Conventional Isoforms Research Articles

Absence of developmental and unconventional myosin heavy chain in human suprahyoid muscles

Contradictory reports of the myosin heavy chain (MHC) composition of adult human suprahyoid muscles leave unresolved the extent to which these muscles express developmental and unconventional MHC. By immunohistochemistry, separation sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE)-Coomassie, separation SDS-PAGE-Western blot, and mRNA PCR, we tested for conventional MHCI, MHCIIA, MHCIIX, developmental MHC embryonic and MHC neonatal, and unconventional MHC alpha-cardiac, MHC extraocular, and MHC slow tonic in adult human anterior digastric (AD), geniohyoid (GH), and mylohyoid (MH) muscles. By separation SDS-PAGE-Coomassie and Western blot, only conventional MHC are present. By immunohistochemistry all muscle fibers are positive for MHCI, MHCIIA, or MHCIIX, and fewer than 4 fibers/mm(2) are positive for developmental or unconventional MHC. By PCR, mRNA of MHCI and MHCIIA dominate, with sporadically detectable MHC alpha-cardiac and without detectable mRNA of other developmental and unconventional MHC. We conclude that human suprahyoid muscles AD, GH, and MH are composed almost exclusively of conventional MHC isoforms.

Identification and characterization of a neuron‐specific isoform of protrudin

Protrudin is a membrane protein that regulates polarized vesicular transport. Now, we have identified a novel isoform of protrudin (protrudin-L) that contains an additional seven amino acids between the FFAT motif and the coiled-coil domain compared with the conventional isoform (protrudin-S) as a result of alternative splicing of a microexon (exon L). Protrudin-L mRNA was found to be mostly restricted to the central nervous system in mice, whereas protrudin-S mRNA was detected in all tissues examined. With the use of a splicing reporter minigene that produces two distinct fluorescent proteins in a manner dependent on the splicing pattern of protrudin transcripts, we found that most neurons express protrudin-L, whereas astrocytes express both protrudin isoforms and oligodendrocytes express only protrudin-S. Protrudin-L associated to a greater extent with vesicle-associated membrane protein-associated protein (VAP) than protrudin-S. Expression of protrudin-L in hippocampal neurons of protrudin-deficient mice also promoted neurite outgrowth more efficiently than protrudin-S. Our results suggest that protrudin-L is a neuron-specific protrudin isoform that promotes axonal elongation and contributes to the establishment of neuronal polarity.

P141 Differential expression of conventional and inhibitory VEGFA isoforms in normal and fibrotic fibroblasts–a potential role in IPF pathogenesis?

IntroductionVascular endothelial growth factor (VEGFA) has been implicated in the pathogenesis of Idiopathic Pulmonary Fibrosis (IPF). Two families of endogenous isoforms exist formed by alternative splicing of mRNA transcripts: the...

Free fatty acids and protein kinase C activation induce GPR120 (free fatty acid receptor 4) phosphorylation

GPR120, free fatty acid receptor 4, is a recently deorphanized G protein-coupled receptor that seems to play cardinal roles in the regulation of metabolism and in the pathophysiology of inflammatory and metabolic disorders. In the present work a GPR120-Venus fusion protein was expressed in HEK293 Flp-In T-REx cells and its function (increase in intracellular calcium) and phosphorylation were studied. It was observed that the fusion protein migrated in sodium dodecyl sulfate-polyacrylamide gels as a band with a mass of ≈70–75kDa, although other bands of higher apparent weight (>130kDa) were also detected. Cell stimulation with docosahexaenoic acid or α-linolenic acid induced concentration-dependent increases in intracellular calcium and GPR120 phosphorylation. Activation of protein kinase C with phorbol esters also induced a marked receptor phosphorylation but did not alter the ability of 1µM docosahexaenoic acid to increase the intracellular calcium concentration. Phorbol ester-induced GPR120 phosphorylation, but not that induced with docosahexaenoic acid, was blocked by protein kinase C inhibitors (bis-indolyl-maleimide I and Gö 6976) suggesting that conventional kinase isoforms mediate this action. The absence of effect of protein kinase C inhibitors on agonist-induced GPR120 phosphorylation indicates that this kinase does not play a major role in agonist-induced receptor phosphorylation. Docosahexaenoic acid action was associated with marked GPR120 internalization whereas that induced with phorbol esters was smaller at early times.

