Regulation of ADAM12 cell-surface expression by protein kinase C epsilon

The ADAM (a disintegrin and metalloprotease) family consists of multidomain cell-surface proteins that have a major impact on cell behavior. These transmembrane-anchored proteins are synthesized as proforms that have (from the N terminus): a prodomain; a metalloprotease-, disintegrin-like-, cysteine-rich, epidermal growth factor-like, and transmembrane domain; and a cytoplasmic tail. The 90-kDa mature form of human ADAM12 is generated in the trans-Golgi through cleavage of the prodomain by a furin-peptidase and is stored intracellularly until translocation to the cell surface as a constitutively active protein. However, little is known about the regulation of ADAM12 cell-surface translocation. Here, we used human RD rhabdomyosarcoma cells, which express ADAM12 at the cell surface, in a temporal pattern. We report that protein kinase C (PKC) epsilon induces ADAM12 translocation to the cell surface and that catalytic activity of PKCepsilon is required for this translocation. The following results support this conclusion: 1) treatment of cells with 0.1 microM phorbol 12-myristate 13-acetate (PMA) enhanced ADAM12 cell-surface immunostaining, 2) ADAM12 and PKCepsilon could be co-immunoprecipitated from membrane-enriched fractions of PMA-treated cells, 3) RD cells transfected with EGFP-tagged, myristoylated PKCepsilon expressed more ADAM12 at the cell surface than did non-transfected cells, and 4) RD cells transfected with a kinase-inactive PKCepsilon mutant did not exhibit ADAM12 cell-surface translocation upon PMA treatment. Finally, we demonstrate that the C1 and C2 domains of PKCepsilon both contain a binding site for ADAM12. These studies show that PKCepsilon plays a critical role in the regulation of ADAM12 cell-surface expression.     

Amygdala protein kinase C epsilon controls alcohol consumption

Alcoholism is a progressive disorder that involves the amygdala. Mice lacking protein kinase C epsilon (PKCɛ) show reduced ethanol consumption, sensitivity and reward. We therefore investigated whether PKCɛ signaling in the amygdala is involved in ethanol consumption. Local knockdown of PKCɛ in the amygdala reduced ethanol consumption and preference in a limited-access paradigm. Further, mice that are heterozygous for the PKCɛ allele consume less ethanol compared with wild-type mice in this paradigm. These mice have a >50% reduction in the abundance of PKCɛ in the amygdala compared with wild-type mice. We conclude that amygdala PKCɛ is important for ethanol consumption in mice.     

Mitogen-activated protein kinase-activated protein kinase 2-deficient mice show increased susceptibility to Listeria monocytogenes infection

Mitogen-activated protein kinase-activated protein kinase 2 (MK2) is one of several kinases activated through direct phosphorylation by p38 mitogen-activated protein kinase. MK2 regulates LPS-induced TNF mRNA translation, and targeted mutation of the MK2 gene renders mice more resistant to D-galactosamine plus LPS-induced liver damage. In the present study, we investigated the role of MK2 in immune defense against Listeria monocytogenes infection. MK2-deficient mice displayed diminished resistance to L. monocytogenes due to impaired control of bacterial growth. The increase in bacterial load in MK2(-/-) mice was associated with normal levels of IL-1 beta, IL-6, and IFN-gamma, whereas TNF production was strongly attenuated. In line, MK2-deficient bone marrow-derived macrophages showed impaired release of TNF, but not of IL-1 beta, in response to various bacterial stimuli in addition to decreased phagocytosis of fluorescence-labeled bacteria. Furthermore, spleen cells from MK2(-/-) mice displayed diminished IFN-gamma synthesis after stimulation with L. monocytogenes. In contrast, MK2 deficiency had no effect on macrophage generation of NO or on oxidative burst activity in response to L. moocytogenes. These results indicate an essential role of MK2 in host defense against intracellular bacteria probably via regulation of TNF and IFN-gamma production required for activation of antibacterial effector mechanisms.     

Prostaglandin-mediated suppression of macrophage phagocytosis of Listeria monocytogenes

Suppression of macrophage phagocytosis of Listeria monocytogenes has been shown to be due to a low-molecular-weight component of spleen cell culture supernatant. The possibility that the factor could be a prostaglandin was investigated. When murine peritoneal macrophages were treated with prostaglandin E2 (PGE2), L. monocytogenes was phagocytized at a rate comparable to that phagocytized when treated with a low-molecular-weight fraction of concanavalin A-generated spleen cell culture supernatant. Suppressive activity of the spleen cell culture supernatant was abrogated when supernatant was prepared in the presence of indomethacin, a prostaglandin synthetase inhibitor. Prostaglandins were identified in supernatants with thin-layer and high-pressure liquid chromatography. These results suggest a role for prostaglandins, particularly PGE2, as a modulator of macrophage phagocytosis of L. monocytogenes.     

