstrated that during mitosis PKD1 is activated and associated with centrosomes, spindles, and midbodies. However, these observations did not address whether PKD1 was associated with mitosis regulation. Accordingly, we used rapidly acting PKD-specific inhibitors to examine the contribution of PKD1 the sequence of events in mitosis. We found that although PKD1 overexpression did not affect mitosis progression, suppression of its catalytic activity by two structurally unrelated inhibitors (kb NB 142-70 and CRT 0066101) induced a significant delay in metaphase to anaphase transition time. PKD1 inhibition during mitosis also produced the appearance of abnormal spindles, defects in chromosome alignment, and segregation as well as apoptosis. Thus, these observations indicate that PKD1 activity is associated with mitosis regulation.

PKD1 is a potential biomarker and therapeutic target in triple-negative breast cancer.

Protein Kinase D1 (PKD1) is a serine/threonine kinase encoded by the PRKD1 gene. PKD1 has been previously shown to be a prognostic factor in ERα+ tamoxifen-resistant breast tumors and PKD1 overexpression confers estrogen independence to ERα+ MCF7 cells. In the present study, our goal was to determine whether PKD1 is a prognostic factor and/or a relevant therapeutic target in breast cancer. We analyzed PRKD1 mRNA levels in 527 primary breast tumors. We found that high PRKD1 mRNA levels were significantly and independently associated with a low metastasis-free survival in the whole breast cancer population and in the triple-negative breast cancer (TNBC) subtype specifically. High PRKD1 mRNA levels were also associated with a low overall survival in TNBC. We identified novel PKD1 inhibitors and assessed their antitumor activity in vitro in TNBC cell lines and in vivo in a TNBC patient-derived xenograft (PDX) model. Pharmacological inhibition and siRNA-mediated depletion of PKD1 reduced colony formation in MDA-MB-436 TNBC cells. PKD1 inhibition also reduced tumor growth in vivo in a TNBC PDX model. Together, these results establish PKD1 as a poor prognostic factor and a potential therapeutic target in TNBC.

Pleiotropic Effects of Chronic Phorbol Ester Treatment to Improve Glucose Transport in Insulin-Resistant Cardiomyocytes.

Stimulation of glucose transport is an important determinant of myocardial susceptibility to ischemia and reperfusion. Stimulation of glucose transport is markedly impaired in cardiomyocytes exposed to free fatty acids (FFA). Deactivation of the Focal Adhesion Kinase (FAK) by FFA contributes to glucose transport impairment, and could be corrected by chronic treatment with the phorbol ester TPA. However, TPA must have effects in addition to FAK reactivation to restore stimulated glucose transport. Chronic treatment with TPA improved basal and stimulated glucose transport in FFA-exposed, but not in control cardiomyocytes. Chronic FFA exposure induced the activation of PKCδ and PKCϵ. TPA markedly downregulated the expression of PKCα, PKCδ, and PKCϵ, suggesting that PKCδ or PKCϵ activation could contribute to inhibition of glucose transport by FFA. Rottlerin, a specific PKCδ inhibitor, improved glucose transport in FFA-exposed cardiomyocytes; and PKCδ was reduced in the particulate fraction of FFA + TPA-exposed cardiomyocytes. TPA also activated Protein Kinase D 1(PKD1) in FFA-exposed cardiomyocytes, as assessed by autophosphorylation of PKD1 on Y916. Pharmaceutical inhibition of PKD1 only partially prevented the improvement of glucose transport by TPA. Chronic TPA treatment also increased basal and stimulated glycolysis and favored accumulation of lipid droplets in FFA-exposed cardiomyocytes. In conclusion, basal and stimulated glucose transport in cardiomyocytes is reduced by chronic FFA exposure, but restored by concomitant treatment with a phorbol ester. The mechanism of action of phorbol esters may involve downregulation of PKCδ, activation of PKD1 and a general switch from fatty acid to glucose metabolism. J. Cell. Biochem. 9999: 4716-4727, 2017. © 2017 Wiley Periodicals, Inc.

