Evidence for the phosphorylation of serine259 of histone deacetylase 5 by protein kinase Cδ

Signaling via pro-growth G protein coupled receptors triggers phosphorylation of HDAC5 on two serine residues (Ser₂₅₉ and Ser₄₉₈), resulting in nuclear export of HDAC5 and de-repression of downstream target genes. In the previous paper we reported the important role of PKD isozymes in the regulation of HDAC5 by phosphorylating Ser₄₉₈ of HDAC5 [Q.K. Huynh, T.A. Mckinsey, Arch. Biochem. Biophys. 450 (2006) 141–148]. In the present paper, we provide evidence that PKCδ can directly phosphorylate Ser₂₅₉ of HDAC5. The evidence is based on the following facts (a) isolated kinase fraction from human failing heart tissues contained PKCδ that phosphorylated HDAC5 Ser₂₅₉ peptide and no significant activity was found for the unbound fraction after they were immunoprecipitated with PKCδ specific antibody; (b) specific inhibitors for PKCδ inhibited kinase activity from isolated fraction and recombinant human PKCδ with similar IC₅₀ values; (c) recombinant human PKCδ can directly phosphorylate full length Ser₂₅₉ HDAC5 protein and HDAC5 Ser₂₅₉ peptide. The results suggest that in addition to activation of protein kinase D isozymes by phosphorylating Ser₇₄₄ and Ser₇₄₈ at their activation sites, PKCδ may also play a role in the regulation of HDAC5 by phosphorylation of Ser₂₅₉.     

Phosphorylation at Ser244 by CK1 determines nuclear localization and substrate targeting of PKD2

Protein kinase D2 (PKD2), a member of the PKD family of serine/threonine kinases, is localized in various subcellular compartments including the nucleus where the kinase accumulates upon activation of G-protein-coupled receptors. We define three critical post-translational modifications required for nuclear accumulation of PKD2 in response to activation of the CCK2 receptor (CCK2R): phosphorylation at Ser706 and Ser710 within the activation loop by PKC eta leading to catalytic activity and phosphorylation at Ser244 within the zinc-finger domain, which is crucial for blocking nuclear export of active PKD2 by preventing its interaction with the Crm-1 export machinery. We identify CK1delta and epsilon as upstream activated kinases by CCK2R that phosphorylate PKD2 at Ser244. Moreover, nuclear accumulation of active PKD2 is a prerequisite for efficient phosphorylation of its nuclear substrate, HDAC7. Only nuclear, active PKD2 mediates CCK2R-induced HDAC7 phosphorylation and Nur77 expression. Thus, we define a novel, compartment-specific signal transduction pathway downstream of CCK2R that phosphorylates PKD2 at three specific sites, results in nuclear accumulation of the active kinase and culminates in efficient phosphorylation of nuclear PKD2 substrates in human gastric cancer cells.     

Essential role for protein kinase D family kinases in the regulation of class II histone deacetylases in B lymphocytes

We have taken a knockout approach to interrogate the function of protein kinase D (PKD) serine/threonine kinases in lymphocytes. DT40 B cells express two PKD family members, PKD1 and PKD3, which are both rapidly activated by the B-cell antigen receptor (BCR). DT40 cells with single or dual deletions of PKD1 and/or PKD3 were viable, allowing the role of individual PKD isoforms in BCR signal transduction to be assessed. One proposed downstream target for PKD1 in lymphocytes is the class II histone deacetylases (HDACs). Regulation of chromatin accessibility via class II histone deacetylases is an important mechanism controlling gene expression patterns, but the molecules that control this key process in B cells are not known. Herein, we show that phosphorylation and nuclear export of the class II histone deacetylases HDAC5 and HDAC7 are rapidly induced following ligation of the BCR or after treatment with phorbol esters (a diacylglycerol mimetic). Loss of either PKD1 or PKD3 had no impact on HDAC phosphorylation, but loss of both PKD1 and PKD3 abrogated antigen receptor-induced class II HDAC5/7 phosphorylation and nuclear export. These studies reveal an essential and redundant role for PKD enzymes in controlling class II HDACs in B lymphocytes and suggest that PKD serine kinases are a critical link between the BCR and epigenetic control of chromatin.     

