共查询到20条相似文献,搜索用时 15 毫秒
1.
2.
Constance M. Ely William M. Tomiak Colleen M. Allen Laurel Thomas Gary Thomas Sarah J. Parsons 《Journal of neurochemistry》1994,62(3):923-933
Abstract: Secretion of catecholamines by adrenal chromaffin cells is a highly regulated process that involves serine/threonine and tyrosine phosphorylations. The nonreceptor tyrosine kinase pp60c-sre is expressed at high levels and localized to plasma membranes and secretory vesicle membranes in these cells, suggesting an interaction of this enzyme with components of the secretory process. To test the hypothesis that pp60c-sic is involved in exocytosis, we transiently expressed exogenous c-src cDNA using a vaccinia virus vector in primary cultures of bovine adrenomedullary chromaffin cells. Chromaffin cells infected with a c-src recombinant virus restored the diminished secretory activity accompanying infection by wild type virus alone or a control recombinant virus. The level of enhanced catecholamine release correlated directly with the time and level of exogenous c-src expression. These results could not be attributed to differences in cytopathic effects of wild type versus recombinant viruses as assessed by cell viability assays, nor to differences in norepinephrine uptake or basal release, suggesting that pp60c-src is involved in stimulus-secretion coupling in infected cells. Surprisingly, exogenous expression of an enzymatically inactive mutant c-src also restored catecholamine release, indicating that regions of the introduced c-src protein other than the kinase domain may affect catecholamine release. Secretory activity was elevated by both forms of c-src in response to either nicotine or carbachol (which activate the nicotinic and the nicotinic/muscarinic receptors, respectively). In contrast, release of catecholamines upon membrane depolarization (as elicited by 55 mM K+) or by treatment with the calcium ionophore A23187 was unaffected by either vaccinia infection or increased levels of pp60c-src. These results suggest that pp60c-src affects secretory processes in vaccinia-infected cells that are activated through ligand-gated, but not voltage-gated, ion channels. 相似文献
3.
pp60
c-src
kinase activity can be increased by phosphotyrosine dephosphorylation or growth factor-dependent phosphorylation reactions. Expression of the transmembrane phosphotyrosine phosphatase (PTPase) CD45 has been shown to inhibit growth factor receptor signal transduction (Mooney, RA, Freund, GG, Way, BA and Bordwell, KL (1992) J Biol Chem 267, 23443–23446). Here it is shown that PTPase expression decreased platelet-derived growth factor (PDGF)-dependant activation of pp60
c-src
but failed to increase hormone independent (basal) pp60
c-src
activity. PDGF-dependent tyrosine phosphorylation of its receptor was reduced by approximately 60% in cells expressing the PTPase. In contrast, a change in phosphotyrosine content of pp60
c-src
was not detected in response to PDGF or in PTPase+cells. PDGF increased the intrinsic tyrosine kinase activity of pp60
c-src
in both control and PTPase+cells but the effect was smaller in PTPase+cells. In anin vitro assay, hormone-stimulate pp60
c-src
autophosphorylation from PTPase+ cells was decreased 64±22%, and substrate phosphorylation by pp60
c-src
was reduced 54±16% compared to controls. Hormone-independent pp60
c-src
kinase activity was unchanged by expression of the PTPase. pp60
c-src
was, however, anin vitro substrate for CD45, being dephosphorylated at both the regulatory (Tyr527) and kinase domain (Tyr416) residues. In addition,in vitro dephosphorylation by CD45 increased pp60
c-src
activity. These findings suggest that the PDGF receptor was anin vivo substrate of CD45 but pp60
c-src
was not. The lack of activation of pp60
c-src
in the presence of expressed PTPase may demonstrate the importance of compartmentalization and/or accessory proteins to PTPase-substrate interactions.Abbreviations PTPase
phosphotyrosine phosphatase
- PDGF
platelet-derived growth factor
- PMSF
phenylmethylsulfonyl fluoride
- LCA, CD45
leukocyte common antigen
- PBS
phosphate buffered saline
- SDS
sodium dodecyl sulfate
- PAGE
polyacrylamide gel electrophoresis
- DTT
dithiothreitol
- Na3VO4
sodium orthovanadate
- PV
pervanadate
- -ME
-mercaptoethanol 相似文献
4.
