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1.
Antibodies that specifically recognize signaling proteins (or individual phosphorylation events at specific residues in proteins of interest) have become important tools in the study of signaling pathways. However, the recognition properties of many commercially available antibodies have not been fully characterized. In the course of studies exploring PKC- phosphorylation mechanisms in cardiomyocytes, we have demonstrated that a BD Transduction Laboratories anti-PKC- MAb (generally viewed as an anti-PKC- protein antibody) recognizes PKC- in resting, but not in PMA-treated, cardiomyocytes. The observations that PKC- immunoreactivity is preserved when cultures are treated with PMA in the presence of a the PKC inhibitor GF-109203X and that PKC- immunoreactivity is restored by in vitro acid phosphatase treatment indicate that the epitope recognized by the BD Transduction Laboratories anti-PKC- MAb is masked by phosphorylation. The BD Transduction Laboratories MAb is poorly suited for studies that compare PKC- expression in resting and agonist-activated samples (or in studies of the relationship between PKC- phosphorylation and PKC- downregulation) because it artifactually displays PKC- phosphorylation as a decline in total PKC- protein. Other studies have shown that two anti-PKC--pY311 antibodies (manufactured by Cell Signaling Technology, Beverly, MA, and BioSource International, Camarillo, CA, respectively) specifically recognize stimulus-induced changes in PKC--Y311 phosphorylation on the endogenous PKC- enzyme, but the Cell Signaling Technology anti-PKC--pY311 antibody provides a better measure of Y311 phosphorylation in overexpressed PKC-. Collectively, these studies have identified features of anti-PKC- antibodies that affect the interpretation of immunoblot analysis experiments. These findings related to PKC- may be symptomatic of a more pervasive feature of immunoblot analysis studies of phosphoproteins in general. protein phosphorylation; signal transduction pathways; cardiomyocytes  相似文献   

2.
Protein kinase C (PKC) plays a critical role in diseases such as cancer, stroke, and cardiac ischemia and participates in a variety of signal transduction pathways including apoptosis, cell proliferation, and tumor suppression. Here, we demonstrate that PKC is proteolytically cleaved and translocated to the nucleus in a time-dependent manner on treatment of desferroxamine (DFO), a hypoxia-mimetic agent. Specific knockdown of the endogenous PKC by RNAi (sh-PKC) or expression of the kinase-dead (Lys376Arg) mutant of PKC (PKCKD) conferred modulation on the cellular adaptive responses to DFO treatment. Notably, the time-dependent accumulation of DFO-induced phosphorylation of Ser-139-H2AX (-H2AX), a hallmark for DNA damage, was altered by sh-PKC, and sh-PKC completely abrogated the activation of caspase-3 in DFO-treated cells. Expression of Lys376Arg-mutated PKC-enhanced green fluorescent protein (EGFP) appears to abrogate DFO/hypoxia-induced activation of endogenous PKC and caspase-3, suggesting that PKCKD-EGFP serves a dominant-negative function. Additionally, DFO treatment also led to the activation of Chk1, p53, and Akt, where DFO-induced activation of p53, Chk1, and Akt occurred in both PKC-dependent and -independent manners. In summary, these findings suggest that the activation of a PKC-mediated signaling network is one of the critical contributing factors involved in fine-tuning of the DNA damage response to DFO treatment. DNA damage; caspase-3; Akt  相似文献   

3.
Direct binding of nonmuscle F-actin and the C2-like domain of PKC- (C2-like domain) is involved in hormone-mediated activation of epithelial Na-K-2Cl cotransporter isoform 1 (NKCC1) in a Calu-3 airway epithelial cell line. The goal of this study was to determine the site of actin binding on the 123-amino acid C2-like domain. Truncations of the C2-like domain were made by restriction digestion and confirmed by nucleotide sequencing. His6-tagged peptides were expressed in bacteria, purified, and analyzed with a Coomassie blue stain for predicted size and either a 6xHis protein tag stain or an INDIA His6 probe for expression of the His6 tag. Truncated peptides were tested for competitive inhibition of binding of activated, recombinant PKC- with nonmuscle F-actin. Peptides from the NH2-terminal region, but not the COOH-terminal region, of the C2-like domain blocked binding of activated PKC- to F-actin. The C2-like domain and three NH2-terminal truncated peptides of 17, 83, or 108 amino acids blocked binding, with IC50 values ranging from 1.2 to 2.2 nmol (6–11 µM). NH2-terminal C2-like peptides also prevented methoxamine-stimulated NKCC1 activation and pulled down endogenous actin from Calu-3 cells. The proximal NH2 terminus of the C2-like domain encodes a 1-sheet region. The amino acid sequence of the actin-binding domain is distinct from actin-binding domains in other PKC isotypes and actin-binding proteins. Our results indicate that F-actin likely binds to the 1-sheet region of the C2-like domain in airway epithelial cells. truncation; protein kinase C-; C2-like domain; slot blot assay; inhibitory constant; bumetanide; Na-K-2Cl cotransporter  相似文献   