A phosphorylation state-specific antibody recognizes Hsp27, a novel substrate of protein kinase D

The use of phosphorylation state-specific antibodies has revolutionized the field of cellular signaling by Ser/Thr protein kinases. A more recent application of this technology is the development of phospho-specific antibodies that specifically recognize the consensus substrate phosphorylated motif of a given protein kinase. Here, we describe the development and use of such an antibody which is directed against the optimal phosphorylation motif of protein kinase D (PKD). A degenerate phosphopeptide library with fixed residues corresponding to the consensus LXR(Q/K/E/M)(M/L/K/E/Q/A)S*XXXX was used as an antigen to generate an antibody that recognizes this motif. We characterized the antibody by enzyme-linked immunosorbent assay and with immobilized peptide arrays and also detected immunoreactive phosphoproteins in HeLa cells stimulated with agonists known to activate PKD. Silencing PKD expression using RNA interference validated the specificity of this antibody immunoreactive against putative substrates. The antibody also detected the PKD substrates RIN1 and HDAC5. Knowledge of the PKD consensus motif also enabled us to identify Ser(82) in the human heat shock protein Hsp27 as a novel substrate for PKD. We term this antibody anti-PKD pMOTIF and predict that it will enable the discovery of novel PKD substrate proteins in cells.     

Phosphorylation of Ser 402 impedes phosphatase activity of slingshot 1

By using mass spectrometry, we have identified Ser 402 as a new phosphorylation site within the catalytic domain of human slingshot 1 (SSH1). Phosphorylation at this site inhibits substrate binding and, thus, phosphatase activity in vitro, resulting in enrichment of phosphorylated cofilin in monolayer cell culture. We further demonstrate that protein kinase D (PKD) is upstream from Ser 402 phosphorylation. Accordingly, expression of active PKD in Drosophila phenotypically mimics the loss of SSH activity by inducing accumulation of phosphorylated cofilin and filamentous actin. We thus identify a universal mechanism by which PKD controls SSH1 phosphatase activity.     

Protein kinase D1 and D2 are involved in chemokine release induced by toll-like receptors 2, 4, and 5

The protein kinase D (PKD) family consists of three serine-threonine kinases involved in cellular proliferation, motility, and apoptosis. We previously reported that human toll-like receptor 5 (TLR5) contains a consensus PKD phosphorylation site. Flagellin stimulation of cells activated PKD1, and inhibition of PKD1 reduced flagellin-induced interleukin-8 (IL-8) production in epithelial cells. In the current work, we examined PKD1 and PKD2 involvement downstream of TLR5, TLR4 and TLR2. We found that inhibition of either kinase with shRNA reduced IL-8 and CCL20 release due to TLR4 and TLR2 agonists to a similar extent as previously reported for TLR5. PKD1 and PKD2 inhibition reduced NF-κB activity but not MAPK activation. These results demonstrate that both PKD1 and PKD2 are required for inflammatory responses following TLR2, TLR4, or TLR5 activation, although PKD1 is more strongly involved. These kinases likely act downstream of the TLRs themselves to facilitate NF-κB activation but not MAP kinase phosphorylation.     

Novel function of cardiac protein kinase D1 as a dynamic regulator of Ca2+ sensitivity of contraction

