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1.
We have identified two tyrosine phosphorylation sites, Tyr 1009 and Tyr 1021, in the C-terminal noncatalytic region of the human platelet-derived growth factor (PDGF) receptor beta subunit. Mutant receptors with phenylalanine substitutions at either or both of these tyrosines were expressed in dog epithelial cells. Mutation of Tyr 1021 markedly reduced the PDGF-stimulated binding of phospholipase C (PLC) gamma 1 but had no effect on binding of the GTPase activator protein of Ras or of phosphatidylinositol 3 kinase. Mutation of Tyr 1009 reduced binding of PLC gamma 1 less severely. Mutation of Tyr 1021, or both Tyr 1009 and Tyr 1021, also reduced the PDGF-dependent binding of a transiently expressed fusion protein containing the two Src-homology 2 domains from PLC gamma 1. Mutation of Tyr 1021, or both Tyr 1009 and Tyr 1021, greatly reduced PDGF-stimulated tyrosine phosphorylation of PLC gamma 1 but did not prevent the tyrosine phosphorylation of other cell proteins, including mitogen-activated protein kinase. We conclude that Tyr 1021, and possibly Tyr 1009, is a binding site for PLC gamma 1. 相似文献
2.
SR James A Paterson TK Harden RA Demel CP Downes 《Canadian Metallurgical Quarterly》1997,36(4):848-855
We have examined the influence of surface pressure and phospholipid composition on hydrolysis of phosphatidylinositol (4,5)-bisphosphate (PIP2) by phospholipase C beta 1 (PLC beta 1) and PLC beta 2 in mixed composition phospholipid monolayers. Increasing the monolayer surface pressure from 15 to 36 mN/m reduced the rate at which PIP2 was hydrolyzed by PLC beta 1 and PLC beta 2 by 4-6-fold, although PLC beta 1 was more active than PLC beta 2, even at high surface pressure. Reduced enzyme activity was accompanied by an increase in reaction induction times, suggesting that increasing surface pressure reduced the penetration rate of the enzymes into the monolayer. Quantitation of interfacial enzyme concentration using 35S-labeled PLC beta 1 confirmed that less enzyme was associated with the monolayer at higher pressures. The relationship between PLC activity and substrate concentration was examined at a single surface pressure of 30 mN/m. This relationship was not hyperbolic, and increases in the mole percentage (mol %) of PIP2 in the monolayer resulted in an upwardly-curving increase in PLC activity. Thus, PLC beta 1 activity increased 7-fold and PLC beta 2 activity increased 4-fold when the mol % of PIP2 in the monolayer increased from 17.9% to 29%, increasing further thereafter. Paradoxically, increasing the mol % of PIP2 from 0 to 60% was accompanied by a 3-fold decrease in interfacial enzyme concentrations. Taken together, these data show that the catalytic activity of PLC beta involves some element of penetration of lipid interfaces, and suggest that the organization of the substrate facilitates PLC activity, giving credence to the substrate theory of interfacial activation of phospholipases. We present a hypothesis suggesting that PIP2 molecules coalesce into enriched lateral domains which favor PLC beta activity. 相似文献
3.