《Journal of Lipid Mediators and Cell Signalling》1997,15(2):193-202
Stimulation of rat mesangial cells with platelet-derived growth factor BB (PDGF-BB) enhanced phospholipase D (PLD) activity in a concentration dependent manner. Mesangial cells overexpressing the tyrosine kinase pp60c-src (c-Src) were used to determine the effect of this non transforming protooncogene on PLD activation. Overexpression of c-Src interfered with PDGF-BB-mediated activation of PLD. This modulation was dependent on the tyrosine kinase activity of c-Src, since overexpression of tyrosine kinase-negative mutants of c-Src did not affect PLD activation. No effect of c-Src overexpression was observed, when PLD was activated by ATP or guanosine 5′-3-O-(thio)triphosphate (GTPγS). The results indicate that the tyrosine kinase c-Src specifically interfered with PDGF-mediated but not with ATP- or GTPγS-mediated PLD activation. 相似文献
5.
pp60c-src is a positive regulator of growth factor-induced cell scattering in a rat bladder carcinoma cell line 总被引:3,自引:0,他引:3
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《The Journal of cell biology》1995,131(3):761-773
The NBT-II rat carcinoma cell line exhibits two mutually exclusive responses to FGF-1 and EGF, entering mitosis at cell confluency while undergoing an epithelium-to-mesenchyme transition (EMT) when cultured at subconfluency. EMT is characterized by acquisition of cell motility, modifications of cell morphology, and cell dissociation correlating with the loss of desmosomes from cellular cortex. The pleiotropic effects of EGF and FGF-1 on NBT-II cells suggest that multiple signaling pathways may be activated. We demonstrate here that growth factor activation is linked to at least two intracellular signaling pathways. One pathway leading to EMT involves an early and sustained stimulation of pp60c-src kinase activity, which is not observed during the growth factor-induced entry into the cell cycle. Overexpression of normal c-src causes a subpopulation of cells to undergo spontaneous EMT and sensitizes the rest of the population to the scattering activity of EGF and FGF-1 without affecting their mitogenic responsiveness. Addition of cholera toxin, a cAMP-elevating agent, severely perturbs growth factor induction of EMT without altering pp60c-src activation, therefore demonstrating that cAMP blockade takes place downstream or independently of pp60c-src. On the other hand, overexpression of a mutated, constitutively activated form of pp60c-src does not block cell dispersion while strongly inhibiting growth factor-induced entry into cell division. Moreover, stable transfection of a dominant negative mutant of c-src inhibits the scattering response without affecting mitogenesis induced by the growth factors. Altogether, these results suggest a role for pp60c-src in epithelial cell scattering and indicate that pp60c-src might contribute unequally to the two separate biological activities engendered by a single signal. 相似文献
6.
Substrate Recognition by Osteoclast Precursors Induces C-src/Microtubule Association 总被引:1,自引:0,他引:1
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Yousef Abu-Amer F. Patrick Ross Paul Schlesinger M. Mehrdad Tondravi Steven L. Teitelbaum 《The Journal of cell biology》1997,137(1):247-258
The osteoclast is distinguished from other macrophage polykaryons by its polarization, a feature induced by substrate recognition. The most striking component of the polarized osteoclast is its ruffled membrane, probably reflecting insertion of intracellular vesicles into the bone apposed plasmalemma. The failure of osteoclasts in c-src−/− osteopetrotic mice to form ruffled membranes indicates pp60c-src (c-src) is essential to osteoclast polarization. Interestingly, c-src itself is a vesicular protein that targets the ruffled membrane. This being the case, we hypothesized that matrix recognition by osteoclasts, and their precursors, induces c-src to associate with microtubules that traffic proteins to the cell surface. We find abundant c-src associates with tubulin immunoprecipitated from avian marrow macrophages (osteoclast precursors) maintained in the adherent, but not nonadherent, state. Since the two proteins colocalize only within adherent avian osteoclast-like cells examined by double antibody immunoconfocal microscopy, c-src/tubulin association reflects an authentic intracellular event. C-src/tubulin association is evident within 90 min of cell-substrate recognition, and the event does not reflect increased expression of either protein. In vitro kinase assay demonstrates tubulin-associated c-src is enzymatically active, phosphorylating itself as well as exogenous substrate. The increase in microtubule-associated kinase activity attending adhesion mirrors tubulin-bound c-src and does not reflect enhanced specific activity. The fact that microtubule-dissociating drugs, as well as cold, prevent adherence-induced c-src/tubulin association indicates the protooncogene complexes primarily, if not exclusively, with polymerized tubulin. Association of the two proteins does not depend upon protein tyrosine phosphorylation and is substrate specific, as it is induced by vitronectin and fibronectin but not type 1 collagen. Finally, consistent with cotransport of c-src and the osteoclast vacuolar proton pump to the polarized plasmalemma, the H+-ATPase decorates microtubules in a manner similar to the protooncogene, specifically coimmunoprecipitates with c-src from the osteoclast light Golgi membrane fraction, and is present, with c-src, in preparations enriched with acidifying vesicles reconstituted from the osteoclast ruffled membrane. 相似文献
7.
