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1.
Inbred Fischer 344 rats display airway hyperresponsiveness (AHR) in vivo compared with the normoresponsive Lewis strain. Fischer AHR has been linked with increased airway smooth muscle (ASM) contraction ex vivo and enhanced ASM cell intracellular Ca(2+) mobilization in response to serotonin compared with Lewis. To determine the generality of this association, we tested whether bradykinin (BK) also stimulates greater contraction of Fischer airways and greater Ca(2+) mobilization in Fischer ASM cells. Explants of Fischer intraparenchymal airways constricted faster and to a greater degree in response to BK than Lewis airways. BK also evoked higher Ca(2+) transients in Fischer than in Lewis ASM cells. ASM cell B(2) receptor expression was similar between the two strains. BK activated both phosphatidylinositide-specific phospholipase C (PI-PLC) and phosphatidylcholine-specific PLC to mobilize Ca(2+) in Fischer and Lewis ASM cells. PI-PLC activity, as measured by inositol polyphosphate accumulation, was similar in the two strains. PKC inhibition with GF109203X, Go6973, or Go6983 attenuated BK-mediated Ca(2+) transients in Fischer cells, whereas GF109203X potentiated while Go6976 and Go6983 did not affect Ca(2+) transients in Lewis cells. Enhanced Ca(2+) mobilization in ASM cells can arise from variations in PKC and may be an important component of nonspecific, innate AHR.  相似文献   

2.
Airway smooth muscle (ASM) mass is likely to be an important determinant of airway responsiveness. Highly inbred Fisher rats model innate hyperresponsiveness, and also have more ASM in vivo than control Lewis rats. Platelet derived growth factor (PDGF) is an important endogenous growth factor for ASM, and partially purified PDGF-AB causes enhanced growth of Fisher rat ASM cells, compared to Lewis cells. The aim of the present study was to determine the mitogenic effects of all three recombinant PDGF isoforms on ASM cells, and investigate the mechanisms of enhanced Fisher ASM growth responses. The potential mechanisms assessed include PDGF receptor expression and activation (tyrosine phosphorylation), and intracellular calcium (Ca2+) responses to PDGF isoforms. Fisher ASM cells had a greater mitogenic response to PDGF-AB and -AA, and a greater Ca2+ response to -BB than Lewis ASM cells. A Ca2+ response was not necessary for a mitogenic response, and the effects of PDGF isoforms on Ca2+ were not associated with their effects on growth. Therefore, we suggest that enhanced Fisher mitogenic response to PDGF-AA and -AB is not mediated by differences in Ca2+ signalling. Western analysis of the PDGF receptor indicated a similar expression of beta-PDGF receptor in ASM cells from the two rat strains, but a greater expression of alpha-PDGF receptor in Fisher cells; however, phosphorylation of the PDGF receptor following growth stimulation did not differ between strains. This suggests a role for post-receptor signals, in addition to enhanced receptor expression, in the enhanced growth response of Fisher ASM cells to PDGF-AA and -AB.  相似文献   

