Protein kinase C mediates platelet secretion and thrombus formation through protein kinase D2

Platelets are highly specialized blood cells critically involved in hemostasis and thrombosis. Members of the protein kinase C (PKC) family have established roles in regulating platelet function and thrombosis, but the molecular mechanisms are not clearly understood. In particular, the conventional PKC isoform, PKCα, is a major regulator of platelet granule secretion, but the molecular pathway from PKCα to secretion is not defined. Protein kinase D (PKD) is a family of 3 kinases activated by PKC, which may represent a step in the PKC signaling pathway to secretion. In the present study, we show that PKD2 is the sole PKD member regulated downstream of PKC in platelets, and that the conventional, but not novel, PKC isoforms provide the upstream signal. Platelets from a gene knock-in mouse in which 2 key phosphorylation sites in PKD2 have been mutated (Ser707Ala/Ser711Ala) show a significant reduction in agonist-induced dense granule secretion, but not in α-granule secretion. This deficiency in dense granule release was responsible for a reduced platelet aggregation and a marked reduction in thrombus formation. Our results show that in the molecular pathway to secretion, PKD2 is a key component of the PKC-mediated pathway to platelet activation and thrombus formation through its selective regulation of dense granule secretion.     

Protein kinase C mediates mutant N-Ras-induced developmental abnormalities in normal human erythroid cells

RAS mutations are one of the most frequent molecular abnormalities associated with myeloid leukemia and preleukemia, yet there is a poor understanding of how they contribute to the pathogenesis of these conditions. Here, we describe the consequences of ectopic mutant N-Ras (N-Ras*) expression on normal human erythropoiesis. We show that during early (erythropoietin [EPO]-independent) erythropoiesis, N-Ras* promoted the amplification of a phenotypically primitive but functionally defective subpopulation of CD34(+) erythroblasts. N-Ras* also up-regulated the expression of megakaryocyte antigens on human erythroblasts. Although early erythroblasts expressing N-Ras* were able to respond to erythropoietin and generate mature progeny, this occurred with greatly reduced efficiency, probably explaining the poor colony growth characteristics of these cells. We further report that this oncogene promoted the expression and activation of protein kinase C (PKC) and that the effects of N-Ras* on erythropoiesis could be abrogated or attenuated by inhibition of PKC. Similarly, the effects of this oncogene could be partially mimicked by treatment with PKC agonist. Together, these data suggest that expression of N-Ras* is able to subvert the normal developmental cues that regulate erythropoiesis by activating PKC. This gives rise to phenotypic and functional abnormalities commonly observed in preleukemia, suggesting a direct link between RAS mutations and the pathogenesis of preleukemia.     

Retinoic acid stimulates neurite outgrowth in the amphibian spinal cord.

There is increasing evidence that retinoic acid (RA), a vitamin A metabolite, plays a role in the development of the nervous system. Here we specifically test this notion by examining the effect of RA on neurite outgrowth from explanted segments of the axolotl spinal cord. We show that there is a threshold concentration in the region of 0.1-1 nM above which neurite outgrowth is stimulated 4-5 fold. Retinol, by contrast, only stimulated the migration of glial cells from the explants. Using HPLC we demonstrate that RA and retinol are present endogenously in the axolotl spinal cord. In addition, we have identified by immunocytochemistry with antipeptide antibodies the cells of the spinal cord that contain the binding proteins for RA (cellular RA-binding protein; CRABP) and retinol (cellular retinol-binding protein; CRBP). CRABP is found in the axons and CRBP is found in the ependyma and glial cells. These results provide strong evidence for a role for RA in the developing nervous system, and we propose a specific hypothesis involving CRBP, CRABP, retinol, and RA in the control of axon outgrowth in the spinal cord.     

Low density lipoprotein receptor-related protein mediates apolipoprotein E-dependent neurite outgrowth in a central nervous system-derived neuronal cell line.

The epsilon 4 allele of apolipoprotein E (apoE) is a major risk factor for Alzheimer disease, suggesting that apoE may directly influence neurons in the aging brain. Recent data suggest that apoE-containing lipoproteins can influence neurite outgrowth in an isoform-specific fashion. The neuronal mediators of apoE effects have not been clarified. We show here that in a central nervous system-derived neuronal cell line, apoE3 but not apoE4 increases neurite extension. The effect of apoE3 was blocked at low nanomolar concentrations by purified 39-kDa protein that regulates ligand binding to the low density lipoprotein receptor-related protein (LRP). Anti-LRP antibody also completely abolished the neurite-promoting effect of apoE3. Understanding isoform-specific cell biological processes mediated by apoE-LRP interactions in central nervous system neurons may provide insight into Alzheimer disease pathogenesis.     