Protein kinase C inhibitor G\xf66976 but not G\xf66983 induces the reversion of E- to N-cadherin switch and metastatic phenotype in melanoma: identification of the role of protein kinase D1

BackgroundMelanoma is a highly metastatic type of cancer that is resistant to all standard anticancer therapies and thus has a poor prognosis. Therefore, metastatic melanoma represents a significant clinical problem and requires novel and effective targeted therapies. The protein kinase C (PKC) family comprises multiple isoforms of serine/threonine kinases that possess distinct roles in cancer development and progression. In this study, we determined whether inhibition of PKC could revert a major process required for melanoma progression and metastasis; i.e. the E- to N-cadherin switch.MethodsThe cadherin switch was analyzed in different patient-derived primary tumors and their respective metastatic melanoma cells to determine the appropriate cellular model (aggressive E-cadherin-negative/N-cadherin-positive metastasis-derived melanoma cells). Next, PKC inhibition in two selected metastatic melanoma cell lines, was performed by using either pharmacological inhibitors (Gö6976 and Gö6983) or stable lentiviral shRNA transduction. The expression of E-cadherin and N-cadherin was determined by western blot. The consequences of cadherin switch reversion were analyzed: cell morphology, intercellular interactions, and β-catenin subcellular localization were analyzed by immunofluorescence labeling and confocal microscopy; cyclin D1 expression was analyzed by western blot; cell metastatic potential was determined by anchorage-independent growth assay using methylcellulose as semi-solid medium and cell migration potential by wound healing and transwell assays.ResultsGö6976 but not Gö6983 reversed the E- to N-cadherin switch and as a consequence induced intercellular interactions, profound morphological changes from elongated mesenchymal-like to cuboidal epithelial-like shape, β-catenin translocation from the nucleus to the plasma membrane inhibiting its oncogenic function, and reverting the metastatic potential of the aggressive melanoma cells. Comparison of the target spectrum of these inhibitors indicated that these observations were not the consequence of the inhibition of conventional PKCs (cPKCs), but allowed the identification of a novel serine/threonine kinase, i.e. protein kinase Cμ, also known as protein kinase D1 (PKD1), whose specific inhibition allows the reversion of the metastatic phenotype in aggressive melanoma.ConclusionIn conclusion, our study suggests, for the first time, that while cPKCs don’t embody a pertinent therapeutic target, inhibition of PKD1 represents a novel attractive approach for the treatment of metastatic melanoma.

0379 : Inhibition of glucose transport in cardiac myocytes exposed to free fatty acids: role of FAK, PKCs and PKD in the restoration of glucose transport by chronic phorbol ester treatment

Stimulation of glucose transport is an important determinant of myocardial susceptibility to ischemia and reperfusion. Stimulation of glucose transport of markedly impaired in cardiomyocytes exposed to free fatty acids (FFA), in part due to inhibition of Focal Adhesion Kinase (FAK). To determine whether reactivation of FAK by chronic phenylephrine (PE) or phorbol ester (tetradecanoyl phorbol acetate; TPA) treatment improves glucose transport in cardiomyocytes exposed to FFA. Isolated cultured cardiomyocytes were chronically exposed to FFA±PE or TPA. Glucose transport was measured during acute stimulation either by insulin or by metabolic inhibition with oligomycin resulting in AMPK activation. Chronic treatment with PE or TPA improved basal and stimulated glucose transport in FFA-exposed, but not in control cardiomyocytes. These effects were associated with increased FAK activity. However, the effect of TPA was much more pronounced than that of PE, despite similar FAK reactivation, suggesting additional effects of TPA. Chronic FFA exposure induced the activation of PKCδ and PKCε. TPA markedly downregulated the expression of PKCα, PKCδ and PKCε, suggesting that PKCδ or PKCε activation could contribute to inhibition of glucose transport by FFA. Rottlerin, a specific PKCδ inhibitor, improved glucose transport in FFA-exposed cardiomyocytes; and PKCδ was reduced in the particulate fraction of FFA + TPAexposed cardiomyocytes. TPA also activated Protein Kinase D 1(PKD1) in FFA-exposed cardiomyocytes, as assessed by autophosphorylation of PKD1 on Y916. Pharmaceutical inhibition of PKD1 only partially prevented the improvement of glucose transport by TPA. basal and stimulated glucose transport in cardiomyocytes is reduced by chronic FFA exposure, but restored by concomitant treatment with a phorbol ester. The mechanism of action of phorbol esters may involve FAK activation, downregulation of PKCδ and activation of PKD1. The author hereby declares no conflict of interest

A novel miR-34a target, protein kinase D1, stimulates cancer stemness and drug resistance through GSK3/β-catenin signaling in breast cancer.