VEGF111b, a new member of VEGFxxxb isoforms and induced by mitomycin C, inhibits angiogenesis

Vascular endothelial growth factor (VEGF-A) stimulating angiogenesis is required for tumor growth and progression. The conventional VEGF-A isoforms have been considered as pro-angiogenic factors. Another family of VEGF-A isoforms generated by alternative splicing, termed VEGFxxxb isoforms, has anti-angiogenic property, exemplified by VEGF165b. Here, we identify a new number of VEGFxxx family-VEGF111b induced by mitomycin C, although not detected in mitomycin C-unexposed ovarian cancer cells. SKOV3 cells were transfected with pcDNA3.1 empty vector, pcDNA3.1-VEGF111b or pcDNA3.1-VEGF165b to collect conditioned mediums respectively. VEGF111b overexpression inhibits proliferation, migration and tube formation of endothelial cell by inhibiting VEGF-R2 phosphorylation and its downstream signaling, similar to VEGF165b but slightly lower than VEGF165b. The anti-angiogenic property depends on the six amino acids of exon 8b of the VEGFxxxb isoforms. Our results show that VEGF111b is a novel potent anti-angiogenic agent that can target the VEGF-R2 and its signaling pathway to inhibit ovarian tumor growth.

Expression of conventional and novel glucose transporters, GLUT1, -9, -10, and -12, in vascular smooth muscle cells

Intimal hyperplasia is characterized by exaggerated proliferation of vascular smooth muscle cells (VSMCs). Enhanced VSMC growth is dependent on increased glucose uptake and metabolism. Facilitative glucose transporters (GLUTs) are comprised of conventional GLUT isoforms (GLUT1-5) and novel GLUT isoforms (GLUT6-14). Previous studies demonstrate that GLUT1 overexpression or GLUT10 downregulation contribute to phenotypic changes in VSMCs. To date, the expression profile of all 14 GLUT isoforms has not been fully examined in VSMCs. Using the proliferative and differentiated phenotypes of human aortic VSMCs, the present study has determined the relative abundance of GLUT1-14 mRNAs by quantitative real-time PCR analysis. Twelve GLUT mRNAs excluding GLUT7 and GLUT14 were detectable in VSMCs. In the proliferative phenotype, the relative abundance of key GLUT mRNAs was GLUT1 (∼43%)>GLUT10 (∼26%)>GLUT9 (∼13%)>GLUT12 (∼4%), whereas in the differentiated phenotype the relative abundance was GLUT10 (∼28%)>GLUT1 (∼25%)>GLUT12 (∼20%)>GLUT9 (∼14%), together constituting 86-87% of total GLUT transcripts. To confirm the expression of key GLUT proteins, immunoblot and immunocytochemical analyses were performed using GLUT isoform-specific primary antibodies. The protein bands characteristic of GLUT1, -9, -10, and -12 were detected in VSMCs in parallel with respective positive controls. In particular, GLUT1 protein expression showed different molecular forms representative of altered glycosylation. While GLUT1 protein displayed a predominant distribution in the plasma membrane, GLUT9, -10, and -12 proteins were mostly distributed in the intracellular compartments. The present study provides the first direct evidence for GLUT9 and GLUT12 expression in VSMCs in conjunction with the previously identified GLUT1 and GLUT10.

Enhanced Survival of Wild-Type and Lurcher Purkinje Cells In Vitro Following Inhibition of Conventional PKCs or Stress-Activated MAP Kinase Pathways

Recent studies using both dissociated and organotypic cell cultures have shown that heterozygous Lurcher (Lc/+) Purkinje cells (PCs) grown in vitro share many of the same survival and morphological characteristics as Lc/+ PCs in vivo. We have used this established tissue culture system as a valuable model for studying cell death mechanisms in a relatively simple system where neurodegeneration is induced by a constitutive cation leak mediated by the Lurcher mutation in the δ2 glutamate receptor (GluRδ2). In this study, Ca(++) imaging and immunocytochemistry studies indicate that intracellular levels of Ca(++) are chronically increased in Lc/+ PCs and the concentration and/or distribution of the conventional PKCγ isoform is altered in degenerating Lc/+ PCs. To begin to characterize the molecular mechanisms that regulate Lc/+ PC death, the contributions of conventional PKC pathways and of two MAP kinase family members, JNK and p38, were examined in slice cultures from wild-type and Lc/+ mutant mouse cerebellum. Cerebellar slice cultures from P0 pups were treated with either a conventional PKC inhibitor, a JNK inhibitor, or a p38 inhibitor either from 0 to 14 or 7 to 14 DIV. Treatment with either of the three inhibitors from 0 DIV significantly increased wild type and Lc/+ PC survival through 14 DIV, but only Lc/+ PC survival was significantly increased following treatments from 7 to 14 DIV. The results suggest that multiple PC death pathways are induced by the physical trauma of making organotypic slice cultures, naturally-occurring postnatal cell death, and the GluRδ2 (Lc) mutation.