《Cell communication & adhesion》2013,20(2):91-100
As cells adhere to extracellular matrix proteins, several focal adhesion proteins become tyrosine phosphorylated. One of the most prominent of these has been identified as the tyrosine kinase p125FAK (focal adhesion kinase, FAK). An interaction between FAK and members of the Src family tyrosine kinases p59fyn, pp60v-src, and activated pp60c-src (527F) has been demonstrated, raising the possibility that these kinases may regulate FAK activity. To explore the role of Src family kinases in focal adhesions and in the regulation of FAK activity, we isolated fibroblasts from transgenic mice that lack either pp60c-src p59fyn, or pp62c-yes. These primary fibroblasts, and those of a control mouse, were passaged numerous times and resulted in spontaneously immortalized cell lines without the addition of transforming agents. After confirming the absence of the appropriate nonreceptor tyrosine kinases in the fyc¯, srn¯ and yes¯ fibroblasts, the ability of these fibroblasts to form focal adhesions and stress fibers was assessed by immunofluorescence microscopy and found to be comparable to that of normal fibroblasts. We investigated phosphotyrosine levels in response to adhesion to fibronectin and identified the pp60src substrate p130 as the one major protein with reduced levels of tyrosine phosphorylation in the cells lacking p59fyn and pp62c-yes, and particularly in those lacking pp60c-scr. We examined FAK phosphorylation and kinase activity and found that there were no significant differences between these cells. 相似文献
8.
Activation of the pp60c-src kinase by middle T antigen binding or by dephosphorylation. 总被引:132,自引:13,他引:119
S A Courtneidge 《The EMBO journal》1985,4(6):1471-1477
The transforming protein of polyoma virus, middle T antigen, associates with the protein tyrosine kinase pp60c-src, and analysis of mutants of middle T suggests that this complex plays an important role in transformation by polyoma. It has recently been reported that pp60c-src from polyoma virus-transformed cells has enhanced tyrosine kinase activity in vitro. The data presented here confirm these findings and show that the enhanced kinase activity of pp60c-src is due to an increase in the Vmax of the enzyme. Sucrose density gradient analysis demonstrates that only the form of pp60c-src which is bound to middle T antigen is activated. The difference in enzyme activity between pp60c-src from normal and middle T-transformed cells is more marked when the enzyme is prepared from lysates containing the phosphotyrosine protein phosphatase inhibitor, sodium orthovanadate. pp60c-src from middle T transformed cells is unaffected, but pp60c-src from normal cells has reduced kinase activity if dephosphorylation is prevented. The kinase activity of pp60c-src thus appears to be regulated by its degree of phosphorylation at tyrosine, and data are presented which support this hypothesis. pp60c-src is the first example of a protein tyrosine kinase whose activity is inhibited by phosphorylation at tyrosine. Middle T antigen may increase the kinase activity of pp60c-src by preventing phosphorylation at this regulatory site. 相似文献
9.