One of the properties of human breast cancer cells is cancer stemness, which is characterized by self-renewal capability and drug resistance. Protein kinase D1 (PRKD1) functions as a key regulator of many cellular processes and is downregulated in invasive breast cancer cells. In this study, we found that PRKD1 was upregulated in MCF-7-ADR human breast cancer cells characterized by drug resistance. Additionally, we discovered that PRKD1 expression was negatively regulated by miR-34a binding to the PRKD1 3′-UTR. PRKD1 expression increased following performance of a tumorsphere formation assay in MCF-7-ADR cells. We also found that reduction of PRKD1 by ectopic miR-34a expression or PRKD1 siRNA treatment resulted in suppressed self-renewal ability in breast cancer stem cells. Furthermore, we confirmed that the PRKD1 inhibitor CRT0066101 reduced phosphorylated PKD/PKCμ, leading to suppression of breast cancer stemness through GSK3/β-catenin signaling. PRKD1 inhibition also influenced apoptosis initiation in MCF-7-ADR cells. Tumors from nude mice treated with miR-34a or CRT0066101 showed suppressed tumor growth, proliferation, and induced apoptosis. These results provide evidence that regulation of PRKD1, a novel miR-34a target, contributes to overcoming cancer stemness and drug resistance in human breast cancer.

Protein kinase D1 (PKD1) phosphorylation promotes dopaminergic neuronal survival during 6-OHDA-induced oxidative stress.

Oxidative stress is a major pathophysiological mediator of degenerative processes in many neurodegenerative diseases including Parkinson’s disease (PD). Aberrant cell signaling governed by protein phosphorylation has been linked to oxidative damage of dopaminergic neurons in PD. Although several studies have associated activation of certain protein kinases with apoptotic cell death in PD, very little is known about protein kinase regulation of cell survival and protection against oxidative damage and degeneration in dopaminergic neurons. Here, we characterized the PKD1-mediated protective pathway against oxidative damage in cell culture models of PD. Dopaminergic neurotoxicant 6-hydroxy dopamine (6-OHDA) was used to induce oxidative stress in the N27 dopaminergic cell model and in primary mesencephalic neurons. Our results indicated that 6-OHDA induced the PKD1 activation loop (PKD1S744/S748) phosphorylation during early stages of oxidative stress and that PKD1 activation preceded cell death. We also found that 6-OHDA rapidly increased phosphorylation of the C-terminal S916 in PKD1, which is required for PKD1 activation loop (PKD1S744/748) phosphorylation. Interestingly, negative modulation of PKD1 activation by RNAi knockdown or by the pharmacological inhibition of PKD1 by kbNB-14270 augmented 6-OHDA-induced apoptosis, while positive modulation of PKD1 by the overexpression of full length PKD1 (PKD1WT) or constitutively active PKD1 (PKD1S744E/S748E) attenuated 6-OHDA-induced apoptosis, suggesting an anti-apoptotic role for PKD1 during oxidative neuronal injury. Collectively, our results demonstrate that PKD1 signaling plays a cell survival role during early stages of oxidative stress in dopaminergic neurons and therefore, positive modulation of the PKD1-mediated signal transduction pathway can provide a novel neuroprotective strategy against PD.

Neuregulin Mediates F-actin-driven Cell Migration through Inhibition of Protein Kinase D1 via Rac1 Protein

Neuregulin (NRG; heregulin) is overexpressed in ∼30% of breast cancers and mediates various processes involved in tumor progression, including tumor cell migration and invasion. Here, we show that NRG mediates its effects on tumor cell migration via PKD1. Downstream of RhoA, PKD1 can prevent directed cell migration through phosphorylation of its substrate SSH1L. NRG exerts its inhibitory effects on PKD1 through Rac1/NADPH oxidase, leading to decreased PKD1 activation loop phosphorylation and decreased activity toward SSH1L. The consequence of PKD1 inhibition by NRG is decreased binding of 14-3-3 to SSH1L, localization of SSH1L to F-actin at the leading edge, and increased cofilin activity, resulting in increased reorganization of the actin cytoskeleton and cell motility. Our data provide a mechanism through which the Rho GTPase Rac1 cross-talks with PKD1 signaling pathways to facilitate directed cell migration.

