Stimulation Of Protein Kinase C Research Articles

Differential activation of enkephalin, galanin, somatostatin, NPY, and VIP neuropeptide production by stimulators of protein kinases A and C in neuroendocrine chromaffin cells

Neuropeptides function as peptide neurotransmitters and hormones to mediate cell-cell communication. The goal of this study was to understand how different neuropeptides may be similarly or differentially regulated by protein kinase A (PKA) and protein kinase C (PKC) intracellular signaling mechanisms. Therefore, this study compared the differential effects of treating neuroendocrine chromaffin cells with stimulators of PKA and PKC on the production of the neuropeptides (Met)enkephalin, galanin, somatostatin, NPY, and VIP. Significantly, selective increases in production of these neuropeptides were observed by forskolin or phorbol myristate acetate (PMA) which stimulate PKA and PKC mechanisms, respectively. (Met)enkephalin production was stimulated by up to 2-fold by forskolin treatment, but not by PMA. In contrast, PMA treatment (but not forskolin) resulted in a 2-fold increase in production of galanin and somatostatin, and a 3-fold increase in NPY production. Notably, VIP production was highly stimulated by forskolin and PMA, with increases of 3-fold and 10-15-fold, respectively. Differences in elevated neuropeptides occurred in cell extracts compared to secretion media, which consisted of (i) increased NPY primarily in secretion media, (ii) increased (Met)enkephalin and somatostatin in secretion media (not cell extracts), and (iii) increased galanin and VIP in both cell extracts and secretion media. Involvement of PKA or PKC for forskolin or PMA regulation of neuropeptide biosynthesis, respectively, was confirmed with direct inhibitors of PKA and PKC. The selective activation of neuropeptide production by forskolin and PMA demonstrates that PKA and PKC pathways are involved in the differential regulation of neuropeptide production.

Dopamine transporter endocytic determinants: Carboxy terminal residues critical for basal and PKC-stimulated internalization

Dopamine (DA) reuptake terminates dopaminergic neurotransmission and is mediated by DA transporters (DATs). Acute protein kinase C (PKC) activation accelerates DAT internalization rates, thereby reducing DAT surface expression. Basal DAT endocytosis and PKC-stimulated DAT functional downregulation rely on residues within the 587–596 region, although whether PKC-induced DAT downregulation reflects transporter endocytosis mechanisms linked to those controlling basal endocytosis rates is unknown. Here, we define residues governing basal and PKC-stimulated DAT endocytosis. Alanine substituting DAT residues 587–590 1) abolished PKC stimulation of DAT endocytosis, and 2) markedly accelerated basal DAT internalization, comparable to that of wildtype DAT during PKC activation. Accelerated basal DAT internalization relied specifically on residues 588–590, which are highly conserved among SLC6 neurotransmitter transporters. Our results support a model whereby residues within the 587–590 stretch may serve as a locus for a PKC-sensitive braking mechanism that tempers basal DAT internalization rates.

Presenilin regulates extracellular regulated kinase (Erk) activity by a protein kinase C alpha dependent mechanism

Presenilin (PS1 and PS2) mutations cause early-onset familial Alzheimer's disease (AD). In addition to affecting beta-amyloid precursor protein (APP) processing and Abeta generation, PSs regulate a number of signaling pathways. We previously showed that PSs regulate both phospholipase C (PLC) and protein kinase C (PKC) alpha and gamma activities. We also reported that PS double knockout mouse embryonic fibroblasts (MEFs) have reduced levels of PKCalpha and enhanced levels of PKCdelta. Here, we determined whether the PS modulation of PLC/PKC has consequences for extracellular regulated kinase (Erk) signaling. Erk has been suggested to be important in AD pathology by modulating APP processing and tau phosphorylation. We found that knocking out PS1 or PS2 alone resulted in increased Erk activity and that this effect could be reversed by the PKCalpha inhibitor Gö6976. We also found that Erk activity following either PLC or PKC stimulation was significantly lower in PS double knockout cells and that treatment with the PKC activator phorbol 12,13-dibutyrate (PdBu) down-regulated total-Erk levels in all cells except PS double knockouts. These results demonstrate that PSs regulate Erk activity through a PKCalpha dependent pathway and that disruption of PLC/PKC signaling in the absence of both PS1 and PS2 results in lower downstream activation of Erk.