3.
Maintenance of pregnancy in the rat requires the corpus luteum. At a time when rat placental lactogens (rPLs) are required to support progesterone production by the corpus luteum and when relaxin expression is initiated, expression of a specific protein kinase C (PKC) isoform, PKC delta, is dramatically increased. We therefore assessed whether prolactin (PRL) receptor activation promotes activation of PKC delta in a luteinized granulosa cell model. We also assessed the activation status of PKC delta in corpora lutea obtained when the corpus luteum is exposed to chronically high concentrations of rPLs. The activity of PKC delta was assessed by two means: an immune complex (IC) assay and Western blotting with a phospho-epitope-specific antibody that detects PKC delta phosphorylated on serine 662. PKC delta activation in the IC kinase assay was determined by the ability of immunoprecipitated PKC delta to phosphorylate the PKC delta-preferential substrate small heat shock protein (HSP-27). Treatment of luteinized rat granulosa cells with phorbol myristate acetate, a known activator of PKC, promoted a 7-fold increase in HSP-27 phosphorylation by PKC delta. Similarly, immunoreactivity with the phospho-epitope-specific PKC delta antibody was increased in extracts prepared from luteinized granulosa cells treated with phorbol myristate acetate or following in vitro activation of recombinant PKC delta. Using these assays, we assessed whether PRL receptor agonists were capable of activating PKC delta in luteinized granulosa cells. PRL receptor agonists induced translocation PKC delta from the cytosolic to the Triton-soluble membrane fraction and increased PKC delta activity assessed by both IC kinase assay and Western blotting with phospho-epitope-specific PKC delta antibody. Analysis of PKC delta activity in corpora lutea obtained during pregnancy by both the IC kinase assay and Western blotting with the phospho-epitope-specific PKC delta antibody revealed that PKC delta activity was increased throughout the second half of pregnancy. These results demonstrate that PRL receptor activation promotes the acute activation of PKC delta in luteinized rat granulosa cells. At a time when the rat is exposed to chronically high concentrations of rPLs, PKC delta is increasingly expressed and active.  相似文献   

4.
Epidermal growth factor (EGF)-stimulated proliferation of renal epithelial cells plays an important role in the recovery of kidney tubule epithelia following exposure to insult. Numerous studies have demonstrated that tyrosine phosphorylation of the focal adhesion protein paxillin mediates in part the effects of growth factors on cell growth, migration, and organization of the actin-based cytoskeleton. The experiments in this report were designed to determine the effect of EGF on paxillin phosphorylation in normal rat kidney (NRK) epithelial cells. Interestingly, treatment of NRK cells with EGF stimulated paxillin serine/threonine phosphorylation, which caused a reduction in the mobility of paxillin on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The EGF-stimulated mobility shift of paxillin was independent of an intact cytoskeleton, phosphatidylinositol 3-kinase (PI 3-kinase) activation, protein kinase C (PKC) activation, and cellular adhesion. However, inhibitors of the mitogen-activated protein kinase/extracellular signal-regulated kinase kinase abrogated the EGF-stimulated change in paxillin mobility. In addition, the EGF-stimulated change in paxillin serine/threonine phosphorylation was not accompanied by a profound reorganization of the actin cytoskeleton. These results identify paxillin as a component EGF signaling in renal epithelial cells and implicate members of the MAP kinase pathway as critical regulators of paxillin serine/threonine phosphorylation.  相似文献   

5.
The mechanisms underlying control of cell growth and differentiation in epithelial tissues are poorly understood. Protein kinase C (PKC) isozymes, members of a large family of serine/threonine kinases of fundamental importance in signal transduction, have been increasingly implicated in the regulation of cell growth, differentiation, and function. Using the rat intestinal epithelium as a model system, we have examined PKC-specific activity as well as individual PKC isozyme expression and distribution (i.e., activation status) in epithelial cells in situ. Increased PKC activity was detected in differentiating and functional cells relative to immature proliferating crypt cells. Immunofluorescence and Western blot analysis using a panel of isozyme- specific antibodies revealed that PKC alpha, beta II, delta, epsilon, and zeta are expressed in rat intestinal epithelial cells and exhibit distinct subcellular distribution patterns along the crypt-villus unit. The combined morphological and biochemical approach used permitted analysis of the activation status of specific PKC isozymes at the individual cell level. These studies showed that marked changes in membrane association and level of expression for PKC alpha, beta II, delta, and zeta occur as cells cease division in the mid-crypt region and begin differentiation. Additional changes in PKC activation status are observed with acquisition of mature function on the villus. These studies clearly demonstrate naturally occurring alterations in PKC isozyme activation status at the individual cell level within the context of a developing tissue. Direct activation of PKC in an immature intestinal crypt cell line was shown to result in growth inhibition and coincident translocation of PKC alpha from the cytosolic to the particulate subcellular fraction, paralleling observations made in situ and providing further support for a role of intestinal PKC isozymes in post-mitotic events. PKC isozymes were also found to be tightly associated with cytoskeletal elements, suggesting participation in control of the structural organization of the enterocyte. Taken together, the results presented strongly suggest an involvement of PKC isoforms in cellular processes related to growth cessation, differentiation, and function of intestinal epithelial cells in situ.  相似文献   