ES Oh A Woods ST Lim AW Theibert JR Couchman 《Canadian Metallurgical Quarterly》1998,273(17):10624-10629
Phosphatidylinositol 4,5-bisphosphate (PIP2) is involved in the organization of the actin cytoskeleton by regulating actin-associated proteins. The transmembrane heparan sulfate proteoglycan syndecan-4 also plays a critical role in protein kinase C (PKC) signaling in the formation of focal adhesions and actin stress fibers. The cytoplasmic domain of syndecan-4 core protein directly interacts with and potentiates PKCalpha activity, and it can directly interact with the phos- phoinositide PIP2. We, therefore, investigated whether the interaction of inositol phosphates and inositol phospholipids with syndecan-4 could regulate PKC activity. Data from in vitro kinase assays using purified PKCalpha beta gamma show that in the absence of phosphatidylserine and diolein, PIP2 increased the extent of autophosphorylation of PKCalpha beta gamma and partially activated it to phosphorylate both histone III-S and an epidermal growth factor receptor peptide. This activity was dose-dependent, and its calcium dependence varied with PKC isotype/source. Addition of the cytoplasmic syndecan-4 peptide, but not equivalent syndecan-1 or syndecan-2 peptides, potentiated the partial activation of PKCalpha beta gamma by PIP2, resulting in activity greater than that observed with phosphatidylserine, diolein, and calcium. This study indicates that syndecan-4 cytoplasmic domain may bind both PIP2 and PKCalpha, localize them to forming focal adhesions, and potentiate PKCalpha activity there. 相似文献
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H Ishihara Y Shibasaki N Kizuki T Wada Y Yazaki T Asano Y Oka 《Canadian Metallurgical Quarterly》1998,273(15):8741-8748
Type I phosphatidylinositol 4-phosphate (PtdIns(4)P) 5-kinases (PIP5K) catalyze the synthesis of phosphatidylinositol 4, 5-bisphosphate, an essential lipid molecule in various cellular processes. Here, we report the cloning of the third member (PIP5Kgamma) and the characterization of members of the type I PIP5K family. Type I PIP5Kgamma has two alternative splicing forms, migrating at 87 and 90 kDa on SDS-polyacrylamide gel electrophoresis. The amino acid sequence of the central portion of this isoform shows approximately 80% identity with those of the alpha and beta isoforms. Northern blot analysis revealed that the gamma isoform is highly expressed in the brain, lung, and kidneys. Among three isoforms, the beta isoform has the greatest Vmax value for the PtdIns(4)P kinase activity and the gamma isoform is most markedly stimulated by phosphatidic acid. By analyzing deletion mutants of the three isoforms, the minimal kinase core sequence of these isoforms were determined as an approximately 380-amino acid region. In addition, carboxyl-terminal regions of the beta and gamma isoforms were found to confer the greatest Vmax value and the highest phosphatidic acid sensitivity, respectively. It was also discovered that lysine 138 in the putative ATP binding motif of the alpha isoform is essential for the PtdIns(4)P kinase activity. As was the case with the alpha isoform reported previously (Shibasaki, Y., Ishihara, H., Kizuki, N., Asano, T., Oka, Y., Yazaki, Y. (1997) J. Biol. Chem. 272, 7578-7581), overexpression of either the beta or the gamma isoform induced an increase in short actin fibers and a decrease in actin stress fibers in COS7 cells. Surprisingly, a kinase-deficient substitution mutant also induced an abnormal actin polymerization, suggesting a role of PIP5Ks via structural interactions with other molecules. 相似文献
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YY Chang SJ Kim TK Park SS Kang MJ Ha JF Mushinski JS Chun 《Canadian Metallurgical Quarterly》1998,45(6):1139-1148
This study was performed to examine effects of the overexpression of protein kinase C (PKC) isoforms (i.e., beta I, beta II, gamma, delta, eta, and zeta) on mitogen-activated protein (MAP) kinase (Erk-1 and -2) signaling and growth characteristics of NIH3T3 cells. Phorbol ester (PMA) activated endogenous and ectopically expressed PKC alpha, beta I, beta II, gamma, delta, epsilon, and eta. Overexpression of the examined PKC isoforms enhanced PMA-induced MAP kinase activation. Potentiation of MAP kinase activation was also observed upon stimulation of cells with platelet-derived growth factor (PDGF) although there was no indication for the activation PKC isoforms by PDGF. Inhibition of PKC blocked PMA- but not PDGF-induced MAP kinase activation. Thus, potentiation of PDGF-induced MAP kinase activation appears to be independent to PKC activity, while PMA-induced MAP kinase activation requires PKC activity. The ability of PKC isoforms to potentiate MAP kinase activation is not related to the growth characteristics of cells because individual PKC isoforms differentially regulated maximum density and proliferation of cells. 相似文献
9.