The last residue of the Na, K‐ATPase catalytic subunit isoform specific region (ISR) plays a critical role in Na, K‐ATPase membrane trafficking

The Isoform Specific Region (ISR) is a region of structural heterogeneity among the 4 isoforms of the Na, K‐ATPase α‐subunit. Replacing α1 ISR (K489NPNASEPKHL499) by α2 ISR sequence (E487REDSPQS‐HV496) results in a doubled response to PKC, suggesting that the ISR participates in isoform‐specific PKC regulation. PKC regulation of α1 involves membrane recycling via clathrin‐coated vesicles. In α1, L499 is immediately followed by another L, making it a potential di‐Leucine (LL) target motif for clathrin adaptor proteins. Replacing α1 ISR by α2 ISR disrupts this LL motif. To test whether this explains the change in PKC regulation observed when tampering with ISR, we compared a L499V mutant of α1 to the full length α1 in a model of heterologous expression of rat constructs in opossum kidney cells. In the absence of PKC stimulation, total expression and Na, K‐ATPase mediated 86Rb+ uptake were comparable in α1L499V and α1 expressing cells. However, biotinylation experiments revealed a higher surface expression of the mutant, suggesting that the mutation affected the steady state level of surface expression of the protein. Upon PKC stimulation, activation of Na, K‐ATPase mediated 86Rb+ uptake was doubled in mutant‐expressing cells. This suggests that the last residue of the ISR plays a critical role in Na,K‐ATPase isoform‐specific response to PKC activation and in steady‐state Na,K‐ATPase expression at the cell surface.

Protective Effects of K6PC-5, A Sphingosine Kinase Activator, Against 1-methyl-4-phenylpyridinium-induced Dopaminergic Neuronal Cell Death

Sphingosine-1-phosphate (S1P), a bioactive sphingolipid metaolite, regulates multiple cellular responses such as Ca signaling, cellular growth and survival, and differentiation. Sphingosine kinase (SphK) is a key enzyme in modulating the levels of S1P and is emerging as an important regulating enzyme. Here we have investigated whether K6PC-5, a newly synthesized SphK activator, plays a neuroprotective role by activating cell survival systems such as protein kinase C, phosphatidylinositol-3-kinase (PI3K), and acting on the anti-apoptotic and the pro-apoptotic genes in SN4741 dopaminergic cells. 1-methyl-4-phenylpyridinium is a neurotoxin that selectively inhibits the mitochondrial functions of dopaminergic neurons in the substantia nigra pars compacta. In the present study, we found that MPP induced a decrease in SN4741 mouse dopaminergic cell viability. K6PC-5 restored the reduced phospho-PKC and phospho-PI3K activities caused by MPP + toxicity. In addition, gene expression analysis revealed that K6PC-5 prevented both the MPP + induced expression of the pro-apoptotic gene mRNA, Bax, and the decrease of the anti-apoptotic gene mRNA, bcl-w. These results suggest that the neuroprotective mechanism of K6PC-5 against MPP-induced apoptotic cell death includes stimulation of PKC and PI3K, and modulation of cell survival and death genes.

Renoprotection with vitamin D: Specific for diabetic nephropathy?

Zhang and co-workers report on the renoprotective role of the vitamin D receptor (VDR) in diabetic nephropathy using the method of streptozotocin-induced hyperglycemia in wild-type and VDR(-/-) mice. Also, experiments with cultured mesangial cells and podocytes confirm the effect of the active vitamin D metabolite 1,25(OH)(2)D(3) on inhibition of the renin-angiotensin system (RAS) in vitro. The authors conclude that the higher activation of the intrarenal RAS is the key factor to induce more severe diabetic nephropathy in VDR(-/-) mice.

Mice Lacking Pleckstrin or Pleckstrin-2 Each Have Unique Platelet Secretion Defects.