Abstract 230: Protein Kinase D1 Mediates Vegf-induced Vascular Permeability In Vitro And In Vivo By Promoting Endothelial Cell Stress Fiber Formation

Background Vascular hyperpermeability is critical components of many human diseases, including inflammation, cancer and diabetic retinopathy. Vascular endothelial growth factor (VEGF) is a potent vascular permeability factor. Better understanding of the molecular mechanisms by which VEGF induces vascular permeability may provide novel therapeutic targets for these diseases. We and others have recently demonstrated that protein kinase D1 (PKD1), a newly discovered serine/threonine protein kinase, is implicated in VEGF signaling. We hypothesized that PKD1 plays a crucial role in VEGF-induced vascular permeability in vivo and vitro by promoting endothelial cell stress fiber formation. Methods and results Using Tie2-Cre transgenic mice and PKD1 flox mice, we generated endothelium-specific PKD1 knockout (PKD1-ecKO) mice. PKD1-ecKO mice were viable and born at the expected Mendelian ratio. Comparing with wild type mice, PKD1-ecKO mice exhibited the dramatic reduction of VEGF-induced vascular permeability in lung and ear with Miles assay, expressed as Evans blue leakage. Selective pharmacological PKD inhibitor also significantly attenuated in vivo vascular permeability induced by VEGF intravenously injection in mice. In cultured endothelial monolayer, down-regulation of PKD1 by siRNA, GFP-PKD1 dominant negative mutant plasmid or PKD inhibitor significantly reduced VEGF-induced endothelial permeability, measured by bovine serum albumin leakage through endothelial monolayer cultured on transwell system. Moreover, we found that the inhibition of PKD1 diminished F-actin stress fiber formation and the dismantlement of endothelial adherens junctions that lead to intercellular gap formation and leakage in response to VEGF. We further observed that PKD1 acted as a downstream mediator of Rho activation in VEGF-induced stress fiber formation. Specifically, Rho inhibitor Y27632 blocked VEGF-induced PKD1 phosphorylation, while down-regulation of PKD1 expression and activation did not affect VEGF-stimulated Rho activation in endothelial cells. Conclusion PKD1 is required for VEGF-induced vascular permeability in vivo and in vitro through promoting F-actin stress fiber formation and disrupting endothelial adherens junctions.

689 PROTEIN KINASE D1 INDUCES STEMNESS IN PROSTATE CANCER CELLS THROUGH SNAIL PHOSPHORYLATION

You have accessJournal of UrologyProstate Cancer: Basic Research V1 Apr 2010689 PROTEIN KINASE D1 INDUCES STEMNESS IN PROSTATE CANCER CELLS THROUGH SNAIL PHOSPHORYLATION Cheng Du, Md. Helal Uddin Biswas, Chuanyou Zhang, and K.C. Balaji Cheng DuCheng Du More articles by this author , Md. Helal Uddin BiswasMd. Helal Uddin Biswas More articles by this author , Chuanyou ZhangChuanyou Zhang More articles by this author , and K.C. BalajiK.C. Balaji More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2010.02.1088AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES There is increasing evidence that a subset of cancer cell population often referred to as cancer stem cells (CSC) or progenitor cells are treatment refractory and contribute to disease recurrence, progression and death. We have previously demonstrated that Protein Kinase D1 (PKD1), a novel serine-threonine kinase, is down regulated in advanced prostate cancer (PC), interacts and phosphorylates E-cadherin-beta-catenin complex of proteins known to play a role in epithelial differentiation. In this study, we explored role and mechanism of PKD1 in de-differentiation of PC cells to stem like cells. METHODS Standard gene cloning and molecular biological tools including Western blotting, luciferase promoter assay, Yeast-2-hybrid, FACS sorting, in vitro phosphorylation and immunoflorescence imaging were used for in vitro experiments using PC and breast cancer cell lines. Anchorage independent growth assay was used to study prostosphere formation. RESULTS Down regulation of Snail, a major transcriptional repressor of E-cadherin mimicked epithelial mesenchymal transition (EMT) protein expression pattern similar to PKD1 down regulation suggesting Snail could be a PKD1 substrate. PKD1 phosphorylates Snail at serine11 residue and nuclear export of Snail is blocked by lack of S11 phosphorylation leading to E-cadherin repression and EMT. The PKD1 mediated Snail nuclear export is dependent on 14-3-4 sigma protein. Stable transfection of S11 non-phosphorylation mutant increased expression of CD44hi/CD24hi stem cell markers expressing cells in MCF7 breast cancer cells. Transfection of DU145 PC cells with PKD1 decreased PC stem cell markers CD44hi/CD133low expressing cells by 3 fold (1.39% to 0.41%), whereas inhibition of PKD1 using selective inhibitor in LNCaP cells increased stem cell like population by 8 fold (0.32% to 2.35%). Transfection of aggressive PC cell line PC3 with PKD1 totally abolished anchorage independent growth and prostatospheres formation in vitro. CONCLUSIONS Loss of PKD1 function in PC leads to stemness, which provides novel insights in to disease progression. S11 non-phosphorylation mutants could be used for enrichment of stem cell population in PC, which can be used to study stem cell biology and targeting in PC. Worcester, MA© 2010 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 183Issue 4SApril 2010Page: e269 Peer Review Report Advertisement Copyright & Permissions© 2010 by American Urological Association Education and Research, Inc.MetricsAuthor Information Cheng Du More articles by this author Md. Helal Uddin Biswas More articles by this author Chuanyou Zhang More articles by this author K.C. Balaji More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