Protein kinase C of smooth muscle.

The primary mechanism of regulation of smooth muscle contraction involves the phosphorylation of myosin catalyzed by Ca2+/calmodulin-dependent myosin light chain kinase. However, additional mechanisms, both Ca(2+)-dependent and Ca(2+)-independent, can modulate the contractile state of smooth muscle. Protein kinase C was first implicated in the regulation of smooth muscle contraction with the observation that phorbol esters induce slowly developing, sustained contractions. Protein kinase C occurs in at least four Ca(2+)-dependent (alpha, beta I, beta II, and gamma) and four Ca(2+)-independent (delta, epsilon, zeta, and eta) isoenzymes. Only the alpha, beta, epsilon, and zeta isoenzymes have been identified in smooth muscle. Both classes of isoenzymes have been implicated in the regulation of smooth muscle contraction. However, the physiologically important protein substrates of protein kinase C have not yet been identified. Specific isoenzymes may be activated by different contractile agonists, and individual isoenzymes exhibit some degree of substrate specificity. Prolonged activation of protein kinase C can result in its proteolysis to the constitutively active catalytic fragment protein kinase M, which would dissociate from the sarcolemma and phosphorylate proteins such as myosin that are inaccessible to membrane-bound protein kinase C. Protein kinase M induces relaxation of demembranated smooth muscle fibers contracted at submaximal Ca2+ concentrations. We suggest that protein kinase C plays two distinct roles in regulating smooth muscle contractility. Stimuli triggering phosphoinositide turnover or phosphatidylcholine hydrolysis induce translocation of protein kinase C (probably specific isoenzymes) to the sarcolemma, phosphorylation of protein, and a slow contraction. Prolonged association of the kinase with the membrane may lead to proteolysis and release into the cytosol of protein kinase M, resulting in myosin phosphorylation and relaxation.     

Protein kinase C epsilon mediates angiotensin II-induced activation of beta1-integrins in cardiac fibroblasts

OBJECTIVE: Angiotensin II (AII) promotes cardiac fibrosis by increased extracellular matrix production and enhanced interaction of matrix proteins with their cellular receptors, including integrins. AII and other growth factors augment beta(1)-integrin-dependent adhesion and spreading by "inside-out signaling" without affecting the total number of integrin receptors. "Inside-out signaling" involves specific signaling pathways, including protein kinase C (PKC), leading to activation of beta1-integrins. In the present study we investigated the mechanisms involved in AII-increased adhesion of adult rat cardiac fibroblasts (CFBs), obtained from Sprague-Dawley rats, to collagen I mediated by beta1-integrin. METHODS AND RESULTS: Treatment of CFBs with AII induced a concentration-dependent increase in adhesion to collagen I (2.2-fold, p<0.01) within 3-6 h of treatment. This effect was mediated by beta1-integrin via the angiotensin AT1 receptor and was significantly reduced by protein kinase C inhibition. AII significantly induced phosphorylation of PKC epsilon (PKCepsilon), which is involved in beta1-integrin-dependent adhesion and motility, and phosphorylation of the cytoplasmatic tail of beta1-integrin at threonine 788/789, required for adhesion. Phosphorylation of beta1-integrin and an increase in adhesion was also induced by l-alpha-phosphatidylinositol-3,4,5-triphosphate (l-alpha-PIP3), an activator of endogenous PKCepsilon. AII failed to increase adhesion in myofibroblasts obtained from PKCepsilon (-/-) mice, but not in cells obtained from control mice. Co-immunoprecipitation and double immunofluorescence demonstrated that AII induced a close association of PKCepsilon with beta1-integrin in CFBs. CONCLUSION: The present study demonstrates that AII increased beta1-integrin-dependent adhesion to collagen I in cardiac fibroblasts by inside-out signaling via PKCepsilon and phosphorylation of the cytoplasmatic tail of the beta1-integrin.     