Approximately 1% of total cellular protein within platelets and leukocytes is pleckstrin, a protein best known for containing the two prototypic Pleckstrin Homology (PH) domains. Following platelet activation, PKC rapidly phosphorylates pleckstrin, inducing it to bind membrane bound phospholipids such as phosphatidylinositol 4,5 bisphosphate (PIP2). Platelets also contain a widely expressed paralog of pleckstrin, called pleckstrin-2. Although the activity of pleckstrin is regulated through protein phosphorylation, pleckstrin-2 is not a phosphoprotein, but is instead activated by binding a specific PI3K generated phospholipid, phosphatidylinositol 3,4 bisphosphate (PI3,4P2). To define the role of individual pleckstrin isoforms in vivo, we generated mice containing loss of function mutations within either the pleckstrin or pleckstrin-2 genes. Mice lacking either isoform exhibited no spontaneous hemorrhage. Studies of pleckstrin knockout platelets demonstrated that they displayed normal aggregation and secretion in response to collagen and thrombin. In contrast, pleckstrin null platelets had no secretion of dense or alpha granules following exposure to the PKC stimulant, PMA. This demonstrates that pleckstrin is the critical effector for PKC-mediated platelet secretion. Our findings also suggest that an alternative pathway in pleckstrin knockout platelets is able to compensate for the secretion defect when the cells are stimulated with an agonist such as thrombin. We reasoned that the compensatory pathway might utilize another PIP2-dependent second messenger. Since the phosphorylation of PIP2 by PI3K generates second messengers that also contribute to platelet secretion, we tested whether a PI3K-dependent pathway compensated for the loss of pleckstrin. We found that pleckstrin knockout platelets completely failed to secrete in response to stimulation of the thrombin receptor in the presence of a PI3K inhibitors (LY294002 or wortmannin.) This demonstrates that second messengers generated by PI3K are able to compensate for the secretion defect induced by the loss of pleckstrin. In contrast to the findings observed with pleckstrin knockout platelets, we have found that platelets lacking pleckstrin-2 aggregated and secreted normally in response to thrombin, collagen, and PMA. Additionally, unlike the effect seen on pleckstrin knockout platelets, inhibitors of PI3K had no effect on the aggregation or secretion of pleckstrin-2 knockout platelets. However, again in contrast to pleckstrin knockout platelets, pleckstrin-2 null platelets failed to secrete in response to thrombin when they were exposed to inhibitors of either PLC or PKC. This demonstrates that pleckstrin-2 knockout platelets compensate for their secretion defect by a pathway dependent on PLC and PKC. It is notable that PI3K or PKC inhibitors only minimally affected the thrombin-induced secretion of wild-type platelets unless both inhibitors were used together. Together, our results show that both pleckstrin and pleckstrin-2 are essential components of thrombin-mediated platelet secretion. These data also demonstrate that thrombin-induced platelet secretion can be mediated by one of two parallel pathways, the first involving PKC and pleckstrin, and the second involving PI3K and pleckstrin-2.

Modulation of the human equilibrative nucleoside transporter1 (hENT1) activity by IL-4 and PMA in B cells from chronic lymphocytic leukemia

Nucleoside transporters (NTs) are essential for the uptake of therapeutic nucleoside analogs, broadly used in cancer treatment. The mechanisms responsible for NT regulation are largely unknown. IL-4 is a pro-survival signal for chronic lymphocytic leukemia (CLL) cells and has been shown to confer resistance to nucleoside analogs. The aim of this study was to investigate whether IL-4 is able to modulate the expression and function of the human equilibrative NT1 (hENT1) in primary cultures of CLL cells and, consequently, to affect cytotoxicity induced by therapeutic nucleosides analogs. We found that treatment with IL-4 (20ng/ml for 24h) increased mRNA hENT1 expression in CLL cells without affecting that of normal B cells. Given that the enhanced mRNA levels of hENT1 in CLL cells did not result in increased transport activity, we examined the possibility that hENT1 induced by IL-4 may require post-translational modifications to become active. We found that the acute stimulation of PKC in IL-4-treated CLL cells by short-term incubation with PMA significantly increased hENT1 transport activity and favoured fludarabine-induced apoptosis. By contrast, and in line with previous reports, IL-4 plus PMA protected CLL cells from a variety of cytotoxic agents. Our findings indicate that the combined treatment with IL-4 and PMA enhances hENT1 activity and specifically sensitizes CLL cells to undergo apoptosis induced by fludarabine.