Circulation Research Thematic Synopsis

<i>Circulation Research</i> Thematic Synopsis

Protein Kinase Cθ C2 Domain Is a Phosphotyrosine Binding Module That Plays a Key Role in Its Activation

Protein kinase Cθ (PKCθ) is a novel PKC that plays a key role in T lymphocyte activation. To understand how PKCθ is regulated in T cells, we investigated the properties of its N-terminal C2 domain that functions as an autoinhibitory domain. Our measurements show that a Tyr(P)-containing peptide derived from CDCP1 binds the C2 domain of PKCθ with high affinity and activates the enzyme activity of the intact protein. The Tyr(P) peptide also binds the C2 domain of PKCδ tightly, but no enzyme activation was observed with PKCδ. Mutations of PKCθ-C2 residues involved in Tyr(P) binding abrogated the enzyme activation and association of PKCθ with Tyr-phosphorylated full-length CDCP1 and severely inhibited the T cell receptor/CD28-mediated activation of a PKCθ-dependent reporter gene in T cells. Collectively, these studies establish the C2 domain of PKCθ as a Tyr(P)-binding domain and suggest that the domain may play a major role in PKCθ activation via its Tyr(P) binding.

PKC Activation is Required for TSH-mediated Lipolysis via Perilipin Activation

Adipocytes express TSH receptors, and TSH can stimulate cAMP-dependent protein kinase, perilipin phosphorylation, and lipolysis in human and mouse 3T3-L1 adipocytes. TSH activates PKC in thyrocytes. Since PKC has been implicated in lipolysis in adipocytes, we examined whether the family of conventional isoforms of PKC (cPKC) is a target of TSH in adipocytes, and whether cPKC is required for TSH-stimulated lipolysis. Differentiated 3T3-L1 and subcutaneous abdominal human adipocytes in culture were treated with TSH in the presence or absence of either PKC inhibitor Gö6976 (inhibits PKCα, βI) or Gö6983 (inhibits PKCα, βI, βII, γ, δ). Activation of cPKC was assessed by phospho-(ser) PKC substrate antibody immunoblot analysis. Perilipin phosphorylation was measured by SDS-PAGE electromobility shift followed by anti-perilipin immunoblot analysis. Lipolysis was quantified by the amount of nonesterified fatty acids (NEFAs) released into the medium. TSH strongly and significantly activated cPKC in differentiated human and 3T3-L1 adipocytes from undetectable levels in control conditions. This cPKC stimulation in human adipocytes by TSH was reduced significantly by 40% or 48% in the presence of PKC inhibitor Gö6983 or Gö6976, respectively. Gö6976 inhibited TSH-stimulated human adipocyte perilipin phosphorylation and NEFA release by 80% and 50%, respectively. We conclude that cPKC is activated by TSH in human differentiated adipocytes. Based on the effects of cPKC inhibition, cPKC activation is required for TSH-stimulated perilipin phosphorylation and lipolysis in human differentiated adipocytes.