4.
Activation of PLC-delta1 by Gi/o-coupled receptor agonists   总被引:1,自引:0,他引:1  
The mechanism of phospholipase (PLC)- activation by G protein-coupled receptor agonists was examined in rabbit gastric smooth muscle. Ca2+ stimulated an eightfold increase in PLC-1 activity in permeabilized muscle cells. Treatment of dispersed or cultured muscle cells with three Gi/o-coupled receptor agonists (somatostatin, -opioid agonist [D-Pen2,D-Pen5]enkephalin, and A1 agonist cyclopentyl adenosine) caused delayed increase in phosphoinositide (PI) hydrolysis (8- to 10-fold) that was strongly inhibited by overexpression of dominant-negative PLC-1(E341R/D343R; 65–76%) or constitutively active RhoA(G14V). The response coincided with capacitative Ca2+ influx and was not observed in the absence of extracellular Ca2+, but was partly inhibited by nifedipine (16–30%) and strongly inhibited by SKF-96365, a blocker of store-operated Ca2+ channels. Treatment of the cells with a Gq/13-coupled receptor agonist, CCK-8, caused only transient, PLC-1-mediated PI hydrolysis. Unlike Gi/o-coupled receptor agonists, CCK-8 activated RhoA and stimulated RhoA:PLC-1 association. Inhibition of RhoA activity with C3 exoenzyme or by overexpression of dominant-negative RhoA(T19N) or G13 minigene unmasked a delayed increase in PI hydrolysis that was strongly inhibited by coexpression of PLC-1(E341R/D343R) or by SKF-96365. Agonist-independent capacitative Ca2+ influx induced by thapsigargin stimulated PI hydrolysis (8-fold), which was partly inhibited by nifedipine (25%) and strongly inhibited by SKF-96365 (75%) and in cells expressing PLC-1(E341R/D343R). Agonist-independent Ca2+ release or Ca2+ influx via voltage-gated Ca2+ channels stimulated only moderate PI hydrolysis (2- to 3-fold), which was abolished by PLC-1 antibody or nifedipine. We conclude that PLC-1 is activated by Gi/o-coupled receptor agonists that do not activate RhoA. The activation is preferentially mediated by Ca2+ influx via store-operated Ca2+ channels. phospholipase C; G protein  相似文献   

5.
Airway goblet cell mucin secretion is controlled by agonist activation of P2Y2 purinoceptors, acting through Gq/PLC, inositol-1,4,5-trisphosphate (IP3), diacylglycerol, Ca2+ and protein kinase C (PKC). Previously, we showed that SPOC1 cells express cPKC, nPKC, nPKC, and nPKC; of these, only nPKC translocated to the membrane in correlation with mucin secretion (Abdullah LH, Bundy JT, Ehre C, Davis CW. Am J Physiol Lung Physiol 285: L149–L160, 2003). We have verified these results and pursued the identity of the PKC effector isoform by testing the effects of altered PKC expression on regulated mucin release using SPOC1 cell and mouse models. SPOC1 cells overexpressing cPKC, nPKC, and nPKC had the same levels of ATPS- and phorbol-1,2-myristate-13-acetate (PMA)-stimulated mucin secretion as the levels in empty retroviral vector expressing cells. Secretagogue-induced mucin secretion was elevated only in cells overexpressing nPKC (14.6 and 23.5%, for ATPS and PMA). Similarly, only SPOC1 cells infected with a kinase-deficient nPKC exhibited the expected diminution of stimulated mucin secretion, relative to wild-type (WT) isoform overexpression. ATPS-stimulated mucin secretion from isolated, perfused mouse tracheas was diminished in P2Y2-R null mice by 82% relative to WT mice, demonstrating the utility of mouse models in studies of regulated mucin secretion. Littermate WT and nPKC knockout (KO) mice had nearly identical levels of stimulated mucin secretion, whereas mucin release was nearly abolished in nPKC KO mice relative to its WT littermates. We conclude that nPKC is the effector isoform downstream of P2Y2-R activation in the goblet cell secretory response. The translocation of nPKC observed in activated cells is likely not related to mucin secretion but to some other aspect of goblet cell biology. protein kinase C; mucins; goblet cells; exocytosis; airways; epithelium; lung  相似文献   