RA Klinghoffer B Duckworth M Valius L Cantley A Kazlauskas 《Canadian Metallurgical Quarterly》1996,16(10):5905-5914
Upon binding of platelet-derived growth factor (PDGF), the PDGF beta receptor (PDGFR) undergoes autophosphorylation on distinct tyrosine residues and binds several SH2-domain-containing signal relay enzymes, including phosphatidylinositol 3-kinase (PI3K), phospholipase C gamma (PLC gamma), the GTPase-activating protein of Ras (RasGAP), and the tyrosine phosphatase SHP-2. In this study, we have investigated whether PDGF-dependent PI3K activation is affected by the other proteins that associate with the PDGFR. We constructed and characterized a series of PDGFR mutants which contain binding sites for PI3K as well as one additional protein, either RasGAP, SHP-2, or PLC gamma. While all of the receptors had wild-type levels of PDGF-stimulated tyrosine kinase activity and associated with comparable amounts of PI3K activity, their abilities to trigger accumulation of PI3K products in vivo differed dramatically. The wild-type receptor, as well as receptors that recruited PI3K or PI3K and SHP-2, were all capable of fully activating PI3K. In contrast, receptors that associated with PI3K and RasGAP or PI3K and PLC gamma displayed a greatly reduced ability to stimulate production of PI3K products. When this series of receptors was tested for their ability to activate Ras, we observed a strong positive correlation between Ras activation and PI3K activation. Further investigation of the relationship between Ras and PI3K indicated that Ras was upstream of PI3K. Thus, activation of PI3K requires not only binding of PI3K to the tyrosine-phosphorylated PDGFR but accumulation of GTP-bound Ras as well. Furthermore, PLC gamma and RasGAP negatively modulate PDGF-dependent PI3K activation. Finally, PDGF-stimulated signal relay can be regulated by altering the ratio of SH2-domain-containing enzymes that are recruited to the PDGFR. 相似文献
10.
BACKGROUND & AIMS: Long-term ethanol intake suppresses liver regeneration in vivo and ethanol interferes with epidermal growth factor (EGF)-induced DNA synthesis in vitro. Therefore, the effects of long-term ethanol treatment on EGF-activated signaling reactions in rat hepatocytes were investigated. METHODS: Hepatocytes from long-term ethanol-fed rats and pair-fed controls were stimulated with EGF (0.5-20 nmol/L) for 15-120 seconds. Tyrosine phosphorylation of EGF receptor (EGFR), Shc, and phospholipase-C gamma1 (PLC gamma), and growth factor receptor binding protein 2 (Grb2) coprecipitation with EGFR and Shc were analyzed by Western blotting. RESULTS: EGFR autophosphorylation was suppressed at all EGF concentrations in ethanol-fed cells compared with pair-fed cells, without significant differences in total EGFR protein or EGFR tyrosine kinase activity detected in cell lysates, suggesting that intracellular factors suppressed EGFR function. EGF-induced PLC gamma tyrosine phosphorylation and inositol 1,4,5-trisphosphate (InsP3) formation were suppressed, but cytosolic [Ca2+]c elevation was little affected, indicating enhanced InsP3-mediated intracellular Ca2+ release in ethanol-fed cells. Grb2 binding to EGFR was suppressed, but EGF-induced Shc tyrosine phosphorylation and Grb2 association with Shc were not significantly decreased. CONCLUSIONS: Long-term ethanol feeding suppressed EGF-induced receptor autophosphorylation in rat hepatocytes with differential inhibition of downstream signaling processes mediated by PLC gamma, Shc, and Grb2. Altered patterns of downstream signals emanating from EGFR may contribute to deficient liver regeneration in chronic alcoholism. 相似文献
11.