Increased response to morphine in mice lacking protein kinase C epsilon

The protein kinase C (PKC) family of serine-threonine kinases has been implicated in behavioral responses to opiates, but little is known about the individual PKC isozymes involved. Here, we show that mice lacking PKCepsilon have increased sensitivity to the rewarding effects of morphine, revealed as the expression of place preference and intravenous self-administration at very low doses of morphine that do not evoke place preference or self-administration in wild-type mice. The PKCepsilon null mice also show prolonged maintenance of morphine place preference in response to repeated testing when compared with wild-type mice. The supraspinal analgesic effects of morphine are enhanced in PKCepsilon null mice, and the development of tolerance to the spinal analgesic effects of morphine is delayed. The density of mu-opioid receptors and their coupling to G-proteins are normal. These studies identify PKCepsilon as a key regulator of opiate sensitivity in mice.     

Signal transduction of constitutively active protein kinase C epsilon

The protein kinase C (PKC) family is the most prominent target of tumor-promoting phorbol esters. For the PKCε isozyme, different intracellular localizations and oncogenic potential in several but not all experimental systems have been reported. To obtain information about PKCε-signaling, we investigated the effects of constitutively active rat PKCε (PKCεA/E, alanine 159 is replaced by glutamic acid) in HeLa cells in a doxycycline-inducible vector. Upon induction of PKCεA/E expression by doxycycline, the major part of PKCεA/E was localized to the Golgi. This led (i) to phosphorylations of PKCε^S729, Elk-1^S383, PDK1^S241 and Rb^S807/S811, (ii) to elevated expression of receptor of activated C kinase 2 (RACK2) after 12 h, and (iii) increased colony formation in soft agar, increased cell migration and invasion, but not to decreased doubling time. Following induction of PKCεA/E-expression by doxycycline for 24 h and additional short-term treatment with 12-O-tetradecanoylphorbol-13-acetate (TPA), PKCεA/E translocated to the plasma membrane and increased phosphorylation of MARCKS^S152/156. Treatment with doxycycline/TPA or TPA alone increased phosphorylations of Elk-1^S383, PDK1^S241, Rb^S807/S811, PKCδ^T505, p38MAPK^T180/Y182, MEK1/2^S217/S221 and ERK2^T185/T187. MARCKS was not phosphorylated after treatment with TPA alone, demonstrating that in this system it is phosphorylated only by PKCε localized to the plasma membrane but not by PKCα or δ, the other TPA-responsive PKC isozymes in HeLa cells. These results demonstrate that PKCε can induce distinctly different signaling from the Golgi and from the plasma membrane.     

Induction of macrophage interleukin-1 production by Listeria monocytogenes hemolysin.

Listeriolysin O produced by a hemolytic strain of Listeria monocytogenes was purified from the ammonium sulfate precipitate of a culture supernatant through the steps of ion-exchange chromatography and gel filtration. The purified hemolysin finally gave a single band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis with a molecular weight of 58,000. When peritoneal exudate macrophages were stimulated with purified hemolysin, we found a high level of IL-1 activity as determined by thymocyte costimulator assay in the culture supernatant. Cell-associated and intracellular IL-1 activity was also detected. The activity in the supernatant or membrane was blocked by polyclonal antibody to murine IL-1 alpha. Moreover, IL-1-specific mRNA expression could be detected in the macrophages stimulated with listeriolysin O by Northern blot analysis. Possible contamination by LPS of the listeriolysin O preparation did not seem to contribute to the induction of macrophage IL-1 production.     