6.
ATP, a purinergic receptor agonist, has been shown to be involved in vascular smooth muscle (VSM) cell DNA synthesis and cell proliferation during embryonic and postnatal development, after injury, and in atherosclerosis. One mechanism that ATP utilizes to regulate cellular function is through activation of ERK1/2. In the present study, we provide evidence that ATP-dependent activation of ERK1/2 in VSM cells utilizes specific isoforms of the multifunctional serine/threonine kinases, PKC, and Ca2+/calmodulin-dependent protein kinase II (CaMKII) as intermediates. Selective inhibition of PKC- activity with rottlerin, or adenoviral overexpression of kinase-negative PKC-, attenuated the ATP- and phorbol 12,13-dibutyrate (PDBu)-stimulated ERK1/2 activation. Inhibition of PKC- activity with Gö-6976, or adenoviral overexpression of kinase-negative PKC-, was ineffective. Alternatively, treatment with KN-93, a selective inhibitor of CaMKII activation, or adenoviral overexpression of kinase-negative CaMKII-2, inhibited ATP-dependent activation of ERK1/2 but had no effect on PDBu- or PDGF-stimulated ERK1/2. In addition, adenoviral overexpression of dominant-negative ras (Ad.HA-RasN17) partially inhibited the ATP- and PDBu-induced activation of ERK1/2 and blocked ionomycin- and EGF-stimulated ERK1/2, and inhibition of tyrosine kinases with AG-1478, an EGFR inhibitor, or the src family kinase inhibitor PP2 attenuated ATP-stimulated ERK1/2 activation. Taken together, these data indicate that PKC- and CaMKII-2 coordinately mediate ATP-dependent transactivation of EGF receptor, resulting in increased ERK1/2 activity in VSM cells. protein kinase C-; calcium/calmodulin-dependent protein kinase II- 2; extracellular signal-regulated kinase 1/2; epidermal growth factor receptor transactivation; adenovirus  相似文献   

7.
Previous studies demonstrated a requirement for multifunctional Ca2+/calmodulin-dependent protein kinase II (CaMKII) in PDGF-stimulated vascular smooth muscle (VSM) cell migration. In the present study, molecular approaches were used specifically to assess the role of the predominant CaMKII isoform (2 or C) on VSM cell migration. Kinase-negative (K43A) and constitutively active (T287D) mutant forms of CaMKII2 were expressed using recombinant adenoviruses. CaMKII activities were evaluated in vitro by using a peptide substrate and in intact cells by assessing the phosphorylation of overexpressed phospholamban on Thr17, a CaMKII-selective phosphorylation site. Expression of kinase-negative CaMKII2 inhibited substrate phosphorylation both in vitro and in the intact cell, indicating a dominant-negative function with respect to exogenous substrate. However, overexpression of the kinase-negative mutant failed to inhibit endogenous CaMKII2 autophosphorylation on Thr287 after activation of cells with ionomycin, and in fact, these subunits served as a substrate for the endogenous kinase. Constitutively active CaMKII2 phosphorylated substrate in vitro without added Ca2+/calmodulin and in the intact cell without added Ca2+-dependent stimuli, but it inhibited autophosphorylation of endogenous CaMKII2 on Thr287. Basal and PDGF-stimulated cell migration was significantly enhanced in cells expressing kinase-negative CaMKII2, an effect opposite that of KN-93, a chemical inhibitor of CaMKII activation. Expression of the constitutively active CaMKII2 mutant inhibited PDGF-stimulated cell migration. These studies point to a role for the CaMKII2 isoform in regulating VSM cell migration. An inclusive interpretation of results using both pharmacological and molecular approaches raises the hypothesis that CaMKII2 autophosphorylation may play an important role in PDGF-stimulated VSM cell migration. calcium/calmodulin-dependent protein kinase II; cell migration; adenovirus; autophosphorylation; chemotaxis; platelet-derived growth factor  相似文献   