OX2R activation induces PKC-mediated ERK and CREB phosphorylation

Deficiencies in brain orexins and components of mitogen activated protein kinase (MAPK) signaling pathway have been reported in either human depression or animal model of depression. Brain administration of orexins affects behaviors toward improvement of depressive symptoms. However, the documentation of endogenous linkage between orexin receptor activation and MAPK signaling pathway remains to be insufficient. In this study, we report the effects of orexin 2 receptor (OX2R) activation on cell signaling in CHO cells over-expressing OX2R and in mouse hypothalamus cell line CLU172. Short-term extracellular signal-regulated kinase (ERK) phosphorylation and long-term cyclic adenosine monophosphate (cAMP) response element binding protein (CREB) phosphorylation were subsequently observed in CHO cells that over-express OX2R while 20min of ERK phosphorylation was significantly detected in mouse adult hypothalamus neuron cell line CLU172. Orexin A, which can also activate OX2R, mediated ERK phosphorylation was as the same as orexin B in CHO cells. A MAPK inhibitor eliminated ERK phosphorylation but not CREB phosphorylation in CHO cells. Also, ERK and CREB phosphorylation was not mediated by protein kinase A (PKA) or calmodulin kinase (CaMK). However, inhibition of protein kinase C (PKC) by GF 109203X eliminated the phosphorylation of ERK and CREB in CHO cells. A significant decrease in ERK and CREB phosphorylation was observed with 1μM GF 109203X pre-treatment indicating that the conventional and novel isoforms of PKC are responsible for CREB phosphorylation after OX2R activation. In contrast, ERK phosphorylation induced by orexin B in CLU172 cells cannot be inhibited by 1μM of protein kinase C inhibitor.From above observation we conclude that OX2R activation by orexin B induces ERK and CREB phosphorylation and orexin A played the same role as orexin B. Several isoforms of PKC may be involved in prolonged CREB phosphorylation. Orexin B induced ERK phosphorylation in mouse hypothalamus neuron cells differs from CHO cell line and cannot be inhibited by PKC inhibitor GF 109203X. And hypothalamus neuron cells may use different downsteam pathway for orexin B induced ERK phosphorylation. This result supports findings that orexins might have anti-depressive roles.

Identification and characterization of novel factors regulating kinesin‐1 activation and mitochondria dynamics

The Ran‐binding protein 2 (RanBP2) is a mosaic and pleiotropic protein, which associates in vitro and in vivo with the conventional microtubule‐based motor and kinesin‐1 isoforms, KIF5B/KIF5C. We showed previously that the kinesin‐binding domain (KBD) together with the flanking Ran‐binding domains 2 and 3 (RBD2, RBD3) of RanBP2 directly jump‐start and boost the ATPase motor activity of kinesin‐1, a motor implicated in mitochondria transport. The goal of this study is to define the roles of RBD2 and RBD3 of RanBP2 and Ran GTPase in kinesin‐1 activation in vitro and multiple biophysical parameters of mitochondria motility in live cells. We show that the activation of kinesin‐1 by RBD2‐KBDRBD3 is Ran GTPase independent; yet mutations in RBD2‐KBDRBD3 and abolishing its interaction with Ran GTPase suppress the Michaelis‐Menton and partially affect the allosteric activation phases of kinesin‐1. These effects lead to a ~20% reduction of kinesin‐1 activity (kcat). In live cells, KBD alone stimulates the motility, displacement, frequency of change in direction, motility persistence and anterograde velocity of mitochondria, whereas a mutant KBD construct has no effect. In contrast, RBD2‐KBDRBD3 of RanBP2, but not mutant constructs, suppress all these biophysical parameters of mitochondria motility. Further, there is stereochemical specificity between RBD2 and RBD3 in certain parameters of regulation of mitochondria motility. These studies uncover novel Ran GTPase‐independent functions of RBD2, RBD3 and KBD of RanBP2 in kinesin‐1 activation and regulation of mitochondria motility.NIH support GM083165 &amp; EY019492 and RPB

Nitric oxide reduces NADPH oxidase 5 (Nox5) activity by reversible S-nitrosylation

The NADPH oxidases (Noxs) are a family of transmembrane oxidoreductases that produce superoxide and other reactive oxygen species (ROS). Nox5 was the last of the conventional Nox isoforms to be identified and is a calcium-dependent enzyme that does not depend on accessory subunits for activation. Recently, Nox5 was shown to be expressed in human blood vessels and therefore the goal of this study was to determine whether nitric oxide (NO) can modulate Nox5 activity. Endogenously produced NO potently inhibited basal and stimulated Nox5 activity and this inhibition was reversible with chronic, but not acute, exposure to L-NAME. Nox5 activity was reduced by NO donors, iNOS, and eNOS and in endothelial cells and LPS-stimulated smooth muscle cells in a manner dependent on NO concentration. ROS production was diminished by NO in an isolated enzyme activity assay replete with surplus calcium and NADPH. There was no evidence for NO-dependent changes in tyrosine nitration, glutathiolation, or phosphorylation of Nox5. In contrast, there was evidence for the increased nitrosylation of Nox5 as determined by the biotin-switch assay and mass spectrometry. Four S-nitrosylation sites were identified and of these, mutation of C694 dramatically lowered Nox5 activity, NO sensitivity, and biotin labeling. Furthermore, coexpression of the denitrosylation enzymes thioredoxin 1 and GSNO reductase prevented NO-dependent inhibition of Nox5. The potency of NO against other Nox enzymes was in the order Nox1 ≥ Nox3 > Nox5 > Nox2, whereas Nox4 was refractory. Collectively, these results suggest that endogenously produced NO can directly S-nitrosylate and inhibit the activity of Nox5.