6.
The c-raf kinase has been shown to be activated following stimulation of several tyrosine kinase growth factor receptors. We examined changes in c-raf following engagement of the T cell receptor for antigen (TCR), a stimulus which activates both a non-receptor tyrosine kinase and protein kinase C (PKC). We found that activation of the T-cell receptor on the T cell hybridoma 2B4 causes a rapid and stoichiometric hyperphosphorylation of c-raf and an increase in c-raf-associated kinase activity. Phosphoamino acid analysis showed that the phosphorylation was entirely on serine residues. High-resolution phosphopeptide mapping showed the appearance of a single major new phosphopeptide with TCR stimulation. That phosphopeptide was shown to comigrate with the major new phosphopeptide induced in response to phorbol ester. When cells were depleted of PKC by pretreatment with high concentrations of phorbol ester, TCR stimulation was no longer capable of inducing c-raf-associated kinase activity. To determine whether activation of the tyrosine kinase alone would activate c-raf, we examined the 2B4 variant cell line FL.8. In response to Thy-1 stimulation, these cells activate the tyrosine kinase but not protein kinase C due to a deficiency in TCR eta chain expression. We found that in contrast to Thy-1 stimulation of 2B4 cells, stimulation of FL.8 cells does not lead to the induction of c-raf-associated kinase activity, although phorbol ester activates the kinase to an equivalent degree in both cells. We conclude that T cell receptor activation of c-raf occurs via phosphorylation by the serine/threonine kinase PKC. Activation of c-raf through PKC represents a mechanism distinct from that reported for tyrosine kinase growth factor receptors.  相似文献   

7.
Protein kinase C (PKC) is a family of serine/threonine protein kinases that plays a central role in transducing extracellular signals into a variety of intracellular responses ranging from cell proliferation to apoptosis. Nine PKC genes have been identified in the human genome, which encode 10 proteins. Each member of this protein kinase family displays distinct biochemical characteristics and is enriched in different cellular and subcellular locations. Activation of PKC has been implicated in the regulation of cell growth and differentiation. This review summarizes works of the past years in the field of PKC biochemistry that covers regulation and activation mechanism of different PKC isoforms.  相似文献   

8.
Protein kinase C (PKC) is a heterogeneous family of serine/threonine protein kinases that have different biological effects in normal and neoplastic melanocytes (MCs). To explore the mechanism behind their differential response to PKC activation, we analyzed the expression profile of all nine PKC isoforms in normal human MCs, HPV16 E6/E7 immortalized MCs, and a panel of melanoma cell lines. We found reduced PKCβ and increased PKCζ and PKCι expression at both the protein and mRNA levels in immortalized MCs and melanoma lines. We focused on PKCβ as it has been functionally linked to melanin production and oxidative stress response. Re-expression of PKCβ in melanoma cells inhibited colony formation in soft agar, indicating that PKCβ loss in melanoma is important for melanoma growth. PKCβII, but not PKCβI, was localized to the mitochondria, and inhibition of PKCβ significantly reduced UV-induced reactive oxygen species (ROS) in MCs with high PKCβ expression. Thus alterations in PKCβ expression in melanoma contribute to their neoplastic phenotype, possibly by reducing oxidative stress, and may constitute a selective therapeutic target.  相似文献   