8.
Transforming growth factor- (TGF-) stimulates myofibroblast transdifferentiation, leading to type I collagen accumulation and fibrosis. We investigated the function of Src in TGF--induced collagen I accumulation. In human mesangial cells, PTyr416 Src (activated Src) was 3.3-fold higher in TGF--treated cells than in controls. Src activation by TGF- was blocked by rottlerin and by a dominant negative mutant of protein kinase C (PKC), showing that TGF- activates Src by a PKC-based mechanism. Pharmacological inhibitors and a dominant negative Src mutant prevented the increase in collagen type I secretion in cells exposed to TGF-. Similarly, on-target Src small interference RNA (siRNA) prevented type I collagen secretion in response to TGF-, but off-target siRNA complexes had no effect. It is well established in mesangial cells that upregulation of type I collagen by TGF- requires extracellular signal-regulated kinase 1/2 (ERK1/2), and we found that activation of ERK1/2 by TGF- requires Src. In conclusion, these results suggest that stimulation of collagen type I secretion by TGF- requires a PKC-Src-ERK1/2 signaling motif. mesangial cells; fibrosis; glomerulus; transforming growth factor-  相似文献   

9.
There is accumulating evidence that Ca2+-dependent signaling pathways regulate proliferation and migration of vascular smooth muscle (VSM) cells, contributing to the intimal accumulation of VSM that is a hallmark of many vascular diseases. In this study we investigated the role of the multifunctional serine/threonine kinase, calmodulin (CaM)-dependent protein kinase II (CaMKII), as a mediator of Ca2+ signals regulating VSM cell proliferation. Differentiated VSM cells acutely isolated from rat aortic media express primarily CaMKII gene products, whereas passaged primary cultures of de-differentiated VSM cells express primarily CaMKII2, a splice variant of the gene. Experiments examining the time course of CaMKII isoform modulation revealed the process was rapid in onset following initial dispersion and primary culture of aortic VSM with a significant increase in CaMKII2 protein and a significant decrease in CaMKII protein within 30 h, coinciding with the onset of DNA synthesis and cell proliferation. Attenuating the initial upregulation of CaMKII2 in primary cultured cells using small-interfering RNA (siRNA) resulted in decreased serum-stimulated DNA synthesis and cell proliferation in primary culture. In passaged VSM cells, suppression of CaMKII2 activity by overexpression of a kinase-negative mutant, or suppression of endogenous CaMKII content using multiple siRNAs, significantly attenuated serum-stimulated DNA synthesis and cell proliferation. Cell cycle analysis following either inhibitory approach indicated decreased proportion of cells in G1, an increase in proportion of cells in G2/M, and an increase in polyploidy, corresponding with accumulation of multinucleated cells. These results indicate that CaMKII2 is specifically induced during modulation of VSM cells to the synthetic phenotypic and is a positive regulator of serum-stimulated proliferation. calmodulin kinase II; phenotype modulation  相似文献   

10.
The involvement of PKC, the isoforms of which are categorized into three subtypes: conventional (, I, II, and ), novel [, , , and µ (also known as PKD),], and atypical ( and /), in the regulation of endothelial monolayer integrity is well documented. However, isoform activity varies among different cell types. Our goal was to reveal isoform-specific PKC activity in the microvascular endothelium in response to phorbol 12-myristate 13-acetate (PMA) and diacylglycerol (DAG). Isoform activity was demonstrated by cytosol-to-membrane translocation after PMA treatment and phosphorylation of the myristoylated alanine-rich C kinase substrate (MARCKS) protein after PMA and DAG treatment. Specific isoforms were inhibited by using both antisense oligonucleotides and pharmacological agents. The data showed partial cytosol-to-membrane translocation of isoforms , I, and and complete translocation of PKC and PKD in response to PMA. Furthermore, antisense treatment and pharmacological studies indicated that the novel isoform PKC and PKD are both required for PMA- and DAG-induced MARCKS phosphorylation and hyperpermeability in pulmonary microvascular endothelial cells, whereas isoforms , I, and were dispensable with regard to these same phenomena. signal transduction; permeability; myristolated alanine-rich C kinase substrate; microvasculature; pulmonary endothelium  相似文献   