Although the function of protein kinase D1 (PKD) in cardiac cells has remained enigmatic, recent work has shown that PKD phosphorylates the nuclear regulators HDAC5/7 (histone deacetylase 5/7) and CREB, implicating this kinase in the development of dysfunction seen in heart failure. Additional studies have shown that PKD also phosphorylates multiple sarcomeric substrates to regulate myofilament function. Initial studies examined PKD through adenoviral vector expression of wild type PKD, constitutively active PKD (caPKD), or dominant negative PKD in cultured adult rat ventricular myocytes. Confocal immunofluorescent images of these cells reveal a predominant distribution of all PKD forms in a non-nuclear, Z-line localized, striated reticular pattern, suggesting the importance of PKD in Ca(2+) signaling in heart. Consistent with an established role of PKD in targeting cardiac troponin I (cTnI), caPKD expression led to a marked decrease in contractile myofilament Ca(2+) sensitivity with an unexpected electrical stimulus dependence to this response. This desensitization was accompanied by stimulus-dependent increases in cTnI phosphorylation in control and caPKD cells with a more pronounced effect in the latter. Electrical stimulation also provoked phosphorylation of regulatory site Ser(916) on PKD. The functional importance of this phospho-Ser(916) event is demonstrated in experiments with a phosphorylation-defective mutant, caPKD-S916A, which is functionally inactive and blocks stimulus-dependent increases in cTnI phosphorylation. Dominant negative PKD expression resulted in sensitization of the myofilaments to Ca(2+) and blocked stimulus-dependent increases in cTnI phosphorylation. Taken together, these data reveal that localized PKD may play a role as a dynamic regulator of Ca(2+) sensitivity of contraction in cardiac myocytes.     

Protein kinase D1 mediates stimulation of DNA synthesis and proliferation in intestinal epithelial IEC-18 cells and in mouse intestinal crypts

We examined whether protein kinase D1 (PKD1), the founding member of a new protein kinase family, plays a critical role in intestinal epithelial cell proliferation. Our results demonstrate that PKD1 activation is sustained, whereas that of PKD2 is transient in intestinal epithelial IEC-18 stimulated with the G(q)-coupled receptor agonists angiotensin II or vasopressin. PKD1 gene silencing utilizing small interfering RNAs dramatically reduced DNA synthesis and cell proliferation in IEC-18 cells stimulated with G(q)-coupled receptor agonists. To clarify the role of PKD1 in intestinal epithelial cell proliferation in vivo, we generated transgenic mice that express elevated PKD1 protein in the intestinal epithelium. Transgenic PKD1 exhibited constitutive catalytic activity and phosphorylation at the activation loop residues Ser(744) and Ser(748) and on the autophosphorylation site, Ser(916). To examine whether PKD1 expression stimulates intestinal cell proliferation, we determined the rate of crypt cell DNA synthesis by detection of 5-bromo-2-deoxyuridine incorporated into the nuclei of crypt cells of the ileum. Our results demonstrate a significant increase (p < 0.005) in DNA-synthesizing cells in the crypts of two independent lines of PKD1 transgenic mice as compared with non-transgenic littermates. Morphometric analysis showed a significant increase in the length and in the total number of cells per crypt in the transgenic PKD1 mice as compared with the non-transgenic littermates (p < 0.01). Thus, transgenic PKD1 signaling increases the number of cells per crypt by stimulating the rate of crypt cell proliferation. Collectively, our results indicate that PKD1 plays a role in promoting cell proliferation in intestinal epithelial cells both in vitro and in vivo.     

Protein kinase D isoforms are activated in an agonist-specific manner in cardiomyocytes