Tyrosine phosphorylation and protein tyrosine kinase (PTK) activity in the growth cone membrane-associated glycoprotein (GCGP) fraction of 1-day-old rat brain were examined. Using immunoblotting and immunoprecipitation techniques, pp60c-src was identified as one of the major PTKs associated with GCGPs. Furthermore, only GCGP-associated src that was also tyrosine phosphorylated was active. Immunoprecipitation experiments using various src antibodies revealed that pp60c-src contributed partially to the PTK activity detected in GCGPs, and that it is associated with several proteins of Mr 140 K, 120 K, 85 K and 50 K. This association of src protein with GCGPs was specific, and another src family member p59fyn, which is also abundant in the brain, did not exhibit such an association. In addition to pp60c-src, the GCGP fraction contained several major phosphotryosine-containing proteins of Mr 140 K, and a 97/90 K doublet that corresponded to the beta subunits of IGF-I/ insulin receptors. These studies show that pp60c-src associated with GCGPs is an active PTK that could be involved in neuronal growth and development, transmembrane signalling, and in recognition and/or adhesive events. © 1992 John Wiley & Sons, Inc. 相似文献
10.
Early activation of endogenous pp60src kinase activity during neuronal differentiation of cultured human neuroblastoma cells. 总被引:3,自引:0,他引:3
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C Bjelfman G Meyerson C A Cartwright K Mellstr?m U Hammerling S P?hlman 《Molecular and cellular biology》1990,10(1):361-370
The proto-oncogene product pp60c-src is a tyrosine-specific kinase with a still unresolved cellular function. High levels of pp60c-src in neurons and the existence of a neuronal pp60c-src variant, pp60c-srcN, suggest participation in the progress or maintenance of the differentiated phenotype of neurons. We have previously reported that phorbol esters, e.g., 12-O-tetradecanoylphorbol-13-acetate (TPA), stimulate human SH-SY5Y neuroblastoma cells to neuronal differentiation, as monitored by morphological, biochemical, and functional differentiation markers. In this report, we describe activation of the pp60src (pp60c-src and pp60c-srcN) kinase activity observed at 6 h after induction of SH-SY5Y cells with TPA. This phenomenon coincides in time with neurite outgrowth, formation of growth cone-like structures, and an increase of GAP43 mRNA expression, which are the earliest indications of neuronal differentiation in these cells. The highest specific src kinase activity (a three- to fourfold increase 4 days after induction) was noted in cells treated with 16 nM TPA; this concentration is optimal for development of the TPA-induced neuronal phenotype. During differentiation, there was no alteration in the 1:1 ratio of pp60c-src to pp60c-srcN found in untreated SH-SY5Y cells. V8 protease and trypsin phosphopeptide mapping of pp60src from in vivo 32P-labeled cells showed that the overall phosphorylation of pp60src was higher in differentiated than in untreated cells, mainly because of an intense serine 12 phosphorylation. Tyrosine 416 phosphorylation was not detectable in either cell type, and no change during differentiation in tyrosine 527 phosphorylation was observed. 相似文献
11.
12.
Regulation of bone biology by prostaglandin endoperoxide H synthases (PGHS): a rose by any other name.. 总被引:1,自引:0,他引:1
It is well established that prostaglandins are essential mediators of bone resorption and formation. In the early 1990s, it was discovered that enzymatic reactions producing prostaglandins were regulated by two cyclooxygenase enzymes, one producing prostaglandins constitutively in tissues like the stomach, prostaglandin endoperoxide H synthase-1 (PGHS-1 or COX-1), and another induced by mitogens or inflammatory mediators (PGHS-2 or COX-2). This neat distinction has not been maintained because both enzymes act in different cell systems to provide physiological signaling, constitutively or by induction under certain conditions. For example, the regulation patterns of PGHS-1 and PGHS-2 are distinct, but the evidence shows that PGHS-2 functions constitutively in the skeleton. PGHS-2 has quickly been established, therefore, as a key regulator of bone biology, capable of rapid and transient expression in bone cells, and mediating osteoclastogenesis, mechanotransduction, bone formation and fracture repair. The goal of this review is to summarize the current state of our knowledge of PGHS regulation of bone metabolism and to identify some of the key unresolved challenges and questions that require further study. 相似文献
13.