Oxytocin-Induced Activation of Eukaryotic Elongation Factor 2 in Myometrial Cells Is Mediated by Protein Kinase C

The nonapeptide oxytocin (OT) mediates a wide spectrum of biological action, many of them related to reproduction. Recently, we have shown that OT exerts a trophic effect on uterine smooth muscle cells and induces dephosphorylation, and thus activation, of the translation elongation factor eukaryotic elongation factor 2 (eEF2). The present study was designed to elucidate the mechanisms underlying this novel action of OT in the well-characterized human myometrial cell line hTERT-C3. Pathways known to induce eEF2 dephosphorylation are mammalian target of rapamycin (mTOR), and the MAPKs ERK1/2 and p38. Using a panel of chemical inhibitors of specific signaling pathways, we determined that none of these pathways played a role in OT-mediated eEF2 dephosphorylation. Because the OT receptor is a G protein-coupled receptor linked to Galphaq, we tested the possibility that this OT action was mediated via protein kinase C (PKC). PKC activity was blocked by application of the general PKC chemical inhibitor Go6983 or by incubation with the cell-permeable PKC inhibitor peptide myr-psi PKC. With either approach, the effect of OT on eEF2 dephosphorylation was suppressed, indicating that the PKC pathway is essential for this OT action. Consistent with this idea, we also found that direct stimulation of PKC with the phorbol ester phorbol 12-myristate 13-acetate induced eEF2 dephosphorylation. Moreover, we observed that the stimulatory effect of OT on [(35)S]methionine incorporation into nascent proteins was blocked by PKC inhibition. Overall, these results define a novel hormonal signaling pathway that leads to eEF2 dephosphorylation and activation of protein synthesis.

Abstract 388: Postconditioning Beyond The Heart: Postconditioning The Ischemic Kidney Attenuates Renal Reperfusion Injury

Objective : Postconditioning (Poc), defined as brief cycles of arterial occlusion and reflow applied at the onset of reperfusion (R), has proven to reduce multiple consequences of myocardial ischemia-reperfusion injury (I/R). The protective effects of Poc in the kidney I/R are not known. Therefore, we tested the hypothesis that Poc attenuates renal I/R injury via adenosine receptor (AR) activation and protein kinase C (PKC) signaling. Methods : The in vivo single kidney rat model was used. Rats underwent right nephrectomy and were randomized to 5 groups. A sham group (n=8) underwent the surgical and perfusion protocol without other interventions. In all remaining groups, the left renal artery is occluded for 45min and the left kidney was reperfused for 24 hours. Control group (n=8) received no intervention at R; Poc (n=8) underwent 4 cycles of 45 seconds R and 45 seconds of reocclusion at the onset of R. PC + ARi (n=6): the AR inhibitor 8-SPT (10mg/kg) was administered i.v. 5 minutes before Poc; Poc + PKCi: the PKC inhibitor, chelerytherine (5mg/kg), was administered i.v. 5 min before R. After 24 hours, renal function was assessed by plasma blood urea nitrogen (BUN) and creatinine (Cr), and the kidneys were harvested; apoptotic cells were quantified by TUNEL stain and morphological injury by H&E staining. Results: Compared to sham, BUN and Cr were greater in control I/R group, which was significantly reduced in Poc (table ). Inhibition of AR or PKC stimulation reversed the reductions in BUN and Cr achieved by Poc. In addition, renal I/R increased the percentage of TUNEL positive cells in ten high powered fields vs Sham, which was reduced by Poc. Blockade of AR and PKC reversed the apoptosis reduction achieved by Poc. Conclusion: Postconditioning attenuates renal dysfunction and apoptosis induced by R injury and involves AR and PKC signaling. Poc suggests that R injury is triggered in early moments of reflow.