11.
To elucidate signaling pathways activated by IL-1 and IL-6 that contribute to increased expression of plasminogen activator inhibitor-1 (PAI-1), we studied human hepatoma (HepG2) cells and primary mouse hepatocytes. HepG2 cell PAI-1 mRNA increased in response to IL-1, IL-6, and IL-1 plus IL-6 as shown by real-time PCR. Activity of the transiently transfected PAI-1 promoter (–829 to +36 bp) increased as well. Systematic promoter deletion assays showed that the region from –239 to –210 bp containing a putative CCAAT-enhancer binding protein (C/EBP) binding site was critical. Point mutations in this region abolished the IL-1 and IL-6 responses. Antibody interference electrophoretic mobility shift assays showed that C/EBP (but not C/EBP or C/EBP) binding and protein were increased by IL-1, IL-6, and IL-1 plus IL-6 in HepG2 cells. IL-1 and IL-6 increased expression of both PAI-1 mRNA and C/EBP mRNA in mouse primary hepatocytes as well. Downregulation of C/EBP induced with small interfering RNA (siRNA) decreased secretion of PAI-1. As judged from results obtained with inhibitors, signal transduction in all three of the mitogen-activated protein kinase pathways was involved in IL-1-inducible PAI-1 expression. By contrast, JAK signaling was responsible for the IL-6-induced inducible expression. Thus IL-1 and IL-6 exert directionally similar effects on PAI-1 expression, but the induction involves distinct signaling pathways with a final common mediator, C/EBP. CCAAT-enhancer binding protein; interleukin-1; interleukin-6; statins; thrombosis  相似文献   

12.
Protein kinase C (PKC) is involved in the process of ischemic preconditioning (IPC), although the precise mechanism is still a subject of debate. Using specific PKC inhibitors, we investigated which PKC isoforms were involved in IPC of the human atrial myocardium sections and to determine their temporal relationship to the opening of mitochondrial potassium-sensitive ATP (mitoKATP) channels. Right atrial muscles obtained from patients undergoing elective cardiac surgery were equilibrated and then randomized to receive any of the following protocols: aerobic control, 90-min simulated ischemia/120-min reoxygenation, IPC using 5-min simulated ischemia/5-min reoxygenation followed by 90-min simulated ischemia/120-min reoxygenation and finally, PKC inhibitors were added 10 min before and 10 min during IPC followed by 90-min simulated ischemia/120-min reoxygenation. The PKC isoforms inhibitors investigated were V1–2 peptide, GO-6976, rottlerin, and LY-333531 for PKC-, -, - and -, respectively. To investigate the relation of PKC isoforms to mitoKATP channels, PKC inhibitors found to be involved in IPC were added 10 min before and 10 min during preconditioning by diazoxide followed by 90-min simulated ischemia/120-min reoxygenation in a second experiment. Creatine kinase leakage and methylthiazoletetrazolium cell viability were measured. Phosphorylation of PKC isoforms after activation of the sample by either diazoxide or IPC was detected by using Western blot analysis and then analyzed by using Scion image software. PKC- and - inhibitors blocked IPC, whereas PKC- and - inhibitors did not. The protection elicited by diazoxide, believed to be via mitoKATP channels opening, was blocked by the inhibition of PKC- but not - isoforms. In addition, diazoxide caused increased phosphorylation of PKC- to the same extent as IPC but did not affect the phosphorylation of PKC-, a process believed to be critical in PKC activation. The results demonstrate that PKC- and - are involved in IPC of the human myocardium with PKC- being upstream and PKC- being downstream of mitoKATP channels. cardioprotection; protein kinase C isoforms  相似文献   