pp60c-src tyrosine kinase, myristylation, and modulatory domains are required for enhanced mitogenic responsiveness to epidermal growth factor seen in cells overexpressing c-src. 总被引:15,自引:8,他引:7
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In previous studies examining the potential role of pp60c-src in cellular proliferation, we demonstrated that C3H10T1/2 murine embryo fibroblasts overexpressing transfected chicken genomic c-src displayed an epidermal growth factor (EGF)-induced mitogenic response which was 200 to 500% of the response exhibited by parental control cells (Luttrell et al., Mol. Cell. Biol. 8:497-501, 1988). In order to examine specific structural and functional requirements for pp60c-src in this event, 10T1/2 cells were transfected with chicken c-src genes encoding pp60c-src deficient in tyrosine kinase activity (pm430), myristylation, (pm2A), or a domain hypothesized to modulate the interaction with substrates or regulatory components (dl155). Neomycin-resistant clonal cell lines overexpressing each of the mutated c-src genes were assayed for EGF mitogenic responsiveness by measuring [3H]thymidine incorporation into acid-precipitable material or into labeled nuclei. The results were compared with those obtained with lines overexpressing the cDNA form of wild-type (wt) c-src or control cells transfected with the neomycin resistance gene only. As previously described for cells overexpressing wt genomic c-src (Luttrell et al., 1988), clones overexpressing wt cDNA c-src also exhibited enhanced EGF mitogenic responses ranging from approximately 300 to 400% of the control cell response. In contrast, clones overexpressing unmyristylated, modulation-defective, or kinase-deficient c-src not only failed to support an augmented response to EGF but also exhibited EGF responses lower than that of the control cells. Furthermore, there were no significant differences in the mitogenic responses to 10% fetal calf serum among any of the cells tested. These results indicate that pp60(c-scr) can potentiate mitogenic signaling generated by EGF but not all growth factors. This potentiation requires the utilization of pp60(c-scr) myristylation, and modulatory and tyrosine kinase domains and can me mediated by cDNA-encoded as well as by genome-encoded wt pp60(c-scr). 相似文献
14.
Analysis of pp60c-src protein kinase activity in hamster embryo cells transformed by simian virus 40, human adenoviruses, and bovine papillomavirus 1. 总被引:4,自引:2,他引:2
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We have examined the effect of DNA tumor virus transformation of primary hamster embryo cells on the tyrosyl kinase activity of pp60c-src. Our present study demonstrates that some clones of hamster embryo cells transformed by simian virus 40, adenovirus type 2, adenovirus type 12, or bovine papillomavirus 1 can possess elevated pp60c-src kinase activity when compared with normal hamster embryo cells. However, other clones of hamster embryo cells transformed by these same viruses were found to have normal levels of pp60c-src kinase activity. In those clones of transformed cells where pp60c-src kinase activity was elevated, the increased levels of kinase activity were the result of an apparent increase in the specific activity of the pp60c-src phosphotransferase rather than an increase in the amount of the src gene product. Additionally, pp60c-src was not found to be physically associated with tumor antigens known to be encoded by these viruses. These results indicate that elevated levels of pp60c-src kinase activity can be found in hamster embryo cells transformed by several different DNA tumor viruses and suggest that the molecular mechanism by which pp60c-src kinase activity is elevated may differ from that previously observed in polyomavirus-transformed cells. These results also imply that elevation of pp60c-src kinase activity is not required for the transformation of hamster cells by these viruses. 相似文献
15.