Effects of protein kinase C activation on cardiac repolarization and arrhythmogenesis in Langendorff-perfused rabbit hearts

Cardiac arrhythmias are still a major cause of mortality in western countries. Currently available antiarrhythmic drugs are limited by a low efficacy and proarrhythmic effects. The role of the protein kinase C (PKC) signalling pathway in arrhythmogenesis is still unclear. The goal of the present study was to test the effects of PKC stimulation on whole heart electrophysiology and its pro-/antiarrhythmic activity. Left ventricular (LV) action potential duration (APD 90%) was determined in 27 Langendorff-perfused rabbit hearts, using Tyrode solution plus the PKC agonist phorbol-12-myristate-13-acetate (PMA; 100 nM) alone (nine rabbits), Verapamil alone (n = 6), or PMA in combination with Verapamil (0.25 mg/L, six rabbits), or bisindolylmaleimide (0.5 microM, n = 6). Intermittent programmed extra-stimulation was performed to induce ventricular arrhythmias. Administration of PMA alone led to a significant shortening of repolarization (APD 90%, 157 +/- 8 vs. 128 +/- 5 ms, P<0.05). Non-sustained ventricular fibrillation (VF) could be induced in seven out of nine animals. After perfusion of Verapamil (156 +/- 6 vs. 169 +/- 4 ms, P>0.05) or bisindolylmaleimide, a selective inhibitor of PKC (136 +/- 4 vs. 146 +/- 4 ms, P>0.05), PMA-induced shortening of repolarization could be inhibited, and induction of VF failed. Verapamil alone did not affect APD and VF could not be induced. Activation of PKC facilitates induction of VF, which is most likely due to a shortening of repolarization and a prominent calcium influx. These findings demonstrate involvement of the PKC-signalling pathway in arrhythmogenesis.

Direct Protein Kinase C-dependent Phosphorylation Regulates the Cell Surface Stability and Activity of the Potassium Chloride Cotransporter KCC2

The potassium chloride cotransporter KCC2 plays a major role in the maintenance of transmembrane chloride potential in mature neurons; thus KCC2 activity is critical for hyperpolarizing membrane currents generated upon the activation of gamma-aminobutyric acid type A and glycine (Gly) receptors that underlie fast synaptic inhibition in the adult central nervous system. However, to date an understanding of the cellular mechanism that neurons use to modulate the functional expression of KCC2 remains rudimentary. Using Escherichia coli expression coupled with in vitro kinase assays, we first established that protein kinase C (PKC) can directly phosphorylate serine 940 (Ser(940)) within the C-terminal cytoplasmic domain of KCC2. We further demonstrated that Ser(940) is the major site for PKC-dependent phosphorylation for full-length KCC2 molecules when expressed in HEK-293 cells. Phosphorylation of Ser(940) increased the cell surface stability of KCC2 in this system by decreasing its rate of internalization from the plasma membrane. Coincident phosphorylation of Ser(940) increased the rate of ion transport by KCC2. It was further evident that phosphorylation of endogenous KCC2 in cultured hippocampal neurons is regulated by PKC-dependent activity. Moreover, in keeping with our recombinant studies, enhancing PKC-dependent phosphorylation increased the targeting of KCC2 to the neuronal cell surface. Our studies thus suggest that PKC-dependent phosphorylation of KCC2 may play a central role in modulating both the functional expression of this critical transporter in the brain and the strength of synaptic inhibition.

PKC-dependent autoregulation of membrane kainate receptors

Agonists of kainate receptors (KARs) cause both the opening of the associated ion channels and the activation of signalling pathways driven by G-proteins and PKC. Here we report the existence of an unknown mechanism of KAR autoregulation, involving the interplay of this two signalling mechanisms. Repetitive activation of native KARs evoked the rundown of the ionotropic responses in a manner that was dependent on the activation of PKC. Experiments on recombinant GluR5 expressed in neuroblastoma cells indicated that KARs trigger the activation of PKC and induce the internalization of membrane receptors. This phenomenon depends on the PKC-mediated phosphorylation of serines 879 and 885 of the GluR5-2b subunits, since mutation of these two residues abolished internalization. These results reveal that the non-canonical signalling of KARs is associated with a sensitive mechanism that detects afferent activity. Such a mechanism represents an active way to limit overactivation of the KAR system, by regulating the number of KARs in the cell membrane.