13.
We investigated the involvement of PKC- in apical actin remodeling in carbachol-stimulated exocytosis in reconstituted rabbit lacrimal acinar cells. Lacrimal acinar PKC- cosedimented with actin filaments in an actin filament binding assay. Stimulation of acini with carbachol (100 µM, 2–15 min) significantly (P 0.05) increased PKC- recovery with actin filaments in two distinct biochemical assays, and confocal fluorescence microscopy showed a significant increase in PKC- association with apical actin in stimulated acini as evidenced by quantitative colocalization analysis. Overexpression of dominant-negative (DN) PKC- in lacrimal acini with replication-defective adenovirus (Ad) resulted in profound alterations in apical and basolateral actin filaments while significantly inhibiting carbachol-stimulated secretion of bulk protein and -hexosaminidase. The chemical inhibitor GF-109203X (10 µM, 3 h), which inhibits PKC-, -, -, and -, also elicited more potent inhibition of carbachol-stimulated secretion relative to Gö-6976 (10 µM, 3 h), which inhibits only PKC- and -. Transduction of lacrimal acini with Ad encoding syncollin-green fluorescent protein (GFP) resulted in labeling of secretory vesicles that were discharged in response to carbachol stimulation, whereas cotransduction of acini with Ad-DN-PKC- significantly inhibited carbachol-stimulated release of syncollin-GFP. Carbachol also increased the recovery of secretory component in culture medium, whereas Ad-DN-PKC- transduction suppressed its carbachol-stimulated release. We propose that DN-PKC- alters lacrimal acinar apical actin remodeling, leading to inhibition of stimulated exocytosis and transcytosis. lacrimal gland; acinar epithelial cell; exocytosis; polymeric immunoglobulin A receptor  相似文献   

14.
Protein kinase D (PKD) is a novel protein serine kinase that has recently been implicated in diverse cellular functions, including apoptosis and cell proliferation. The purpose of our present study was 1) to define the activation of PKD in intestinal epithelial cells treated with H2O2, an agent that induces oxidative stress, and 2) to delineate the upstream signaling mechanisms mediating the activation of PKD. We found that the activation of PKD is induced by H2O2 in both a dose- and time-dependent fashion. PKD phosphorylation was attenuated by rottlerin, a selective PKC- inhibitor, and by small interfering RNA (siRNA) directed against PKC-, suggesting the regulation of PKD activity by upstream PKC-. Activation of PKD was also blocked by a Rho kinase (ROK)-specific inhibitor, Y-27632, as well as by C3, a Rho protein inhibitor, demonstrating that the Rho/ROK pathway also mediates PKD activity in intestinal cells. In addition, H2O2-induced PKC- phosphorylation was inhibited by C3 treatment, further suggesting that PKC- is downstream of Rho/ROK. Interestingly, H2O2-induced intestinal cell apoptosis was enhanced by PKD siRNA. Together, these results clearly demonstrate that oxidative stress induces PKD activation in intestinal epithelial cells and that this activation is regulated by upstream PKC- and Rho/ROK pathways. Importantly, our findings suggest that PKD activation protects intestinal epithelial cells from oxidative stress-induced apoptosis. These findings have potential clinical implications for intestinal injury associated with oxidative stress (e.g., necrotizing enterocolitis in infants). Rho kinase; protein kinase C-  相似文献   

15.
We studied the functions of -subunits of Gi/o protein using the Xenopus oocyte expression system. Isoproterenol (ISO) elicited cAMP production and slowly activating Cl currents in oocytes expressing 2-adrenoceptor and the protein kinase A-dependent Cl channel encoded by the cystic fibrosis transmembrane conductance regulator (CFTR) gene. 5-Hydroxytryptamine (5-HT), [D-Ala2, D-Leu5]-enkephalin (DADLE), and baclofen enhanced ISO-induced cAMP levels and CFTR currents in oocytes expressing 2-adrenoceptor-CFTR and 5-HT1A receptor (5-HT1AR), -opioid receptor, or GABAB receptor, respectively. 5-HT also enhanced pituitary adenylate cyclase activating peptide (PACAP) 38-induced cAMP levels and CFTR currents in oocytes expressing PACAP receptor, CFTR and 5-HT1AR. The 5-HT-induced enhancement of Gs-coupled receptor-mediated currents was abrogated by pretreatment with pertussis toxin (PTX) and coexpression of G transducin (Gt). The 5-HT-induced enhancement was further augmented by coexpression of the G-activated form of adenylate cyclase (AC) type II but not AC type III. Thus -subunits of Gi/o protein contribute to the enhancement of Gs-coupled receptor-mediated responses. 5-HT and DADLE did not elicit any currents in oocytes expressing 5-HT1AR or -opioid receptor alone. They elicited Ca2+-activated Cl currents in oocytes coexpressing these receptors with the G-activated form of phospholipase C (PLC)-2 but not with PLC-1. These currents were inhibited by pretreatment with PTX and coexpression of Gt, suggesting that -subunits of Gi/o protein activate PLC-2 and then cause intracellular Ca2+ mobilization. Our results indicate that -subunits of Gi/o protein participate in diverse intracellular signals, enhancement of Gs-coupled receptor-mediated responses, and intracellular Ca2+ mobilization. G protein-coupled receptor; cystic fibrosis transmembrane conductance regulator gene; cross talk; electrophysiology  相似文献   