9.
The spatio-temporal changes of signaling molecules in response to G protein-coupled receptors (GPCR) stimulation is a poorly understood process in intestinal epithelial cells. Here we investigate the dynamic mechanisms associated with GPCR signaling in living rat intestinal epithelial cells by characterizing the intracellular translocation of protein kinase D (PKD), a serine/threonine protein kinase involved in mitogenic signaling in intestinal epithelial cells. Analysis of the intracellular steady-state distribution of green fluorescent protein (GFP)-tagged PKD indicated that in non-stimulated IEC-18 cells, GFP-PKD is predominantly cytoplasmic. However, cell stimulation with the GPCR agonist vasopressin induces a rapid translocation of GFP-PKD from the cytosol to the plasma membrane that is accompanied by its activation via protein kinase C (PKC)-mediated process and posterior plasma membrane dissociation. Subsequently, active PKD is imported into the nuclei where it transiently accumulates before being exported into the cytosol by a mechanism that requires a competent Crm1 nuclear export pathway. These findings provide evidence for a mechanism by which PKC coordinates in intestinal epithelial cells the translocation and activation of PKD in response to vasopressin-induced GPCR activation.  相似文献   

10.
4-aminopyridine (4AP) is a general blocker of voltage-dependent K+ channels. This pyridine derivative has also been shown to inhibit T cell proliferation, to modulate immune responses and to alleviate some of the symptoms associated with neurological disorders such as multiple sclerosis, myasthenia gravis and Alzheimer's disease. 4AP triggers a Ca2+ response in lymphocytes, astrocytes, neurons and muscle cells but little is known about the regulation of the 4AP response in these cells. We report that 4AP induced a non-capacitative transplasma membrane influx of Ca2+ in Jurkat T lymphocytes. The influx of Ca2+ was not affected by activation or inhibition of protein kinase A (PKA). In contrast, activation of protein kinase C (PKC) by phorbol myristyl acetate (PMA), mezerein or 1-oleoyl-2-acetyl-sn-glycerol (OAG) inhibited the influx of Ca2+ triggered by 4AP. The inhibitory effect of PKC could be prevented by prior exposure of the cells to the PKC inhibitor GF 109203X. Under these conditions, mezerein and OAG no longer inhibited the 4AP-dependent Ca2+ response. Inhibition of serine and threonine protein phosphatases PP1 and PP2A by treating the cells with calyculin A (CalA) reduced the Ca2+ response to 4AP. Okadaic acid (OA) had no effect, suggesting an involvement of PP1. A combination of CalA and OAG (or PMA) abolished the influx of Ca2+ induced by 4AP, adding further evidence to the importance of protein phosphorylation in the modulation of the 4AP response. Our data suggest that the transplasma membrane influx of Ca2+ triggered by 4AP in Jurkat T cells can be modulated by the opposite actions of PKC and protein serine and threonine phosphatase(s).  相似文献   

11.
We have examined the phosphorylation of the serine threonine kinase, the product of c-raf proto-oncogene in response to insulin or platelet-derived growth factor in intact cells. Both insulin and platelet-derived growth factor stimulated phosphorylation of the c-raf protein about 2- to 3-fold. The phosphorylation occurred exclusively on serine and threonine residues; phosphotyrosine was not detected. In immune-complex kinase assays, treatment with insulin, and platelet-derived growth factor increased autophosphorylation of the c-raf kinase, suggesting activation of its kinase activity. To investigate whether the phosphorylation of the c-raf protein in intact cells results from an autophosphorylation event or from the phosphorylation by other cellular kinase(s), we replaced lysine 375 in the putative ATP-binding domain of the c-raf protein with alanine using oligonucleotide site-directed mutagenesis and expressed the mutated protein in NIH3T3 cells. The substitution resulted in the inactivation of the serine/threonine-specific autophosphorylation in immune-complex kinase assays. In intact cells, however, although phosphorylation of the mutant protein in response to insulin and platelet-derived growth factor occurred to a lesser extent than that of the wild-type protein, the phosphopeptide maps were indistinguishable. These results suggest that serine threonine phosphorylation might be responsible for the activation of c-raf kinase upon treatment of cells with insulin and platelet-derived growth factor, and most of the phosphate associated with the c-raf protein results from its phosphorylation by as yet uncharacterized cellular serine/threonine kinase(s).  相似文献   