Aquaporin-1 Channel Function Is Positively Regulated by Protein Kinase C

Aquaporin-1 (AQP1) channels contribute to osmotically induced water transport in several organs including the kidney and serosal membranes such as the peritoneum and the pleura. In addition, AQP1 channels have been shown to conduct cationic currents upon stimulation by cyclic nucleotides. To date, the short term regulation of AQP1 function by other major intracellular signaling pathways has not been studied. In the present study, we therefore investigated the regulation of AQP1 by protein kinase C. AQP1 wild type channels were expressed in Xenopus oocytes. Water permeability was assessed by hypotonic challenges. Activation of protein kinase C (PKC) by 1-oleoyl-2-acetyl-sn-glycerol (OAG) induced a marked increase of AQP1-dependent water permeability. This regulation was abolished in mutated AQP1 channels lacking both consensus PKC phosphorylation sites Thr(157) and Thr(239) (termed AQP1 DeltaPKC). AQP1 cationic currents measured with double-electrode voltage clamp were markedly increased after pharmacological activation of PKC by either OAG or phorbol 12-myristate 13-acetate. Deletion of either Thr(157) or Thr(239) caused a marked attenuation of PKC-dependent current increases, and deletion of both phosphorylation sites in AQP1 DeltaPKC channels abolished the effect. In vitro phosphorylation studies with synthesized peptides corresponding to amino acids 154-168 and 236-250 revealed that both Thr(157) and Thr(239) are phosphorylated by PKC. Upon stimulation by cyclic nucleotides, AQP1 wild type currents exhibited a strong activation. This regulation was not affected after deletion of PKC phosphorylation sites in AQP1 DeltaPKC channels. In conclusion, this is the first study to show that PKC positively regulates both water permeability and ionic conductance of AQP1 channels. This new pathway of AQP1 regulation is independent of the previously described cyclic nucleotide pathway and may contribute to the PKC stimulation of AQP1-modulated processes such as endothelial permeability, angiogenesis, and urine concentration.

Protein kinase C (PKC) role in bovine oocyte maturation and early embryo development

The aims of the present study were to determine the role of protein kinase C (PKC) on meiotic resumption and its effects on pronuclear formation and cleavage in the bovine. Oocytes were matured in the presence of 0, 1, 10 and 100 nM of phorbol 12-myristate 13-acetate (PMA), to evaluate the percentage of germinal vesicle breakdown. To study pronuclear formation and cleavage, oocytes were randomly distributed in four groups and matured in modified TCM-199 with LH and FSH (negative control); 10% of estrous cow serum (positive control); 100 nM of PMA (treatment); 100 nM of 4α-PDD (phorbol ester control). Oocytes were also matured in positive control medium, fertilized and transferred to KSOM with increasing concentrations of a PKC inhibitor. The protein profile and the presence of PKC at the end of maturation period were determined by SDS-PAGE followed by Silver Stain and Western blot, respectively. PMA stimulated meiotic resumption in a concentration-dependent manner. PKC stimulation during oocyte maturation caused an increase in pronuclear formation and did not cause parthenogenetic activation. Inhibitor of PKC (MyrPKC) inhibited cleavage in a dose-dependent and irreversible manner. A protein band around 74 kDa was not detected in PMA-treated oocytes and PKC was not detected by Western blot at the end of the maturation period. In conclusion, meiotic resumption was accelerated and the rate of oocytes with two pronuclei was increased when PKC was activated during oocyte maturation. Moreover, cleavage was inhibited in the presence of PMA.