16.
Several related isoforms of p38MAPK have been identified and cloned in many species. Although they all contain the dual phosphorylation motif TGY, the expression of these isoforms is not ubiquitous. p38 and -2 are ubiquitously expressed, whereas p38 and - appear to have more restricted expression. Because there is evidence for selective activation by upstream kinases and selective preference for downstream substrates, the functions of these conserved proteins is still incompletely understood. We have demonstrated that the renal mesangial cell expresses the mRNA for all the isoforms of p38MAPK, with p38 mRNA expressed at the highest level, followed by p38 and the lowest levels of expression by p382 and -. To determine the functional effects of these proteins on interleukin (IL)-1-induced inducible nitric oxide synthase (iNOS) expression, we transduced TAT-p38 chimeric proteins into renal mesangial cells and assessed the effects of wild-type and mutant p38 isoforms on ligand induced iNOS expression. We show that whereas p38 and - had minimal effects on iNOS expression, p38 and -2 significantly altered its expression. p38 mutant and p382 wild-type dose dependently inhibited IL-1-induced iNOS expression. These data suggest that p38 and 2 have reciprocal effects on iNOS expression in the mesangial cell, and these observations may have important consequences for the development of selective inhibitors targeting the p38MAPK family of proteins. TAT proteins; p38 MAPK; inducible nitric oxide synthase; mesangial cell; interleukin-1  相似文献   

17.
We showed previously that enteropathogenic Escherichia coli (EPEC) infection of intestinal epithelial cells induces inflammation by activating NF-B and upregulating IL-8 expression. We also reported that extracellular signal-regulated kinases (ERKs) participate in EPEC-induced NF-B activation but that other signaling molecules such as PKC may be involved. The aim of this study was to determine whether PKC is activated by EPEC and to investigate whether it also plays a role in EPEC-associated inflammation. EPEC infection induced the translocation of PKC from the cytosol to the membrane and its activation as determined by kinase activity assays. Inhibition of PKC by the pharmacological inhibitor rottlerin, the inhibitory myristoylated PKC pseudosubstrate (MYR-PKC-PS), or transient expression of a nonfunctional PKC significantly suppressed EPEC-induced IB phosphorylation. Although PKC can activate ERK, MYR-PKC-PS had no effect on EPEC-induced stimulation of this pathway, suggesting that they are independent events. PKC can regulate NF-B activation by interacting with and activating IB kinase (IKK). Coimmunoprecipitation studies showed that the association of PKC and IKK increased threefold 60 min after infection. Kinase activity assays using immunoprecipitated PKC-IKK complexes from infected intestinal epithelial cells and recombinant IB as a substrate showed a 2.5-fold increase in IB phosphorylation. PKC can also regulate NF-B by serine phosphorylation of the p65 subunit. Serine phosphorylation of p65 was increased after EPEC infection but could not be consistently attenuated by MYR-PKC-PS, suggesting that other signaling events may be involved in this particular arm of NF-B regulation. We speculate that EPEC infection of intestinal epithelial cells activates several signaling pathways including PKC and ERK that lead to NF-B activation, thus ensuring the proinflammatory response. inflammation; enteropathogenic Escherichia coli; nuclear factor-B; protein kinase C; IB kinase; extracellular signal-regulated kinase  相似文献   