12.
Adhesion of fibroblasts to extracellular matrices via integrin receptors is accompanied by extensive cytoskeletal rearrangements and intracellular signaling events. The protein kinase C (PKC) family of serine/threonine kinases has been implicated in several integrin-mediated events including focal adhesion formation, cell spreading, cell migration, and cytoskeletal rearrangements. However, the mechanism by which PKC regulates integrin function is not known. To characterize the role of PKC family kinases in mediating integrin-induced signaling, we monitored the effects of PKC inhibition on fibronectin-induced signaling events in Cos7 cells using pharmacological and genetic approaches. We found that inhibition of classical and novel isoforms of PKC by down-regulation with 12-0-tetradeconoyl-phorbol-13-acetate or overexpression of dominant-negative mutants of PKC significantly reduced extracellular regulated kinase 2 (Erk2) activation by fibronectin receptors in Cos7 cells. Furthermore, overexpression of constitutively active PKCalpha, PKCdelta, or PKCepsilon was sufficient to rescue 12-0-tetradeconoyl-phorbol-13-acetate-mediated down-regulation of Erk2 activation, and all three of these PKC isoforms were activated following adhesion. PKC was required for maximal activation of mitogen-activated kinase kinase 1, Raf-1, and Ras, tyrosine phosphorylation of Shc, and Shc association with Grb2. PKC inhibition does not appear to have a generalized effect on integrin signaling, because it does not block integrin-induced focal adhesion kinase or paxillin tyrosine phosphorylation. These results indicate that PKC activity enhances Erk2 activation in response to fibronectin by stimulating the Erk/mitogen-activated protein kinase pathway at an early step upstream of Shc.  相似文献   

13.
PKC-α和PKC-δ在不同发育阶段小鼠睾丸中的差异性表达   总被引:2,自引:0,他引:2  
蛋白激酶C(PKC)作为一类重要的蛋白激酶 ,通过对底物蛋白的Ser Thr残基的磷酸化 ,参与细胞的短期反应与长期反应的调节 ,具有介导细胞生长和增殖 ,调节核基因的表达 ,影响细胞周期等生理作用 ,从而参与调控多种细胞系 ,如NIH3T3细胞、造血干细胞等细胞系的增殖与分化[1,2 ] .在精子形成这样一个独特的细胞分化过程中 ,PKC的生理功能仍未十分明确 .睾丸生殖细胞的增殖与分化 (精子发生 )是一个独特复杂的细胞分化过程 ,主要分为 3个阶段 :A型精原细胞增殖和B型精原细胞分化为初级精母细胞 ;初级精母细胞通过减数分裂形成…  相似文献   

14.
Protein kinase C alpha (PKC alpha) is a serine/threonine kinase and a member of the conventional (classical) PKCs (cPKCs), which have four conserved (C1 to C4) regions. This ubiquitously expressed PKC isotype is activated in response to many different kinds of stimuli and translocates from cytosol to the specialized cellular compartments (nucleus, focal adhesion, caveolae, etc.) where it is presumed to work. Therefore, PKC alpha has been implicated in a variety of cellular functions including proliferation, apoptosis, differentiation, motility, and inflammation. However, the responses induced by activation or overexpression of PKC alpha vary depending on the types, and sometimes conditions, of cells. For example, in some types of cells, PKC alpha is implicated in cell growth. In contrast, it may play a role in cell cycle arrest and differentiation in other types of cells. Therefore, alterations of cell responses induced by PKC alpha are not an intrinsic property of this isoform. The responses are modulated by dynamic interactions with cell-type specific factors: substrates, modulators and anchoring proteins.  相似文献   

15.
16.
Staurosporine, a protein kinase C (PKC) inhibitor, has been reported to regulate the phosphorylation of ERK1/2 in several cell lines. It is still unknown, however, whether its derivative staurosporine aglycone (SA) has the same effect on ERK1/2 activation. In this study, we investigated the effect of SA on ERK1/2 activity in rat pulmonary arteries and pulmonary arterial smooth muscle cells (PASMCs). The pulmonary arteries and PASMCs were treated with SA at different time points and concentrations, and the activation of ERK1/2 was analyzed by Western blotting. The results showed that SA at nanomolar concentrations suppressed ERK1/2 phosphorylation through the PKC pathway alone, but SA at 30 micromol/L for 2 h enhanced the phosphorylation of ERK1/2. The activation of ERK1/2 was inhibited by the MAPK/ERK kinase inhibitor PD98059 or the protein kinase A (PKA) activator isoproterenol. Together, these results suggest that SA has a strong dual regulating effect on ERK1/2 through the PKC and (or) PKA pathways in rat PASMCs.  相似文献   