Myosin Heavy Chain Composition of the Human Genioglossus Muscle

The human tongue muscle genioglossus (GG) is active in speech, swallowing, respiration, and oral transport, behaviors encompassing a wide range of tongue shapes and movement speeds. Studies demonstrate substantial diversity in patterns of human GG motor unit activation, but whether this is accompanied by complex expression of muscle contractile proteins is not known. The authors tested for conventional myosin heavy chain (MHC) MHCI, MHCIIA, MHCIIX, developmental MHCembryonic and MHCneonatal and unconventional MHCαcardiac, MHCextraocular, and MHCslow tonic in antero-superior (GG-A) and posterior (GG-P) adult human GG. SDS-PAGE, Western blot, and immunohistochemistry were used to describe MHC composition of GG-A and GG-P and the prevalence of muscle fiber MHC phenotypes in GG-A. By SDS-PAGE, only conventional MHC are present with ranking from most to least prevalent MHCIIA > MHCI > MHCIIX in GG-A and MHCI > MHCIIA > MHCIIX in GG-P. By immunohistochemistry, many muscle fibers contain MHCI, MHCIIA, and MHCIIX, but few contain developmental or unconventional MHC. GG-A is composed of 5 phenotypes (MHCIIA > MHCI-IIX > MHCI > MHCI-IIA > MHCIIX). Phenotypes MHCI, MHCIIA, and MHCI-IIX account for 96% of muscle fibers. Despite activation of GG during kinematically diverse behaviors and complex patterns of GG motor unit activity, the human GG is composed of conventional MHC isoforms and 3 primary MHC phenotypes.

CRM1 Protein-mediated Regulation of Nuclear Clusterin (nCLU), an Ionizing Radiation-stimulated, Bax-dependent Pro-death Factor

Expression of the clusterin (CLU) gene results in the synthesis of a conventional secretory isoform set (pre- and mature secretory clusterin proteins, psCLU/sCLU), as well as another set of intracellular isoforms, appearing in the cytoplasm (pre-nuclear CLU, pnCLU) and in the nucleus as an ∼55-kDa mature nuclear clusterin (nCLU) form. These two isoform sets have opposing cell functions: pro-survival and pro-death, respectively. Although much is known about the regulation and function of sCLU as a pro-survival factor, the regulation and function of endogenous nCLU in cell death are relatively unexplored. Here, we show that depletion of endogenous nCLU protein using siRNA specific to its truncated mRNA increased clonogenic survival of ionizing radiation (IR)-exposed cells. nCLU-mediated apoptosis was Bax-dependent, and lethality correlated with accumulation of mature nCLU protein. nCLU accumulation was regulated by CRM1 because binding between CRM1 and nCLU proteins was significantly diminished by leptomycin B (LMB), and nuclear levels of nCLU protein were significantly enhanced by LMB and IR co-treatment. Moreover, LMB treatment significantly enhanced IR-induced nCLU-mediated cell death responses. Importantly, bax(-/-) and bax(-/-)/bak(-/-) double knock-out cells were resistant to nCLU-mediated cell death, whereas bak(-/-) or wild-type bax(+/+)/bak(+/+) cells were hypersensitive. The regulation of nCLU by CRM1 nuclear export/import may explain recent clinical results showing that highly malignant tumors have lost the ability to accumulate nCLU levels, thereby avoiding growth inhibition and cell death.

Phospholipase D: enzymology, functionality, and chemical modulation.