Activation of focal adhesion kinase by protein kinase C epsilon in neonatal rat ventricular myocytes

Focal adhesion kinase (FAK) is a nonreceptor protein tyrosine kinase critical for both cardiomyocyte survival and sarcomeric assembly during endothelin (ET)-induced cardiomyocyte hypertrophy. ET-induced FAK activation requires upstream activation of one or more isoenzymes of protein kinase C (PKC). Therefore, with the use of replication-defective adenoviruses (Adv) to overexpress constitutively active (ca) and dominant negative (dn) mutants of PKCs, we examined which PKC isoenzymes are necessary for FAK activation and which downstream signaling components are involved. FAK activation was assessed by Western blot analysis with an antibody specific for FAK autophosphorylated at Y397 (Y397pFAK). ET (10 nmol/l; 2-30 min) resulted in the time-dependent activation of FAK which was inhibited by chelerythrine (5 micromol/l; 1 h pretreatment). Adv-caPKC epsilon, but not Adv-caPKC delta, activated FAK compared with a control Adv encoding beta-galactosidase. Conversely, Adv-dnPKC epsilon inhibited ET-induced FAK activation. Y-27632 (10 micromol/l; 1 h pretreatment), an inhibitor of Rho-associated coiled-coil-containing protein kinases (ROCK), prevented ET- and caPKC epsilon-induced FAK activation as well as cofilin phosphorylation. Pretreatment with cytochalasin D (1 micromol/l, 1 h pretreatment) also inhibited ET-induced Y397pFAK and cofilin phosphorylation and caPKC epsilon-induced Y397pFAK. Neither inhibitor, however, interfered with ET-induced ERK1/2 activation. Finally, PP2 (50 micromol/l; 1 h pretreatment), a highly selective Src inhibitor, did not alter basal or ET-induced Y397pFAK. PP2 did, however, reduce basal and ET-induced phosphorylation of other sites on FAK, namely, Y576, Y577, Y861, and Y925. We conclude that the ET-induced signal transduction pathway resulting in downstream Y397pFAK is partially dependent on PKC epsilon, ROCK, cofilin, and assembled actin filaments, but not ERK1/2 or Src.     

Membrane perforations inhibit lysosome fusion by altering pH and calcium in Listeria monocytogenes vacuoles

Listeria monocytogenes (Lm) evade microbicidal defences inside macrophages by secreting a pore-forming cytolysin listeriolysin O (LLO), which allows Lm to escape vacuoles. LLO also inhibits Lm vacuole fusion with lysosomes, which indicates LLO alters vacuole chemistry prior to release of Lm into cytoplasm. Using fluorescent probes to measure membrane permeability, calcium and pH, we identified small membrane perforations in vacuoles containing wild-type but not LLO-deficient (hly-) Lm. The small membrane perforations released small fluorescent molecules and persisted for several minutes before expanding to allow exchange of larger fluorescent molecules. Macropinosomes and hly- Lm vacuoles acidified and increased their calcium content ([Ca2+]vac) within minutes of formation; however, the small perforations made by LLO-expressing bacteria increased vacuolar pH and decreased [Ca2+]vac shortly after infection. Experimental increases in vacuolar pH inhibited Lm vacuole fusion with lysosomes. The timing of perforation indicated that LLO-dependent delays of Lm vacuole maturation result from disruption of ion gradients across vacuolar membranes.     

Utilization of multiple substrates by butyrate kinase from Listeria monocytogenes

Listeria monocytogenes, the causative agent of listeriosis, can build up to dangerous levels in refrigerated foods potentially leading to expensive product recalls. An important aspect of the bacterium's growth at low temperatures is its ability to increase the branched-chain fatty acid anteiso C15:0 content of its membrane at lower growth temperatures, which imparts greater membrane fluidity. Mutants in the branched-chain α-keto dehydrogenase (bkd) complex are deficient in branched-chain fatty acids (BCFAs,) but these can be restored by feeding C4 and C5 branched-chain carboxylic acids (BCCAs). This suggests the presence of an alternate pathway for production of acyl CoA precursors for fatty acid biosynthesis. We hypothesize that the alternate pathway is composed of butyrate kinase (buk) and phosphotransbutyrylase (ptb) encoded in the bkd complex which produce acyl CoA products by their sequential action through the metabolism of carboxylic acids. We determined the steady state kinetics of recombinant His-tagged Buk using 11 different straight-chain and BCCA substrates in the acyl phosphate forming direction. Buk demonstrated highest catalytic efficiency with pentanoate as the substrate. Low product formation observed with acetate (C2) and hexanoate (C6) as the substrates indicates that Buk is not involved in either acetate metabolism or long chain carboxylic acid activation. We were also able to show that Buk catalysis occurs through a ternary complex intermediate. Additionally, Buk demonstrates a strong preference for BCCAs at low temperatures. These results indicate that Buk may be involved in the activation and assimilation of exogenous carboxylic acids for membrane fatty acid biosynthesis.     