Protein kinase C mediates insulin-inhibited Ca2+ transport and contraction of vascular smooth muscle

Insulin acutely inhibits contraction of primary cultured vascular smooth muscle (VSM) cells from canine femoral artery by inhibiting contractile agonist-induced Ca²⁺ influx. Insulin also inhibits contraction at step(s) distal to intracellular Ca²⁺ concentration (Ca²⁺_i) by stimulating cyclic guanosine monophosphate (GMP) production. We wished to see whether these effects of insulin are mediated by protein kinase C (PKC). Ca²⁺ influx was assessed by measuring the rate of fluorescence quenching of intracellular fura 2 by extracellular Mn²⁺. We found that 10 μmol/L serotonin (5-HT) stimulated Mn²⁺ influx 3-fold, and 1 nmol/L insulin inhibited the 5-HT–stimulated component of Mn²⁺ influx by 63% (P < .05), but insulin had no effect in the presence of 1 μmol/L staurosporine, an inhibitor of PKC. In the absence of insulin, preincubating cells with 0.1 μmol/L phorbol 12-myristate 13-acetate (PMA) for 5 min inhibited the 5-HT–stimulated component of Mn²⁺ influx by 69% (P < .05). Insulin inhibited cell contraction induced by raising Ca²⁺_i to supraphysiologic levels with ionomycin by 75% (P < .05). We also noted that 10⁻⁶ mol/L calphostin C, another PKC inhibitor, or 16-h preincubation with PMA completely blocked this effect of insulin. Finally, 10-min exposure to insulin or PMA increased cyclic GMP production in ionomycin-treated cells by 50% and 64%, respectively (both P < .05). We conclude that insulin inhibits VSM cell contraction by inhibiting 5-HT–stimulated Ca²⁺ influx and also at step(s) distal to Ca²⁺_i by a PKC-dependent mechanism.     

Protein kinase C stimulatory activity in the pseudopregnant rat ovary.

Ovarian cytosol from pseudopregnant rats was heated to 80-90 degrees C for 2 min and precipitated proteins removed by centrifugation. The supernatant of the heated ovarian cytosol contained no protein kinase C activity but when added to a control preparation containing protein kinase C, enzyme activity was increased to 200% of control. The stimulatory activity was stable to heating for 10 min, was retained on a centrifugal filtration device with a 100,000 M(r) cut-off, did not affect cAMP-dependent protein kinase, was not extractable in petroleum ether or chloroform/methanol (2:1), and enhanced the phosphorylation of protein kinase C-specific peptide substrates. The stimulatory factor was calcium-dependent and could substitute for phosphatidylserine and diacylglycerol in the protein kinase C assay. This stimulatory factor may provide a mechanism whereby the response of protein kinase C to hormonal activation could be regulated by the cell.     

Protein kinase C isozymes in stroke

Stroke is a devastating neurologic disease and a leading cause of death and disability worldwide. Thrombolytic agents have been used to re-establish circulation in thromboembolic stroke, but their utility is limited by hemorrhage and reperfusion injury. Studies with experimental stroke models, mouse genetics, and selective peptide inhibitors and activators have implicated protein kinase C (PKC) epsilon in ischemic preconditioning and PKCdelta and gamma in tissue injury. PKCdelta, resident both in neutrophils and in the brain, appears particularly essential for reperfusion injury, and recent work using PKCdelta-specific peptide inhibitors suggests that PKCdelta inhibitors could prove useful in attenuating reperfusion injury and improving outcome following thrombolysis.     

Rapid and transient palmitoylation of the tyrosine kinase Lck mediates Fas signaling