Biological and biochemical properties of the c-src+ gene product overexpressed in chicken embryo fibroblasts. 总被引:12,自引:7,他引:5
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The c-src protein isolated from neuronal cells (pp60c-src+) displays a higher level of protein kinase activity than does pp60c-src from nonneural tissues. There are two structural alterations present in the amino-terminal half of pp60c-src+ expressed in neurons which could contribute to the enhanced activity of this form of pp60c-src: (i) a hexapeptide insert located at amino acid 114 of avian pp60c-src+ and (ii) a novel site(s) of serine phosphorylation. We characterized pp60c-src+ expressed in a nonneuronal cell type to identify factors that regulate the activity of the c-src+ protein and the importance of the neuronal environment on this regulation. The c-src+ protein overexpressed in chicken embryo fibroblasts (CEFs) displayed higher kinase activity than did pp60c-src. The major sites of phosphorylation of the c-src+ protein were Ser-17 and Tyr-527. The unique site(s) of serine phosphorylation originally identified in pp60c-src+ expressed in neurons was not detected in the c-src+ protein overexpressed in CEFs. Therefore, the hexapeptide insert is sufficient to cause an elevation in the tyrosine protein kinase activity of pp60c-src+. Our data also indicate that CEFs infected with the Rous sarcoma virus (RSV)c-src+ display phenotypic changes that distinguish them from cultures producing pp60c-src and that pp60c-src+-expressing cells are better able to grow in an anchorage-independent manner. The level of total cellular tyrosine phosphorylation in RSVc-src+-infected cultures was moderately higher than the level observed in cultures infected with RSVc-src. This level was not as pronounced as that observed in cells infected with RSVv-src or oncogenic variants of RSVc-src. Thus, pp60c-src+ could be considered a partially activated c-src variant protein much like other c-src proteins that contain mutations in the amino-terminal domain. 相似文献
16.
The sites of phosphorylation by protein kinase C and an intact SH2 domain are required for the enhanced response to beta-adrenergic agonists in cells overexpressing c-src. 总被引:6,自引:3,他引:3
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Previously we demonstrated that C3H10T1/2 murine fibroblasts overexpressing avian c-src exhibit elevated levels of cyclic AMP (cAMP) in response to beta-adrenergic agonists compared with that in control cells and that this enhanced response requires c-src kinase activity (W. A. Bushman, L. K. Wilson, D. K. Luttrell, J. S. Moyers, and S. J. Parsons, Proc. Natl. Acad. Sci. USA 87:7462-7466, 1990). However, it is not yet known which components of the beta-adrenergic receptor pathway, if any, interact with pp60c-src. It has recently been shown that immune complexes of pp60c-src phosphorylate recombinant G alpha proteins in vitro to stoichiometric levels, resulting in alterations of GTP binding and GTPase activity (W. P. Hausdorff, J. A. Pitcher, D. K. Luttrell, M. E. Linder, H. Kurose, S. J. Parsons, M. G. Caron, and R. J. Lefkowitz, Proc. Natl. Acad. Sci. USA 89:5720-5724, 1992), raising the possibility that the Gs alpha protein may be an in vivo target for the interaction with pp60c-src. To further characterize the involvement of pp60c-src in the beta-adrenergic signalling pathway, we have overexpressed, in 10T1/2 cells, pp60c-src containing mutations in several domains which are believed to be important for signalling processes. In this study we show that the sites of phosphorylation by protein kinase C (PKC) (Ser-12 and Ser-48) as well as the SH2 region of pp60c-src are required for the enhanced response of c-src overexpressors to beta-agonist stimulation. Mutation at the site of myristylation (Gly-2) results in a decrease in the enhanced response, while mutation at the site of phosphorylation by cAMP-dependent protein kinase (Ser-17) has no effect. Two-dimensional phosphotryptic analyses indicate that phosphorylation on Ser-12 and Ser-48 in unstimulated cells is associated with the ability of overexpressed pp60c-src to potentiate beta-adrenergic signalling. Cells overexpressing wild-type c-src also exhibit enhanced cAMP accumulation upon treatment with cholera toxin, an effect that is abated in cells overexpressing pp60c-src defective in the kinase or SH2 domains or altered at the sites of phosphorylation by PKC. These studies provide the first evidence for the physiological significance of the pp60c-src sites of PKC phosphorylation. In addition, they show that the SH2, Ser-12/48, and myristylation regions may be important for efficient interaction of pp60c-src with components of the beta-adrenergic pathway. Our data also support the possibility that the Gs alpha protein may be an in vivo target for alteration by pp60c-src. 相似文献
17.