Phosphatidic acid (PA) is a critical phospholipid constituent in eukaryotic cell membranes, that accounts for 1–4 % of the total lipid.1 This lipophilic glycerophospholipid has a phosphate head group, and as such serves not only a structural capacity in lipid bilayers, but also participates as an intermediate in lipid metabolism and as a signaling molecule. Because of the small head group, PA facilitates changes in lipid bilayer curvature that are important for membrane fusion events, such as vesicular trafficking and endocytosis.2 PA is also a precursor to other lipid signaling molecules including diacylglycerol (DAG) and lysophosphatidic acid (LPA). As a lipid second messenger, PA activates signaling proteins and acts as a node within the membrane to which signaling proteins translocate. Several signaling proteins, including Raf-13,4 and mTOR,5 directly bind PA to mediate translocation or activation, respectively. PA has been implicated in signaling cascades involving cell growth, proliferation, and survival. Aberrant PA signaling has been identified in multiple cancers,6 neurodegeneration,7 and platelet aggregation,8 which makes proteins that mediate cellular levels of PA attractive as potential therapeutic targets. PA can be generated de novo9,10,11 by sequential enzyme-catalyzed acylations of glycerol-3-phosphate, or in response to cell signaling pathways (Figure 1). Every glycerophospholipid generated in eukaryotic membranes transitions through PA, a pathway characterized by Eugene Kennedy and his colleagues more than half a century ago.11,12 Signal generated PA is formed by enzymes that modify existing lipids. These enzymes include lysophosphatidic acid acyltransferase (LPAAT) which acylates LPA, DAG kinase which phosphorylates DAG at the sn-3 position, and phospholipase D (PLD) which hydrolyzes the headgroup of a phospholipid, generally phosphatidylcholine (PC), triggering the release of choline. Figure 1 PLD activity, an enzyme catalyzed hydrolysis of a phosphodiester bond, was first described in plants,13,14,15,16 and subsequently many enzymes from a range of viral, prokaryotic and eukaryotic organisms have been described as possessing PLD activity. To date, more than 4000 PLD enzymes have been entered in NCBI GenBank. The majority of these enzymes hydrolyze phosphodiester bonds within phospholipids such as PC (classified as EC 3.1.4.417), but there are other enzymes ascribed to having PLD activity that hydrolyze neutral lipids and even polynucleotide backbone. A large subset of enzymes with PLD activity share a conserved HxKxxxxDx6GSxN motif (HKD),18 or a variation thereof, that is responsible for catalytic activity. These enzymes are members of the PLD superfamily, and are proposed to follow a similar reaction mechanism. Non-HKD enzymes exhibiting PLD activity have divergent structures and catalytic mechanisms. These non-HKD enzymes are discussed here as a means of comparison. In this comprehensive review of the PLD superfamily, specific emphasis is given to the conventional mammalian isoforms, PLD1 and PLD2, and the tools with which these enzymes are studied. The merits of PLD as a potential therapeutic target are also reviewed, as are implications for modulation of PLD activity in cell signaling pathways, whole organisms, and aberrant or disease-related models.

Characterization of Neuroprotective Effects of Biphalin, an Opioid Receptor Agonist, in a Model of Focal Brain Ischemia

Approximately 795,000 people experience a new or recurrent stroke in the United States annually. The purpose of this study was to assess the protective effect of a nonselective opioid receptor agonist, biphalin, in brain edema and infarct damage by using both in vitro and in vivo models of stroke. In an in vivo model of ischemia, biphalin significantly decreased edema (66.6 and 58.3%) and infarct (52.2 and 56.4%) ratios in mouse transient (60-min occlusion/24-h reperfusion) and permanent (6 h) middle cerebral artery occlusion models, respectively. Biphalin administration also showed decreased neurodegeneration in hippocampal, cortical, and striatal brain tissue after ischemia, evidenced by reduced Fluoro-Jade C staining. In addition, biphalin improved neurological function after stroke injury evidenced by neurological score and locomotor activity evaluation. Biphalin significantly decreased penumbral expression of Na(+), K(+), 2Cl(-) cotransporter (NKCC) and the translocation of the conventional isoforms of protein kinase C (PKC). It also reversed the activation of PKC-induced cell volume increase during ischemia in primary neuronal cell cultures exposed to 1 h of oxygen glucose deprivation. These data suggest that opioid receptor activation provides neuroprotection during stroke, and a possible explanation of this mechanism could be the inhibition of NKCC function via the regulation of PKC-dependent cell signaling.