Modulation of hERG potassium currents in HEK‐293 cells by protein kinase C. Evidence for direct phosphorylation of pore forming subunits

The human ether-a-go-go related gene (hERG) potassium channel is expressed in a variety of tissues including the heart, neurons and some cancer cells. hERG channels are modulated by several intracellular signalling pathways and these provide important mechanisms for regulating cellular excitability. In this study, we investigated muscarinic modulation of hERG currents and direct phosphorylation of channel subunits expressed in HEK-293 cells at physiologically relevant temperatures by protein kinase C (PKC). Activation of G(alpha q/11)-coupled M(3)-muscarinic receptors with methacholine, reduced current amplitudes at all potentials with minor effects on the voltage dependence of activation and inactivation. The response to methacholine was insensitive to intracellular BAPTA, but was attenuated by either acute inhibition of PKC with 300 nm bisindolylmaleimide-1 (bis-1) or chronic down-regulation of PKC isoforms by 24 h pretreatment of cells with phorbol 12-myristate 13-acetate (PMA). Stimulation of PKC with 1-oleoyl 2-acetylglycerol (OAG), an analogue of diacylglycerol (DAG), mimicked the actions of muscarinic receptor stimulation. Direct phosphorylation of hERG was measured by [(32)P]orthophosphate labelling of immunoprecipitated protein with an anti-hERG antibody. Basal phosphorylation was high in unstimulated cells and further increased by OAG. The OAG dependent increase was abolished by bis-1 and down-regulation of PKC, but basal levels of phosphorylation were unchanged. Deletion of the amino-terminus of hERG prevented both the modulation of channel activity and the increase of phosphorylation by OAG. Our results are consistent with calcium and/or DAG sensitive isotypes of PKC modulating hERG currents through a mechanism that involves direct phosphorylation of sites on the amino terminus of hERG.

Effectof Pkc Stimulation on Sodium Chloride Cotransporter Function and Surface Expression in Mammalian Cells

Restricted accessAbstractFirst published March 2007Effectof Pkc Stimulation on Sodium Chloride Cotransporter Function and Surface Expression in Mammalian CellsB. Ko, L. Joshi, […], L. Cooke, M. Musch, and R.S. Hoover+2-2View all authors and affiliationsVolume 55, Issue 2https://doi.org/10.1177/108155890705500249

Protein Kinase C regulation of the Na, K‐ATPase α3 isoform in highly metastatic human prostate cells

The Na,K-ATPase is a membrane spanning protein complex that plays a central role in maintaining cellular homeostasis via the exchange of Na+ and K+ ions while consuming energy in the form of cellular ATP. The catalytic subunit of the pump exists in four isoforms (α1, α2, α3, and α4) of which the physiological significance is unknown. One distinction between the α-isoforms may be their regulation by Protein Kinase C (PKC). In many cell types, the α1-isoform typically responds with increased transport when treated with the PKC activator phorbol myristate acetate (PMA). This also appears to be the situation for human prostate cells in culture. In a highly metastatic line (PC3), however, the response of the ATPase-mediated transport to PKC activation over a five minute period is a decrease in activity (−16%). Moreover, PC3 cells express both the α1- and α3-isoforms, but it is unclear which isoforms are being regulated. To address this issue, we expressed exogenous α3 in PC3 cells under conditions in which the endogenous isoforms were inhibited with ouabain. When challenged with PMA, the transfected PC3 cells displayed a decrease in transport indistinguishable from that of the endogenous enzymes (−19%). This indicates that the α3 does indeed respond to PKC stimulation. In addition, it is tempting to suggest that the metastatic state of the PC3 cells may contribute to the altered regulatory response to PKC stimulation. [This project was supported in part by NIH RR-19799 and a Howard Hughes Medical Institute grant through the Undergraduate Science Education Program to Texas Tech University.]

Knockout of the PKC Substrate Pleckstrin Causes Pleomorphic Defects in Platelets, Lymphocytes and Granulocytes.