Targeted gene disruption shows that knobs enable malaria-infected red cells to cytoadhere under physiological shear stress 总被引:1,自引:0,他引:1
BS Crabb BM Cooke JC Reeder RF Waller SR Caruana KM Davern ME Wickham GV Brown RL Coppel AF Cowman 《Canadian Metallurgical Quarterly》1997,89(2):287-296
Sphingosine, which is on the pathway of sphingomyelin degradation, activates phospholipase C (PLC) delta1 moderately. In the liposome assay effect of sphingosine on PLC delta1 activity depends on KCl concentration. Stimulation of PLC delta1 by sphingosine increased as the KCl concentration is increased from 0 to 100 mM, and then diminished with the increasing KCl. In the liposome assay sphingosine diminishes inhibition of PLC delta1 by sphingomyelin. To determine the domain of PLC delta1 which interacts with sphingosine active proteolytic fragments of PLC delta1 were generated by trypsin digestion of the native enzyme. Sphingosine affects the activity of PLC delta1 fragment which lacked the amino-terminal domain (first 60 amino acids) but not the active fragment that has cleaved the domain spanning the X and Y region of PLC delta1. These observations indicate that for interaction of sphingosine with PLC delta1 intact domain that span regions of conservation, designated as X and Y is necessary. When the activity of PLC delta1 was assayed with PIP2 in the erythrocyte membrane as substrate, sphingosine strongly inhibited PLC delta1. The other homolog of sphingosine 4-hydroxysphinganine (phytosphingosine) inhibited PLC delta1 to much lesser extent. The activity of PLC delta1 was inhibited by 68% and 22% in the presence of 20 microM sphingosine and phytosphingosine, respectively. This inhibition was completely abolished by deoxycholate at a concentration of 1.5 mM. These observations suggest that sphingosine may regulate activity of PLC delta1 in the cell. 相似文献
12.
Phosphatidyl inositol-phospholipase C (PI-PLC) in squid retina was studied by immunoblotting and its activities were determined using [3H]phosphatidyl inositol bisphosphate ([3H]PIP2) as substrate. PI-PLC activity was found mostly in soluble fraction when the retina homogenate was treated with 400 mM KCl, but was associated with rhabdomal membranes under low salt conditions (20 mM Hepes). A protein with apparent molecular mass of 130kD was recognized by an antibody against PLC beta 4/norp A in both 400 mM KCl soluble and rhabdomal membrane fractions. A 42 kD protein recognized by antibody against the C-terminus of Gq alpha was also present in these two fractions. GTP gamma S stimulated only the PI-PLC activity associated with membrane and was magnesium dependent. PI-PLC activity was found to be (i) highly dependent upon calcium concentrations, (ii) enhanced by GTP but not by other nucleotides, and (iii) significantly stimulated by light at lower concentrations of GTP gamma S. The stimulation by light was still observed when irradiated membrane was incubated at 10 degrees C for 10 min and then mixed with GTP gamma S. These results suggest that stable metarhodopsin stimulates a PLC beta 4/norp A-like enzyme via a G-protein, Gq. 相似文献
13.
K Oda H Asao M Higuchi N Tanaka S Moffatt M Nakamura K Tabayashi K Sugamura 《Canadian Metallurgical Quarterly》1997,9(9):1303-1310
Our previous study demonstrated that IL-2 suppressed growth of human T cell lines, in which the suppression was observed with members among HTLV-I-infected T cell lines independent of IL-2 for growth. In this study, we examined the molecular mechanism of IL-2-induced growth suppression with two HTLV-I-infected T cell lines; TL-OmI expressing endogenously three subunits, i.e. alpha, beta and gamma chains, of the IL-2 receptor, and an MT-1 transfectant expressing the endogenous alpha and gamma chains and exogenous beta chain. Our analysis revealed that IL-2 induced apoptosis in both T cell lines. Experiments with inhibitors for the proteases responsible for apoptosis signals showed that caspase 1 (IL-1 beta-converting enzyme) was not involved in apoptosis induced by IL-2. Other MT-1 sublines introduced with mutant beta chains demonstrated that IL-2-induced apoptosis required signals from both the serine-rich (S) region and acidic (A) region of the IL-2 receptor beta chain, which are essential but not critical for IL-2-mediated cell growth respectively. Collectively, IL-2 functions not only on growth promotion and prevention of apoptosis but also on induction of apoptosis, which may be implicated in physiological regulation of immune reactions by controlling growth and activation of T cells. 相似文献
14.
Pleckstrin is a 40 kDa substrate for protein kinase C found in platelets and neutrophils. Based upon its sequence, pleckstrin contains two of the recently-described PH domains that are thought to be binding motifs for phosphatidyl 4,5-bisphosphate (PIP2) and/or G protein beta gamma heterodimers (G beta gamma). In the present studies we have examined the interaction between pleckstrin and G beta gamma by incubating pleckstrin fusion proteins with lysates from human platelets. In this analysis, both the N-terminal and C-terminal PH domains from pleckstrin bound G beta gamma in vitro, as did peptides containing as little as the first 30 residues of the C-terminal pleckstrin PH domain. Introduction of a point mutation into this region, analogous to the mutation in the Btk PH domain that causes X-linked immunodeficiency disease (XID) in mice, dramatically disrupted this interaction. We propose that pleckstrin may interact with G beta gamma, and that one potential site for this interaction involves the first 30 residues of pleckstrin's C-terminal PH domain. 相似文献
15.