18.
Activation of NF-B requires the phosphorylation and degradation of its associated inhibitory proteins, IB. Previously, we reported that the extracellular signal-regulated kinase (ERK) is required for IL-1 to induce persistent activation of NF-B in cultured rat vascular smooth muscle cells (VSMCs). The present study examined the mechanism by which the ERK signaling cascade modulates the duration of NF-B activation. In cultured rat VSMCs, IL-1 activated ERK and induced degradation of both IB and IB, which was associated with nuclear translocation of both ribosomal S6 kinase (RSK)1 and NF-B p65. RSK1, a downstream kinase of ERK, was associated with an IB/NF-B complex, which was independent of the phosphorylation status of RSK1. Treatment of VSMCs with IL-1 decreased IB in the RSK1/IB/NF-B complex, an effect that was attenuated by inhibition of ERK activation. Knockdown of RSK1 by small interference RNA attenuated the IL-1-induced IB decrease without influencing ether ERK phosphorylation or the earlier IB degradation. By using recombinant wild-type and mutant IB proteins, both active ERK2 and RSK1 were found to directly phosphorylate IB, but only active RSK1 phosphorylated IB on Ser19 and Ser23, two sites known to mediate the subsequent ubiquitination and degradation. In conclusion, in the ERK signaling cascade, RSK1 is a key component that directly phosphorylates IB and contributes to the persistent activation of NF-B by IL-1. extracellular signal-regulated kinase; in vitro phosphorylation assay; recombinant proteins; small interference RNA; vascular smooth muscle cell  相似文献   

19.
Sex hormone status has emerged as an important modulator of coronary physiology and cardiovascular disease risk in both males and females. Our previous studies have demonstrated that testosterone increases protein kinase C (PKC) expression and activity in coronary smooth muscle (CSMC). Because PKC has been implicated in regulation of proliferation and apoptosis in other cell types, we sought to determine if testosterone modulates CSMC proliferation and/or apoptosis through PKC. Porcine CSMC cultures (passages 2–6) from castrated males were treated with testosterone for 24 h. Testosterone (20 and 100 nM) decreased [3H]thymidine incorporation in proliferating CSMC to 59 ± 5.3 and 33.1 ± 4.5% of control. Flow cytometric analysis demonstrated that testosterone induced G1 arrest in CSMC with a concomitant reduction in the S phase cells. Testosterone reduced protein levels of cyclins D1 and E and phosphorylation of retinoblastoma protein while elevating levels of p21cip1 and p27kip1. There were no significant differences in the levels of cyclins D3, CDK2, CDK4, or CDK6. Testosterone significantly reduced kinase activity of CDK2 and -6, but not CDK4, -7, or -1. PKC small interfering RNA (siRNA) prevented testosterone-mediated G1 arrest, p21cip1 upregulation, and cyclin D1 and E downregulation. Furthermore, testosterone increased CSMC apoptosis in a dose-dependent manner, which was blocked by either PKC siRNA or caspase 3 inhibition. These findings demonstrate that the anti-proliferative, pro-apoptotic effects of testosterone on CSMCs are substantially mediated by PKC. androgens; coronary; smooth muscle; cell cycle  相似文献   

20.
-Syntrophin is a component of the dystrophin glycoprotein complex (DGC). It is firmly attached to the dystrophin cytoskeleton via a unique COOH-terminal domain and is associated indirectly with -dystroglycan, which binds to extracellular matrix laminin. Syntrophin contains two pleckstrin homology (PH) domains and one PDZ domain. Because PH domains of other proteins are known to bind the -subunits of the heterotrimeric G proteins, whether this is also a property of syntrophin was investigated. Isolated syntrophin from rabbit skeletal muscle binds bovine brain G-subunits in gel blot overlay experiments. Laminin-1-Sepharose or specific antibodies against syntrophin, - and -dystroglycan, or dystrophin precipitate a complex with G from crude skeletal muscle microsomes. Bacterially expressed syntrophin fusion proteins and truncation mutants allowed mapping of G binding to syntrophin's PDZ domain; this is a novel function for PDZ domains. When laminin-1 is bound, maximal binding of Gs and G occurs and active Gs, measured as GTP-35S bound, decreases. Because intracellular Ca2+ is elevated in Duchenne muscular dystrophy and Gs is known to activate the dihydropyridine receptor Ca2+ channel, whether laminin also altered intracellular Ca2+ was investigated. Laminin-1 decreases active (GTP-S-bound) Gs, and the Ca2+ channel is inhibited by laminin-1. The laminin 1-chain globular domains 4 and 5 region, the region bound by DGC -dystroglycan, is sufficient to cause an effect, and an antibody that specifically blocks laminin binding to -dystroglycan inhibits G binding by syntrophin in C2C12 myotubes. These observations suggest that DGC is a matrix laminin, G protein-coupled receptor. Duchenne muscular dystrophy; protein G -subunit; pleckstrin homology domain  相似文献   

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