Pleckstrin is a 40 kDa phosphoprotein containing amino- and carboxyl-terminal Pleckstrin Homology (PH) domains separated by a DEP domain. Pleckstrin's expression is restricted to platelets and leukocytes, and represents approximately 1% of total cellular protein within these cells. Following platelet and leukocyte activation, PKC rapidly phosphorylates pleckstrin inducing it to bind membrane bound phospholipids such as phosphatidylinositol 4,5 bisphosphate (PIP2). Heterologously expressed phosphorylated pleckstrin colocalized with integrins and induces cytoskeletal reorganization. To better define the role of pleckstrin in vivo, we introduced a loss-of-function mutation into the murine pleckstrin gene. Pleckstrin-null mice were present in offspring at a frequency consistent with a Mendelian inheritance pattern. Adult pleckstrin −/− mice had 32% lower platelet counts than their littermates, but exhibited no spontaneous hemorrhage. Given the role of PKC and phospholipid second messengers on cytoskeletal dynamics, and our observations of pleckstrin overexpression in cell lines, we analyzed whether loss of pleckstrin affected cell spreading. Pleckstrin −/− platelets spread extremely poorly upon immobilized fibrinogen, and rarely exhibited broad membrane extensions. Granulocytes from pleckstrin −/− mice also have a spreading defect, as well as impaired ability to generate reactive oxygen species in the response to TNFα. Knockout B-cells, CD4-T-cells, and CD8-T-cells all migrated approximately 30% as efficiently as wild type cells in response to a gradient of SDF-1α in a transwell assay. These data suggest that loss of pleckstrin causes cytoskeletal defects in cells of multiple hematopoietic lineages. Analyzing whether this caused a functional defect, we found that pleckstrin −/− platelets exhibited a 22% dense- and 24% alpha-granule exocytosis defect, and a 35% defect in thrombin-induced calcium entry. In spite of these abnormalities, platelets changed shape and aggregated normally after stimulation with thrombin, ADP, or collagen in vitro. Pleckstrin knockout platelets did have a markedly impaired aggregation response following exposure to the PKC stimulant, PMA. This suggested that pleckstrin is a critical effector for PKC-mediated aggregation, but another pathway is able to compensate for this loss of pleckstrin following agonist stimulation. We reasoned that the alternative pathway might also utilize PIP2-dependent second messengers. Since the phosphorylation of PIP2 by PI3K generates second messengers that also contribute to platelet aggregation, we tested whether PI3K compensated for the loss of pleckstrin. We found that the PI3K inhibitor, LY294002 profoundly impaired the aggregation of pleckstrin knockout platelets in response to stimulation of the thrombin receptor. In contrast, the PI3K inhibitor minimally affected wild type platelets. This demonstrates that second messengers generated by PI3K are able to compensate for loss of pleckstrin. This also demonstrates that thrombin-induced platelet aggregation can be mediated by one of two parallel pathways, one involving PKC and pleckstrin, and the other involving PI3K. Together, our results show that pleckstrin is an essential component of PKC-mediated platelet activation and signals directed to the cytoskeleton.

TNF-α decreases hsp 27 in human blood mononuclear cells: Involvement of protein kinase c

Treatment of PBMCs with TNF-α decreased the levels of heat shock protein (HSP) 27, but had little effect on the level of HSP70. Parallel to the decrease of HSP27, TNF-α increased the level of HSP27 in the incubation medium of the cells. The decrease of HSP27 induced by TNF-α was suppressed by the pretreatment of PBMCs with the specific protein kinase C (PKC) inhibitor, GF109203X. Furthermore, phorbol myristate acetate (PMA), a PKC stimulant, but not dibutyryl cyclic AMP, a protein kinase A stimulant, decreased the levels of HSP27. To investigate the effect of TNF-α on the oligomerization state of HSP27 in PBMCs, we performed sucrose density gradient centrifugation with subsequent fractionation and immunoassay. Extract of vehicle-treated PBMCs contained mainly dissociated forms of HSP27. The amounts of dissociated forms of HSP27 in PBMCs was decreased by TNF-α, while the amounts of aggregated form of HSP27 was little changed. In intact PBMCs, HSP27 is constitutively phosphorylated at Ser78, but not at Ser15 or at Ser82. The amount of phosphorylated HSP27 at Ser78 was decreased by TNF-α. These results indicate that TNF-α reduces HSP27 in PBMCs through PKC activation. This decrease may be due to efflux of dissociated form of HSP27, phosphorylated HSP27 at Ser78, from the cells.