17.
Cohen S  Braiman A  Shubinsky G  Isakov N 《FEBS letters》2011,585(20):3208-3215
Members of the protein kinase C (PKC) family of serine/threonine kinases have been implicated in several physiological processes regulating the activation response of platelets. They are involved in processes leading to granule secretion, integrin activation, platelet aggregation and spreading, and procoagulation. The protein kinase C θ (PKCθ) isoform, which was originally identified in T lymphocytes, is also expressed at relatively high levels in platelets, wherein it is involved in the regulation of hemostasis and thrombosis. Recent studies suggest a role for PKCθ in protease-activated receptor (PAR)-, glycoprotein VI (GPVI) receptor- and glycoprotein α(IIb)β(3) integrin receptor-linked signal transduction pathways. The present review focuses on the latest observations relevant to the role of PKCθ in platelet activation.  相似文献   

18.
The role of the protein kinase C (PKC) family of serine/threonine kinases in cellular differentiation, proliferation, apoptosis, and other responses makes them attractive therapeutic targets. The activation of PKCs by ligands in vivo varies depending upon cell type; therefore, methods are needed to screen the potency of PKCs in this context. Here we describe a genetically encoded chimera of native PKCdelta fused to yellow- and cyan-shifted green fluorescent protein, which can be expressed in mammalian cells. This chimeric protein kinase, CY-PKCdelta, retains native or near-native activity in the several biological and biochemical parameters that we tested. Binding assays showed that CY-PKCdelta and native human PKCdelta have similar binding affinity for phorbol 12,13-dibutyrate. Analysis of translocation by Western blotting and confocal microscopy showed that CY-PKCdelta translocates from the cytosol to the membrane upon treatment with ligand, that the translocation has similar dose dependence as that of endogenous PKCdelta, and that the pattern of translocation is indistinguishable from that of the green fluorescent protein-PKCdelta fusion well characterized from earlier studies. Treatment with phorbol ester of cells expressing CY-PKCdelta resulted in a dose-dependent increase in FRET that could be visualized in situ by confocal microscopy or measured fluorometrically. By using this construct, we were able to measure the kinetics and potencies of 12 known PKC ligands, with respect to CY-PKCdelta, in the intact cell. The CY-PKCdelta chimera and the in vivo assays described here therefore show potential for high throughput screening of prospective PKCdelta ligands within the context of cell type.  相似文献   

19.
20.
Protein kinase D (PKD)/protein kinase C (PKC) mu is a serine/threonine protein kinase that can be activated by physiological stimuli like growth factors, antigen-receptor engagement and G protein-coupled receptor (GPCR) agonists via a phosphorylation-dependent mechanism that requires PKC activity. In order to investigate the dynamic mechanisms associated with GPCR signaling, the intracellular translocation of a green fluorescent protein-tagged PKD was analyzed by real-time visualization in fibroblasts and epithelial cells stimulated with bombesin, a GPCR agonist. We found that bombesin induced a rapidly reversible plasma membrane translocation of green fluorescent protein-tagged PKD, an event that can be divided into two distinct mechanistic steps. The first step, which is exclusively mediated by the cysteine-rich domain in the N terminus of PKD, involved its translocation from the cytosol to the plasma membrane. The second step, i.e. the rapid reverse translocation of PKD from the plasma membrane to the cytosol, required its catalytic domain and surprisingly PKC activity. These findings provide evidence for a novel mechanism by which PKC coordinates the translocation and activation of PKD in response to bombesin-induced GPCR activation.  相似文献   

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