JJ Walker 《Canadian Metallurgical Quarterly》1998,16(1):47-55
We have previously reported that gastrin induces a rapid and transient tyrosine phosphorylation of phospholipase C gamma 1 (PLC gamma 1) in association with inositol 1,4,5-trisphosphate (IP3) formation in rat colonic epithelial cells (34). In this study, we demonstrate that gastrin regulates IP3 formation mainly through PLC gamma 1 isozyme. Immunoblotting analysis revealed the expression of PLC beta 3 and -gamma 1, but not PLC beta 1, -beta 2, or -beta 4 in the rat colonic epitheliums. To explore what PLC isozyme(s) modulates gastrin effect on IP3, immunoneutralizing antibody to PLC beta 1, -beta 3, or -gamma 1 was introduced into the colonic cells using a lipid carrier. The gastrin-stimulated increase in IP3 concentration was specifically prevented by anti-PLC gamma 1 but not by anti-PLC beta 1 or -beta 3 antibody. Immunoprecipitation assays have also revealed that gastrin promoted an increase in tyrosine phosphorylation and co-precipitation of a 60 kDa src kinase with PLC gamma 1. Administration of antibody specific to pp60c-src into the colonic cells prevented the gastrin-stimulated increases in IP3. Tyrosine phosphorylation of PLC gamma 1 may be a major mechanism through which gastrin regulates IP3 level in the colonic cells. Pretreatment of cells with the tyrosine kinase inhibitor genistein abrogated gastrin's effect on IP3, while extended pretreatment with pertussis toxin, a G-protein inhibitor, did not affect the ability of gastrin to stimulate IP3 formation. Colonic cells expressed the G alpha i subunits1-3; however, immunoblotting analysis did not reveal any difference in G alpha i proteins' expression between control and gastrin treated cells. The results provide direct evidence that gastrin regulates IP3 level by a signaling mechanism that involves PLC gamma 1 and pp60c-src kinase. 相似文献
16.
Erythropoietin (EPO) is a hormone, as well as a hematopoietic growth factor, that specifically regulates the proliferation and differentiation of erythroid progenitor cells. Although the membrane-bound receptor for EPO has no intrinsic kinase activity, it triggers the activation of protein kinases via phospholipases A2, C, and D. A cascade of serine and threonine kinases, including Raf-1, MAP kinase and protein kinase C (PKC) is activated following tyrosine phosphorylation. In this study, we have examined whether changes in nuclear PKC and 1,2-diacylglycerol (DAG) are induced following EPO treatment of the murine target cell line, B6SUt.EP. Western blot analysis using isoform-specific antibodies demonstrated the presence of PKC beta II, but not PKC alpha, beta I, gamma, epsilon, delta, eta, or zeta in the nuclei of cells stimulated with EPO. The increase in nuclear beta II levels was accompanied by an immediate rise in DAG mass levels with both of the increases peaking by 1 min. These rapid increases in nuclear DAG and PKC beta II expression suggest a mechanism for EPO-induced changes in gene expression necessary for cell proliferation. 相似文献
17.
gamma delta T lymphocytes, which are CD3+ lymphocytes that express gamma delta chains of the T-cell antigen receptor (TCR) on their surface, are functionally distinct from alpha beta T lymphocytes, which express alpha beta chains of the TCR. gamma delta T lymphocytes are thought to differentiate in mouse hepatic sinusoids, to play a role in antitumor action, and to act as natural killer cells. The purpose of this study was to examine whether gamma delta T lymphocytes in the peripheral blood are suppressed when hepatic sinusoids are damaged during transcatheter arterial embolization (TAE). The numbers of alpha beta T lymphocytes and gamma delta T lymphocytes in the peripheral blood were examined with monoclonal antibodies and flow cytometry before and after TAE in 32 patients (from 46 to 78 years of age) with liver cirrhosis and hepatocellular carcinoma. The number of alpha beta T lymphocytes before and after TAE was unchanged. However, the number of gamma delta T lymphocytes and the ratio of gamma delta T lymphocytes to CD3+ lymphocytes were significantly decreased for 3 weeks after TAE treatment. This decrease suggests that TAE suppresses the supply of gamma delta T lymphocytes to the peripheral blood. In addition, TAE may weaken a patient's antitumor immunity, because gamma delta T lymphocytes that have antitumor activity decrease after TAE. 相似文献
18.