Distinct mechanisms of regulation of protein kinase C epsilon by hormones and phorbol diesters

In this study, we examined the effects of T cell activators on the regulation of protein kinase C (PKC) isozymes present in thymocytes. Using affinity-purified anti-PKC antisera, we determined that the major PKC isoforms in murine thymocytes are PKC beta and PKC epsilon. The CD4+/CD8+ thymocyte subset expressed high levels of both PKC beta and PKC epsilon, whereas the CD4-/CD8- subset expressed much less of both. PKC beta was down-regulated following treatment of thymocytes with phorbol 12-myristate acetate (PMA) (2 x 10(-8) M) or ionomycin (0.4 microM). In contrast, PMA did not induce the down-regulation of PKC epsilon. Ionomycin alone, however, induced PKC epsilon down-regulation, similar to its effect on PKC beta. Similar observations were made on a promonocytic cell line, U937, which expresses PKC alpha, PKC beta (Strulovici, B., Daniel-Issakani, S., Oto, E., Nestor, J., Jr., Chan, H., and Tsou, A.-P. (1989) Biochemistry 28, 3569-3576), and PKC epsilon. To facilitate the study of PKC beta and PKC epsilon, we established a Chinese hamster ovary cell line which expresses murine PKC epsilon in addition to endogenous PKC alpha and PKC beta. Both PKC isoforms (beta and epsilon) were mostly in particulate form. PMA treatment left the majority of immunoreactive PKC epsilon intact. By contrast, thrombin treatment caused the disappearance of particulate and cytosolic PKC epsilon (60% by 10 min and 80% by 1 h). PMA and thrombin promoted the down-regulation of PKC beta with similar kinetics (100% down-regulation by 3 h). These results indicate that: 1) thymocytes express PKC epsilon; and 2) this isozyme exhibits a novel form of regulation distinct from the other PKC isozymes.     

The multiple mechanisms of Ca2+ signalling by listeriolysin O, the cholesterol-dependent cytolysin of Listeria monocytogenes

Cholesterol-dependent cytolysins (CDCs) represent a large family of conserved pore-forming toxins produced by several Gram-positive bacteria such as Listeria monocytogenes, Streptococcus pyrogenes and Bacillus anthracis. These toxins trigger a broad range of cellular responses that greatly influence pathogenesis. Using mast cells, we demonstrate that listeriolysin O (LLO), a prototype of CDCs produced by L. monocytogenes, triggers cellular responses such as degranulation and cytokine synthesis in a Ca(2+)-dependent manner. Ca(2+) signalling by LLO is due to Ca(2+) influx from extracellular milieu and release of from intracellular stores. We show that LLO-induced release of Ca(2+) from intracellular stores occurs via at least two mechanisms: (i) activation of intracellular Ca(2+) channels and (ii) a Ca(2+) channels independent mechanism. The former involves PLC-IP(3)R operated Ca(2+) channels activated via G-proteins and protein tyrosine kinases. For the latter, we propose a novel mechanism of intracellular Ca(2+) release involving injury of intracellular Ca(2+) stores such as the endoplasmic reticulum. In addition to Ca(2+) signalling, the discovery that LLO causes damage to an intracellular organelle provides a new perspective in our understanding of how CDCs affect target cells during infection by the respective bacterial pathogens.     

Protective role of protein kinase C epsilon activation in ischemia-reperfusion arrhythmia

PURPOSE: Ischemic heart disease carries an increased risk of malignant ventricular tachycardia (VT), fibrillation (VF), and sudden cardiac death. Protein kinase C (PKC) epsilon activation has been shown to improve the hemodynamics in hearts subjected to ischemia/reperfusion. However, very little is known about the role of epsilon PKC in reperfusion arrhythmias. Here we show that epsilon PKC activation is anti-arrhythmic and its inhibition is pro-arrhythmic. METHOD: Langendorff-perfused isolated hearts from epsilonPKC agonist (epsilonPKC activation), antagonist (epsilonPKC inhibition) transgenic (TG), and wild-type control mice were subjected to 30 min stabilization period, 10 min global ischemia, and 30 min reperfusion. Action potentials (APs) and calcium transients (CaiT) were recorded simultaneously at 37 degrees C using optical mapping techniques. The incidence of VT and VF was assessed during reperfusion. RESULTS: No VT/VF was seen in any group during the stabilization period in which hearts were perfused with Tyrode's solution. Upon reperfusion, 3 out of the 16 (19%) wild-type mice developed VT but no VF. In epsilonPKC antagonist group, in which epsilonPKC activity was downregulated, 10 out of 13 (76.9%) TG mice developed VT, of which six (46.2%) degenerated into sustained VF upon reperfusion. Interestingly, in epsilonPKC agonist mice, in which the activity of epsilonPKC was upregulated, no VF was observed and only 1 out of 12 mice showed only transient VT during reperfusion. During ischemia and reperfusion, CaiT decay was exceedingly slower in the antagonist mice compared to the other two groups. CONCLUSION: Moderate in vivo activation of epsilonPKC exerts beneficial antiarrhythmic effect vis-a-vis the lethal reperfusion arrhythmias. Abnormal CaiT decay may, in part, contribute to the high incidence of reperfusion arrhythmias in the antagonist mice. These findings have important implications for the development of PKC isozyme targeted therapeutics and subsequently for the treatment of ischemic heart diseases.     