Protein kinase D (PKD) exists as a family of structurally related enzymes that are activated through similar phosphorylation-dependent mechanisms involving protein kinase C (PKC). While individual PKD isoforms could in theory mediate distinct biological functions, previous studies identify a high level of functional redundancy for PKD1 and PKD2 in various cellular contexts. This study shows that PKD1 and PKD2 are activated in a stimulus-specific manner in neonatal cardiomyocytes. The α(1)-adrenergic receptor agonist norepinephrine selectively activates PKD1, thrombin and PDGF selectively activate PKD2, and endothelin-1 and PMA activate both PKD1 and PKD2. PKC activity is implicated in the α(1)-adrenergic receptor pathway that activates PKD1 and the thrombin- and PDGF-dependent pathways that activate PKD2. Endothelin-1 activates PKD via both rapid PKC-dependent and more sustained PKC-independent mechanisms. The functional consequences of PKD activation were assessed by tracking phosphorylation of CREB and cardiac troponin I (cTnI), two physiologically relevant PKD substrates in cardiomyocytes. We show that overexpression of an activated PKD1-S744E/S748E transgene increases CREB-Ser(133) and cTnI-Ser(23)/Ser(24) phosphorylation, but agonist-dependent pathways that activate native PKD1 or PKD2 selectively increase CREB-Ser(133) phosphorylation; there is no associated increase in cTnI-Ser(23)/Ser(24) phosphorylation. Gene silencing studies provide unanticipated evidence that PKD1 down-regulation leads to a compensatory increase in PKD2 activity and that down-regulation of PKD1 (alone or in combination with PKD2) leads to an increase in CREB-Ser(133) phosphorylation. Collectively, these studies identify distinct roles for native PKD1 and PKD2 enzymes in stress-dependent pathways that influence cardiac remodeling and the progression of heart failure.     

Protein kinase D regulates RhoA activity via rhotekin phosphorylation

The members of the protein kinase D (PKD) family of serine/threonine kinases are major targets for tumor-promoting phorbol esters, G protein-coupled receptors, and activated protein kinase C isoforms (PKCs). The expanding list of cellular processes in which PKDs exert their function via phosphorylation of various substrates include proliferation, apoptosis, migration, angiogenesis, and vesicle trafficking. Therefore, identification of novel PKD substrates is necessary to understand the profound role of this kinase family in signal transduction. Here, we show that rhotekin, an effector of RhoA GTPase, is a novel substrate of PKD. We identified Ser-435 in rhotekin as the potential site targeted by PKD in vivo. Expression of a phosphomimetic S435E rhotekin mutant resulted in an increase of endogenous active RhoA GTPase levels. Phosphorylation of rhotekin by PKD2 modulates the anchoring of the RhoA in the plasma membrane. Consequently, the S435E rhotekin mutant displayed enhanced stress fiber formation when expressed in serum-starved fibroblasts. Our data thus identify a novel role of PKD as a regulator of RhoA activity and actin stress fiber formation through phosphorylation of rhotekin.     

Regulatory domain determinants that control PKD1 activity

The canonical pathway for protein kinase D1 (PKD1) activation by growth factor receptors involves diacylglycerol binding to the C1 domain and protein kinase C-dependent phosphorylation at the activation loop. PKD1 then autophosphorylates at Ser(916), a modification frequently used as a surrogate marker of PKD1 activity. PKD1 also is cleaved by caspase-3 at a site in the C1-PH interdomain during apoptosis; the functional consequences of this cleavage event remain uncertain. This study shows that PKD1-Δ1-321 (an N-terminal deletion mutant lacking the C1 domain and flanking sequence that models the catalytic fragment that accumulates during apoptosis) and PKD1-CD (the isolated catalytic domain) display high basal Ser(916) autocatalytic activity and robust activity toward CREBtide (a peptide substrate) but little to no activation loop autophosphorylation and no associated activity toward protein substrates, such as cAMP-response element binding protein and cardiac troponin I. In contrast, PKD1-ΔPH (a PH domain deletion mutant) is recovered as a constitutively active enzyme, with high basal autocatalytic activity and high basal activity toward peptide and protein substrates. These results indicate that individual regions in the regulatory domain act in a distinct manner to control PKD1 activity. Finally, cell-based studies show that PKD1-Δ1-321 does not substitute for WT-PKD1 as an in vivo activator of cAMP-response element binding protein and ERK phosphorylation. Proteolytic events that remove the C1 domain (but not the autoinhibitory PH domain) limit maximal PKD1 activity toward physiologically relevant protein substrates and lead to a defect in PKD1-dependent cellular responses.     

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.     