Palmitoylation is the posttranslational modification of proteins with a 16-carbon fatty acid chain through a labile thioester bond. The reversibility of protein palmitoylation and its profound effect on protein function suggest that this modification could play an important role as an intracellular signaling mechanism. Evidence that palmitoylation of proteins occurs with the kinetics required for signal transduction is not clear, however. Here we show that engagement of the Fas receptor by its ligand leads to an extremely rapid and transient increase in palmitoylation levels of the tyrosine kinase Lck. Lck palmitoylation kinetics are consistent with the activation of downstream signaling proteins, such as Zap70 and PLC-γ1. Inhibiting Lck palmitoylation not only disrupts proximal Fas signaling events, but also renders cells resistant to Fas-mediated apoptosis. Knockdown of the palmitoyl acyl transferase DHHC21 eliminates activation of Lck and downstream signaling after Fas receptor stimulation. Our findings demonstrate highly dynamic Lck palmitoylation kinetics that are essential for signaling downstream of the Fas receptor.Palmitoylation of cysteine residues is a common posttranslational protein modification that affects a variety of protein functions by regulating their intracellular localization, trafficking, and stability (1). Compared with other lipid modifications such as myristoylation and prenylation, palmitoylation is the only reversible modification. Furthermore, palmitoylated proteins can undergo multiple cycles of acylation and deacylation (2). The enzymatic control of protein palmitoylation in mammalian cells is mediated by a family of more than 20 protein acyltransferases (PATs) sharing a common DHHC (Asp-His-His-Cys) motif within the catalytic center (2–4). Traditionally, protein palmitoylation is thought to be catalyzed in the Golgi apparatus, and indeed systematic characterization of human DHHC PAT intracellular distribution has confirmed that the majority of these enzymes localized to the Golgi compartment (5); however, several enzymes, including DHHC5, 20, and 21, could be localized at the plasma membrane.The depalmitoylation step is likely mediated by the acyl protein thioesterases APT1 and APT2, the only two enzymes known to reverse palmitoylation of cytosolic proteins in mammalian cells. APT1 was initially identified as a rat liver lysophospholipase widely spread among different tissues (6, 7). Later, its substrate specificity was expanded to a number of palmitoylated proteins, including Ras, heterotrimeric G protein α subunit, RGS4, SNAP-23, and eNOS (8). APT1 was the sole known cytosolic thioesterase until cloning of its close homolog, APT2 (9), which recently was reported to be involved in depalmitoylation of growth-associated protein 43 (10).A number of signaling proteins that play critical roles in the regulation of T-cell function are known to be palmitoylated, including coreceptors CD4 and CD8, kinases Lck and Fyn, and adaptor proteins LAT and Cbp/PAG (11–13). In many cases, palmitoylation of these proteins is essential for their signaling function. Lck, a member of the Src family of tyrosine kinases, is cotranslationally myristoylated at glycine residue 2 and then posttranslationally palmitoylated at cysteine residues 3 and 5 (14, 15). These cysteines are dispensable for the catalytic activity of Lck, and mutagenesis of either site does not prevent palmitoylation or membrane binding (16–18). Simultaneous mutation of cysteine 3 and cysteine 5 produces a palmitoylation-deficient mutant of Lck that is unable to propagate T-cell receptor signaling, demonstrating a critical role for palmitoylation in regulating Lck function (16, 17, 19). The regulatory mechanisms controlling Lck palmitoylation remain largely unknown, however.We have previously demonstrated that Lck is an essential component of the Fas signaling pathway (20). Lck is required for the most proximal Fas signaling events, including PLC-γ1 activation, proapoptotic calcium release, and cell death in T cells (20). Using a pulsed metabolic labeling and detection strategy, here we show that Lck palmitate turnover is very rapid even in resting T cells. Stimulation of T cells with Fas ligand results in a rapid increase and decrease in Lck palmitoylation that closely correlates with the activation state of downstream signaling molecules such as PLC-γ1. Finally, we identify the plasma membrane PAT DHHC21 as the enzyme responsible for Lck palmitoylation and downstream apoptotic calcium release. Our results indicate that agonist-induced protein palmitoylation and depalmitoylation occur with unprecedented kinetics, and suggest that lipidation of signaling proteins may be widely involved in the activation of second messenger signaling cascades.     

Protein kinase C in diabetic nephropathy

Protein kinase C is activated in numerous tissues obtained from diabetic animals and in several cultured cell systems exposed to high media glucose in vitro including glomerular mesangial cells. Several activators of protein kinase C, such as high media glucose, angiotensin II, phorbol ester, low density lipoprotein, and the thromboxane analogue U-46619, increase TGFβ bioactivity or mRNA expression and increase the synthesis of extracellular matrix proteins by mesangial cells in culture. The studies described in the present report support the hypothesis that activation of protein kinase C by thromboxane, an eicosanoid whose production is known to be elevated in diabetes, increases TGFβ production by mesangial cells in culture. TGFβ then acts to increase extracellular matrix protein synthesis through a mechanism that does not require active protein kinase C. Thus, activation of protein kinase C in the glomerulus in diabetes could contribute to mesangial expansion by stimulating active TGFβ production.     

Dopamine induces neurite retraction in retinal horizontal cells via diacylglycerol and protein kinase C.