Chronic kidney disease constitutes an increasing medical burden affecting 26 million people in the United States alone. Diabetes,
hypertension, ischemia, acute injury, and urological obstruction contribute to renal fibrosis, a common pathological hallmark
of chronic kidney disease. Regardless of etiology, elevated TGF-β1 levels are causatively linked to the activation of profibrotic
signaling pathways initiated by angiotensin, glucose, and oxidative stress. Unilateral ureteral obstruction (UUO) is a useful
and accessible model to identify mechanisms underlying the progression of renal fibrosis. Plasminogen activator inhibitor-1
(PAI-1), a major effector and downstream target of TGF-β1 in the progression of several clinically important fibrotic disorders,
is highly up-regulated in UUO and causatively linked to disease severity. SMAD and non-SMAD pathways (pp60c-src, epidermal growth factor receptor [EGFR], mitogen-activated protein kinase, p53) are required for PAI-1 induction by TGF-β1.
SMAD2/3, pp60c-src, EGFR, and p53 activation are each increased in the obstructed kidney. This review summarizes the molecular basis and translational
significance of TGF-β1-stimulated PAI-1 expression in the progression of kidney disease induced by ureteral obstruction. Mechanisms
discussed here appear to be operative in other renal fibrotic disorders and are relevant to the global issue of tissue fibrosis,
regardless of organ site. 相似文献
18.
Ronald W. Walenga Ph.D. Mark Kester Ph.D. Emanuel Coroneos M.D. Sarah Butcher Rohit Dwivedi Christopher Statt 《Prostaglandins & other lipid mediators》1996,52(5):341-359
Primary cultures of human tracheal epithelial (HTE) cells cultured in vitro, in defined serum-free media, express prostaglandin endoperoxide G/H synthase (PGHS) activity and produce prostaglandin E2 (PGE2). In contrast to every other cell type studied to date, HTE cells appear to constitutively express PGHS-2, the ‘inducible’ form of the enzyme, while expressing little or no PGHS-1, the ‘housekeeping’ isoenzyme in vitro. Prostaglandin synthesis in HTE cells was reduced by a selective PGHS-2 inhibitor, N-(2-cyclohexyloyl-4-nitrophenyl] methane-sulfonamide (NS398), with an IC50 of approximately 1 μM. Immunoblotting and immunoprecipitation of enzymatic activity with isozyme-specific antisera revealed only the PGHS-2 isoform. Full length human cDNA probes detected only PGHS-2 message in Northern blots. Neither PGHS-2 activity nor mRNA levels were dependent on, nor stimulated by peptide growth factors present in the defined serum-free growth medium, or by serum. Prolonged maintenance in the absence of retinoic acid, however, lead to a decline in PGHS activity. Phorbol-myristate acetate (PMA) induced PGHS-2 activity and mRNA and neither PMA-induced, nor constitutive PGHS-2 expression was suppressed by corticosteroids. Actinomycin D-treatment for six hours reduced the PGHS-2 activity and mRNA to only 50% that of untreated cells, suggesting that PGHS-2 mRNA is extremely stable in these cells. HTE cells, at least in vitro, appear unique among prostaglandin-producing cells in that they express PGHS-2, constitutively, independent of regulation by growth factors, serum, or corticosteroids and fail to express PGHS-1 under any culture condition studied. 相似文献
19.
20.
At least half the pp60c-src in NIH 3T3-derived c-src overexpresser cells in modified by novel threonine and, possibly, serine phosphorylation within its amino 16 kd region during mitosis. At the same time, the tryptic phosphopeptide containing Ser 17, the site of cyclic AMP-dependent phosphorylation, is either modified or dephosphorylated. While the amount of pp60c-src is not significantly altered, the in vitro-specific kinase activity of modified pp60c-src is enhanced 4- to 7-fold. Modified pp60c-src has the same tyrosine-containing tryptic phosphopeptides as pp60c-src from unsynchronized cells, indicating that activation is independent of Tyr 416/Tyr 527 phosphorylation. Electrophoretic mobility retardations indicated that endogenous pp60c-src and pp60v-src are similarly modified during mitosis. The modifications and enhanced activity disappear near the time of cell division. These results suggest that pp60c-src is regulated by and, in turn, may regulate mitosis-specific events in fibroblasts. 相似文献