Tyrosine hydroxylase phosphorylation in bovine adrenal chromaffin cells: the role of MAPKs after angiotensin II stimulation

Angiotensin II (AII, 100 nM) stimulation of bovine adrenal chromaffin cells (BACCs) produced angiotensin II receptor subtype 1 (AT1)-mediated increases in extracellular regulated protein kinase 1/2 (ERK1/2) and stress-activated p38MAPK (p38 kinase) phosphorylation over a period of 10 min. ERK1/2 and p38 kinase phosphorylation preceded Ser31 phosphorylation on tyrosine hydroxylase (TOH). The inhibitors of mitogen-activated protein kinase kinase 1/2 (MEK1/2) activation, PD98059 (0.1-50 microM) and UO126 (0.1-10 microM), dose-dependently inhibited both ERK2 and Ser31 phosphorylation on TOH in response to AII, suggesting MEK1/2 involvement. The p38 kinase inhibitor SB203580 (20 microM, 30 min) abolished Ser31 and Ser19 phosphorylation on TOH and partially inhibited ERK2 phosphorylation produced by AII. In contrast, 1 microM SB203580 did not affect AII-stimulated TOH phosphorylation, but fully inhibited heat shock protein 27 (HSP27) phosphorylation produced by AII. Also, 1 microM SB203580 fully inhibited Ser19 phosphorylation on TOH and HSP27 phosphorylation in response to anisomycin (30 min, 10 microg/mL). The results suggest that ERKs mediate Ser31 phosphorylation on TOH in response to AII, but p38 kinase is not involved. Previous studies suggesting a role for p38 kinase in the phosphorylation of Ser31 are explained by the non-specific effects of 20 microM SB203580 in BACCs. The p38 kinase pathway is able to phosphorylate Ser19 on TOH in response to anisomycin, but does not do so in response to AII.     

Regulation of the Nuclear–Cytoplasmic Traffic of Class IIa Histone Deacethylases in Rat Soleus Muscle at the Early Stage of Gravitational Unloading

The negative regulation of expression of genes involved in various metabolic pathways in a skeletal muscle is the main function of histone deacetylases 4 and 5 (HDAC4/HDAC5). HDAC4 and HDAC5 seem to be the targets of the AMP-activated protein kinase (AMPK). Earlier, an essential decrease in the level of Thr172-phosphorylated-AMPK in a rat soleus muscle at the first day of gravitational unloading was shown. Possibility of a protein kinase D (PKD) to phosphorylate histone deacetylases 4/5 has been shown, too. We supposed that under the conditions of gravitational unloading, alterations in AMPK phosphorylation level can affect regulation of nuclear-cytoplasmic traffic of class II histone deacetylases and of various skeletal muscle genes expression. To verify the hypothesis, we used administration of an AMPK activator, AICAR, before and during a day-long hindlimb suspension. It was shown that at an early stage of gravitational unloading, HDAC4 is not a PKD target, and its nuclear import is realized due to decrease in AMPK activity. We were the first to show reciprocal relations between AMPK and PKD in a skeletal muscle at early gravitational unloading.     

Uncoupling of protein kinase D from suppression of EGF-dependent c-Jun phosphorylation in cancer cells

Protein kinase D (PKD) has been established as a negative modulator of the c-Jun N-terminal kinase (JNK) signaling pathway. We previously demonstrated that induced expression of constitutively active PKD (PKD-S744/748E) that mimics phosphorylation by PKC is sufficient to attenuate epidermal growth factor (EGF) stimulated c-Jun Ser 63 phosphorylation, a natural substrate of JNK, in HEK 293 cells. Because the JNK pathway has been implicated in sustaining both lung and pancreatic cancerous phenotypes, we have utilized stable inducible expression of PKD-S744/748E in clones of A549 non-small cell lung cancer (NSCLC) and Panc1, pancreatic cancer cells to determine its effects on JNK signaling in the context of the cancerous phenotype. In contrast to HEK 293 cells, induced expression of PKD-S744/748E in either A549 NSCLC or Panc1 cells failed to attenuate EGF dependent phosphorylation of c-Jun, indicating that EGF stimulated JNK phosphorylation of c-Jun is uncoupled from PKD suppression in these cancer cells.