To delineate the specific regions of phospholipase C beta2 (PLC beta2) involved in binding and activation by G protein betagamma subunits, we synthesized peptides corresponding to segments of PLC beta2. Two overlapping peptides corresponding to Asn-564-Lys-583 (N20K) and Glu-574-Lys-593 (E20K) inhibited the activation of PLC beta2 by betagamma subunits (IC50 50 and 150 microM, respectively), whereas two control peptides did not. N20K and E20K, but not the control peptides, inhibited betagamma-dependent ADP-ribosylation of Galphai1 by pertussis toxin and betagamma-dependent activation of phosphoinositide 3-kinase. To demonstrate direct binding of the peptides to betagamma subunits, the peptides were chemically cross-linked to purified beta1gamma2. N20K and E20K cross-linked to both beta1 and gamma2 subunits, whereas the control peptides did not. Cross-linking to beta and gamma was inhibited by incubation with excess PLC beta2 or PLC beta3, whereas cross-linking to gamma but not beta was inhibited by r-myr-alphai1. These data together demonstrate specificity of N20K and E20K for G betagamma binding and inhibition of effector activation by betagamma subunits. The results suggest that an overlapping region of the two active peptides, Glu-574-Lys-583, mimics a region of PLC beta2 that is involved in binding to betagamma subunits. Changing a tyrosine to a glutamine in this overlapping region of the peptides inhibited binding of the peptide to betagamma subunits. Alignment of these peptides with the three-dimensional structure from PLC delta1 identifies a putative alpha helical region on the surface of the catalytic domain of PLC beta2 that could interact with betagamma subunits. 相似文献
19.
We have recently reported increased tyrosine (TYR) phosphorylation of a number of pancreatic acinar cell proteins following antibody ligation of beta 1 integrins (Wrenn and Herman, Biochem, Biophys. Res. Commun. 208, 1995, 978-984). Concurrent with this TYR phosphorylation was a marked activation of protein kinase C (PKC). This led us to investigate phospholipase C gamma 1 (PLC gamma 1), a key enzyme responsible for diacylglycerol generation, as a target for integrin-mediated TYR phosphorylation. Staining with antiphosphotyrosine antibodies revealed increased TYR phosphorylation of immunoprecipitated PLC gamma 1 prepared from beta 1 integrin-ligated acinar cells. Subsequent stripping and reprobing of Western blots with polyclonal anti-PLC gamma 1 was confirmatory. Over this same time period, intracellular [Ca2+] increased from < 100 nM to 600 nM, further suggesting a functional relevance of integrin-linked phosphorylation as a regulatory mechanism in exocrine pancreas. 相似文献
20.
Adenocarcinoma of the prostate is the second leading cause of cancer deaths in men. The protein kinase C (PKC) family of signal transducing kinases has been implicated in neoplastic transformation and progression in other tissues, and some evidence suggests roles for PKC in prostate growth and neoplasia. We have detected expression of eight specific PKC isozyme mRNAs (alpha, beta, gamma, delta, epsilon, eta, theta, and zeta) in normal rat whole prostate and found some of these to be differentially expressed in certain Dunning R-3327 rat prostatic adenocarcinoma sublines. PKC zeta mRNA was detected in normal prostate and Dunning H tumor, whereas an alternatively spliced form of PKC zeta RNA was found in Dunning G tumor and normal brain. Both forms of PKC zeta RNA were markedly reduced in the androgen insensitive, highly metastatic Dunning AT-3, MAT-Lu, and MAT-LyLu tumors. We have cloned and report the sequence of the novel portion of the alternatively spliced form of PKC zeta RNA, which is polyadenylated and present in cytoplasm. 相似文献