Critical Role for CD38-mediated Ca2+ Signaling in Thrombin-induced Procoagulant Activity of Mouse Platelets and Hemostasis

CD38, a multifunctional enzyme that catalyzes the synthesis of intracellular Ca(2+) messengers, cyclic ADP-ribose (cADPR) and nicotinic acid adenine dinucleotide phosphate (NAADP), is known to be expressed on platelets. However, the role of CD38 in platelets remains unclear. Our present results show that treatment of platelets with thrombin results in a rapid and sustained Ca(2+) signal, resulting from a coordinated interplay of Ca(2+)-mobilizing messengers, inositol 1,4,5-trisphosphate, cADPR, and NAADP. By dissecting the signaling pathway using various agents, we delineated that cADPR and NAADP are sequentially produced through CD38 internalization by protein kinase C via myosin heavy chain IIA following phospholipase C activation in thrombin-induced platelets. An inositol 1,4,5-trisphosphate receptor antagonist blocked the thrombin-induced formation of cADPR and NAADP as well as Ca(2+) signals. An indispensable response of platelets relying on cytosolic calcium is the surface exposure of phosphatidylserine (PS), which implicates platelet procoagulant activity. Scrutinizing this parameter reveals that CD38(+/+) platelets fully express PS on the surface when stimulated with thrombin, whereas this response was decreased on CD38(-/-) platelets. Similarly, PS exposure and Ca(2+) signals were attenuated when platelets were incubated with 8-bromo-cADPR, bafilomycin A1, and a PKC inhibitor. Furthermore, in vivo, CD38-deficient mice exhibited longer bleeding times and unstable formation of thrombus than wild type mice. These results demonstrate that CD38 plays an essential role in thrombin-induced procoagulant activity of platelets and hemostasis via Ca(2+) signaling mediated by its products, cADPR and NAADP.

Activation and localization of protein kinase C in Neurospora crassa

The Neurospora crassa protein kinase C (NPKC) is reported to be a regulator of light responsive genes. It phosphorylates the light receptor WC-1 and regulates the levels of the circadian clock protein FRQ and transcription of the light-induced albino-2 gene. In mammals, the conventional and novel isoforms of PKC are activated by diacylglycerol (DAG), which induces PKC translocation from the cytoplasm to membranes. To investigate the interaction of NPKC and DAG in Neurospora, we constructed a strain that expresses a PKC-GFP fusion protein. We found that NPKC localizes to growing tips and sub-apical plasma membrane in actively growing hyphae, and actively participates in septum development. NPKC is activated by exogenous DAG and phorbol esters, and translocates to the plasma membrane from the cytoplasm. We have previously reported that choline depletion of the chol-1 mutant of Neurospora increases DAG levels and lengthens the period of the circadian rhythm of conidiation. We have found that the activity of NPKC is rhythmic, and that NPKC levels are increased on choline depletion. However, over-expression of NPKC did not lengthen the conidiation period, indicating that PKC in Neurospora may not be responsible for the lengthened period in low choline cultures.

Activated PKCδ and PKCϵ Inhibit Epithelial Chloride Secretion Response to cAMP via Inducing Internalization of the Na+-K+-2Cl− Cotransporter NKCC1

The basolateral Na(+)-K(+)-2Cl(-) cotransporter (NKCC1) is a key determinant of transepithelial chloride secretion and dysregulation of chloride secretion is a common feature of many diseases including secretory diarrhea. We have previously shown that activation of protein kinase C (PKC) markedly reduces transepithelial chloride secretion in human colonic T84 cells, which correlates with both functional inhibition and loss of the NKCC1 surface expression. In the present study, we defined the specific roles of PKC isoforms in regulating epithelial NKCC1 and chloride secretion utilizing adenoviral vectors that express shRNAs targeting human PKC isoforms (α, δ, ε) (shPKCs) or LacZ (shLacZ, non-targeting control). After 72 h of adenoviral transduction, protein levels of the PKC isoforms in shPKCs-T84 cells were decreased by ∼90% compared with the shLacZ-control. Activation of PKCs by phorbol 12-myristate 13-acetate (PMA) caused a redistribution of NKCC1 immunostaining from the basolateral membrane to intracellular vesicles in both shLacZ- and shPKCα-T84 cells, whereas the effect of PMA was not observed in shPKCδ- and shPKCε- cells. These results were further confirmed by basolateral surface biotinylation. Furthermore, activation of PKCs by PMA inhibited cAMP-stimulated chloride secretion in the uninfected, shLacZ- and shPKCα-T84 monolayers, but the inhibitory effect was significantly attenuated in shPKCδ- and shPKCε-T84 monolayers. In conclusion, the activated novel isoforms PKCδ or PKCε, but not the conventional isoform PKCα, inhibits transepithelial chloride secretion through inducing internalization of the basolateral surface NKCC1. Our study reveals that the novel PKC isoform-regulated NKCC1 surface expression plays an important role in the regulation of chloride secretion.