Dopamine causes a significant retraction of neurites of bull-head catfish horizontal cells maintained in culture. The effects of dopamine are blocked by haloperidol and SCH 23390, a D1 antagonist, but not by sulpiride, a D2 antagonist. The dopamine-induced morphological changes were mimicked by SKF 38393, a D1 agonist, but not by quinpirole, a D2 agonist. Kainate also caused process retraction, but other neuroactive substances tested including glutamate, 5-hydroxytryptamine, N-methyl-D-aspartate, gamma-aminobutyric acid, and glycine caused only minor changes in neurite length. Cyclic AMP analogues do not induce neurite retraction in horizontal cells, indicating that this effect of dopamine is not mediated by cyclic AMP. However, a protein kinase C activator (phorbol 12-myristate 13-acetate) and synthetic diacylglycerol analogs (1-oleoyl-2-acetyl-sn-glycerol and dioctanoglycerol) caused marked neurite retraction. Their effects, as well as the dopamine-induced changes, were blocked by staurosporine, a potent protein kinase antagonist. The results suggest that dopamine causes neurite retraction by the activation of protein kinase C via diacylglycerol.     

Protein kinase cross-talk: membrane targeting of the beta-adrenergic receptor kinase by protein kinase C.

The beta-adrenergic receptor kinase (betaARK) is the prototypical member of the family of cytosolic kinases that phosphorylate guanine nucleotide binding-protein-coupled receptors and thereby trigger uncoupling between receptors and guanine nucleotide binding proteins. Herein we show that this kinase is subject to phosphorylation and regulation by protein kinase C (PKC). In cell lines stably expressing alpha1B- adrenergic receptors, activation of these receptors by epinephrine resulted in an activation of cytosolic betaARK. Similar data were obtained in 293 cells transiently coexpressing alpha1B- adrenergic receptors and betaARK-1. Direct activation of PKC with phorbol esters in these cells caused not only an activation of cytosolic betaARK-1 but also a translocation of betaARK immunoreactivity from the cytosol to the membrane fraction. A PKC preparation purified from rat brain phospborylated purified recombinant betaARK-1 to a stoichiometry of 0.86 phosphate per betaARK-1. This phosphorylation resulted in an increased activity of betaARK-1 when membrane-bound rhodopsin served as its substrate but in no increase of its activity toward a soluble peptide substrate. The site of phosphorylation was mapped to the C terminus of betaARK-1. We conclude that PKC activates betaARK by enhancing its translocation to the plasma membrane.     

Protein kinase C and Mammalian spermatozoa acrosome reaction.

The presence of protein kinase C (PKC) in mammalian sperm was demonstrated by enzymatic activity assay and immunohistochemistry at the light and electron microscopy levels. The sperm head PKC is localized in the acrosome, equatorial segment, and postacrosomal region. In the flagellum, PKC is associated with the segmented column of the neck and is distributed along the mid, principal, and end pieces. Immunoreactive sites are observed in patches along the axoneme and outer dense fibers and are evenly distributed between these regions. Functional studies suggest the involvement of PKC in flagellar motility and acrosome reaction. The cross-talk between the signaling cascades that operate during sperm activation is discussed. (Trends Endocrinol Metab 1997;8:337-341). (c) 1997, Elsevier Science Inc.     

Protein kinase C isoforms in human erythrocytes

Erythrocytes are the most abundant cells in blood and carry out the vital function of oxygen transport. These cells lack nuclei and do not synthesise new proteins. Their cellular responses are modulated entirely by post-translational modifications in existing proteins. Phosphorylation mediated by protein kinase C (PKC) plays a significant role in red cell physiology. To date PKC alpha and zeta are the only isoforms reported to be expressed in erythrocytes. Upon activation they influence cytoskeletal integrity and erythrocyte functions. In this study we report for the first time the presence of PKC iota and PKC mu in addition to PKC alpha and zeta in human erythrocytes. All isoforms were present only in the cytosolic fraction. PKC alpha was the only isoform that translocated to the membrane upon stimulation with phorbol myristate acetate (PMA). It could thus mediate several of the reported PMA-regulated membrane modifications. Investigations on alterations in PMA-mediated phosphorylation of erythrocyte skeletal components in disorders such as chronic myeloid leukaemia can now focus on PKC alpha, which is the only isoform in erythrocytes, which translocates to the membrane on stimulation of the cells.