CCK causes PKD1 activation in pancreatic acini by signaling through PKC-δ and PKC-independent pathways

Protein kinase D1 (PKD1) is involved in cellular processes including protein secretion, proliferation and apoptosis. Studies suggest PKD1 is activated by various stimulants including gastrointestinal (GI) hormones/neurotransmitters and growth factors in a protein kinase C (PKC)-dependent pathway. However, little is known about the mechanisms of PKD1 activation in physiologic GI tissues. We explored PKD1 activation by GI hormones/neurotransmitters and growth factors and the mediators involved in rat pancreatic acini. Only hormones/neurotransmitters activating phospholipase C caused PKD1 phosphorylation (S916, S744/748). CCK activated PKD1 and caused a time- and dose-dependant increase in serine phosphorylation by activation of high- and low-affinity CCK A receptor states. Inhibition of CCK-stimulated increases in phospholipase C, PKC activity or intracellular calcium decreased PKD1 S916 phosphorylation by 56%, 62% and 96%, respectively. PKC inhibitors GF109203X/Go6976/Go6983/PKC-ζ pseudosubstrate caused a 62/43/49/0% inhibition of PKD1 S916 phosphorylation and an 87/13/82/0% inhibition of PKD1 S744/748 phosphorylation. Expression of dominant negative PKC-δ, but not PKC-ε, or treatment with PKC-δ translocation inhibitor caused marked inhibition of PKD phosphorylation. Inhibition of Src/PI3K/MAPK/tyrosine phosphorylation had no effect. In unstimulated cells, PKD1 was mostly located in the cytoplasm. CCK stimulated translocation of total and phosphorylated PKD1 to the membrane. These results demonstrate that CCK A receptor activation leads to PKD activation by signaling through PKC-dependent and PKC-independent pathways.

Protein Kinase D-Mediated Anterograde Membrane Trafficking Is Required for Fibroblast Motility

Background: Locomoting cells exhibit a constant retrograde flow of plasma membrane (PM) proteins from the leading edge lamellipodium backward, which when coupled to substrate adhesion, may drive forward cell movement. However, the intracellular source of these PM components and whether their continuous retrograde flow is required for cell motility is unknown.Results: To test the hypothesis that the anterograde secretion pathway supplies PM components for retrograde flow that are required for lamellipodial activity and cell motility, we specifically inhibited transport of cargo from the trans-Golgi network (TGN) to the PM in Swiss 3T3 fibroblasts and monitored cell motility using time-lapse microscopy. TGN-to-PM trafficking was inhibited with a dominant-negative, kinase-dead (kd) mutant of protein kinase D1 (PKD) that specifically blocks budding of secretory vesicles from the TGN and does not affect other transport pathways. Inhibition of PKD on the TGN inhibited directed cell motility and retrograde flow of surface markers and filamentous actin, while inhibition of PKD elsewhere in the cell neither blocked anterograde membrane transport nor cell motile functions. Exogenous activation of Rac1 in PKD-kd-expressing cells restored lamellipodial dynamics independent of membrane traffic. However, lamellipodial activity was delocalized from a single leading edge, and directed cell motility was not fully recovered.Conclusions: These results indicate that PKD-mediated anterograde membrane traffic from the TGN to the PM is required for fibroblast locomotion and localized Rac1-dependent leading edge activity. We suggest that polarized secretion transmits cargo that directs localized signaling for persistent leading edge activity necessary for directional migration.