The lipophosphoglycan of Leishmania and macrophage protein kinase C

Lipophosphoglycan (LPG), the major cellsurface glycoconjugate of Leishmania promastigotes, is on essential virulence determinant. One feature of LPG resides in its strong inhibitory effect on the activity of purified protein kinase C (PKC). In this article, Albert Descoteaux and Salvatore Turco briefly review the evidence that LPG effectively inhibits PKC activity in the macrophage, and discuss the implication of such inhibition on Leishmania intramacrophoge survival.     

Non-typeable Haemophilus influenzae decreases cilia beating via protein kinase C epsilon

Background

Haemophilus influenzae infection of the nasal epithelium has long been associated with observations of decreased nasal ciliary beat frequency (CBF) and injury to the ciliated epithelium. Previously, we have reported that several agents that slow CBF also have the effect of activating protein kinase C epsilon (PKCϵ) activity in bronchial epithelial cells. The subsequent auto-downregulation of PKCϵ or the direct inhibition of PKCϵ leads to the specific detachment of the ciliated cells. METHODS: Primary cultures of ciliated bovine bronchial epithelial cells were exposed to filtered conditioned media supernatants from non-typeable H. influenzae (NTHi) cultures. CBF and motile points were measured and PKCϵ activity assayed.

Results

NTHi supernatant exposure significantly and rapidly decreased CBF in a dose-dependent manner within 10 minutes of exposure. After 3 hours of exposure, the number of motile ciliated cells significantly decreased. Direct measurement of PKCϵ activity revealed a dose-dependent activation of PKCϵ in response to NTHi supernatant exposure. Both CBF and PKCϵ activity changes were only observed in fresh NTHi culture supernatant and not observed in exposures to heat-inactivated or frozen supernatants.

Conclusions

Our results suggest that CBF slowing observed in response to NTHi is consistent with the stimulated activation of PKCϵ. Ciliated cell detachment is associated with PKCϵ autodownregulation.     

Amygdala protein kinase C epsilon regulates corticotropin-releasing factor and anxiety-like behavior

Corticotropin-releasing factor (CRF), its receptors, and signaling pathways that regulate CRF expression and responses are areas of intense investigation for new drugs to treat affective disorders. Here, we report that protein kinase C epsilon (PKCɛ) null mutant mice, which show reduced anxiety-like behavior, have reduced levels of CRF messenger RNA and peptide in the amygdala. In primary amygdala neurons, a selective PKCɛ activator, ψɛRACK, increased levels of pro-CRF, whereas reducing PKCɛ levels through RNA interference blocked phorbol ester-stimulated increases in CRF. Local knockdown of amygdala PKCɛ by RNA interference reduced anxiety-like behavior in wild-type mice. Furthermore, local infusion of CRF into the amygdala of PKCɛ^−/− mice increased their anxiety-like behavior. These results are consistent with a novel mechanism of PKCɛ control over anxiety-like behavior through regulation of CRF in the amygdala.     

Characterization of the binding and phosphorylation of cardiac calsequestrin by epsilon protein kinase C.

In this study, we report the cloning of the rat cardiac isoform of calsequestrin on the basis of its interaction with an epsilonprotein kinase C-unique sequence (epsilonV1) derived form the epsilonprotein kinase C regulatory domain. Calsequestrin binds activated epsilonprotein kinase C holoenzyme better than the inactive enzyme and nearly three times better than other protein kinase C isozymes. The interaction between epsilonprotein kinase C and calsequestrin is mediated by sequences in both the regulatory and kinase domains of the epsilonprotein kinase C. Finally, we show that calsequestrin is an epsilonprotein kinase C substrate in vitro and protein kinase C phosphorylation of calsequestrin leads to a decreased binding of epsilonprotein kinase C to calsequestrin.