Multiple forms of phosphoinositide-specific phospholipase C and different modes of activation.
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Animals↗
Biomedical subjects
S G Rhee
Publications and source records attributed to S G Rhee.
Role of phospholipase in generating lipid second messengers in signal transduction.
Many lipids or lipid-derived products generated by phospholipases acting on phospholipids in membranes are implicated as mediators and second messengers in signal transduction. Our current understanding of the primary sequence relationships within the class of extracellular phospholipase A2's and among the numerous forms of the mammalian phosphatidylinositol-specific phospholipase C's is reviewed. New results suggesting roles for these phospholipases as well as other phospholipases such as phospholipase C and D acting on phosphatidlycholine in generating arachidonic acid for eicosanoid biosynthesis, inositol phosphates for Ca2+ mobilization, and diglyceride for protein kinase C activation through receptor-mediated processes, are discussed. In addition, the possible role of phospholipases acting on sphingolipids such as sphinglomyelinase in generating lipid mediators is considered.
Arachidonic Acid↗
The erbB-2 mitogenic signaling pathway: tyrosine phosphorylation of phospholipase C-gamma and GTPase-activating protein does not correlate with erbB-2 mitogenic potency.
The erbB-2 gene product, gp185erbB-2, unlike the structurally related epidermal growth factor (EGF) receptor (EGFR), exhibits constitutive kinase and transforming activity. We used a chimeric EGFR/erbB-2 expression vector to compare the mitogenic signaling pathway of the erbB-2 kinase with that of the EGFR, at similar levels of expression, in response to EGF stimulation. The EGFR/erbB-2 chimera was significantly more active in inducing DNA synthesis than the EGFR when either was expressed in NIH 3T3 cells. Analysis of biochemical pathways implicated in signal transduction by growth factor receptors indicated that both phospholipase C type gamma (PLC-gamma) and the p21ras GTPase-activating protein (GAP) are substrates for the erbB-2 kinase in NIH 3T3 fibroblasts. However, under conditions in which activation of the erbB-2 kinase induced DNA synthesis at least fivefold more efficiently than the EGFR, the levels of erbB-2- or EGFR-induced tyrosine phosphorylation of PLC-gamma and GAP were comparable. In addition, the stoichiometry of tyrosine phosphorylation of these putative substrates by erbB-2 appeared to be at least an order of magnitude lower than that induced by platelet-derived growth factor receptors at comparable levels of mitogenic potency. Thus, our results indicate that differences in tyrosine phosphorylation of PLC-gamma and GAP do not account for the differences in mitogenic activity of the erbB-2 kinase compared with either the EGFR or platelet-derived growth factor receptor in NIH 3T3 fibroblasts.
Animals↗
Cyclic AMP agonists induce the phosphorylation of phospholipase C-tau and of a 76 kDa protein co-precipitated by anti-(phospholipase C-tau) monoclonal antibodies in BALB/c-3T3 cells. Relationship to inositol phosphate formation.
Previous studies have demonstrated enhanced phosphorylation of phospholipase C-tau (PLC-tau), a key regulatory enzyme in phosphoinositide metabolism, in cells treated with platelet-derived growth factor (PDGF) and epidermal growth factor, both of which act via specific receptor tyrosine kinases. Our studies on BALB/c-3T3 cells show that agents that promote cellular cyclic AMP accumulation also increase the phosphorylation, specifically the serine phosphorylation, of this enzyme. Increased phosphorylation of PLC-t (2-3-fold) was evident within 5-10 min of addition of isobutylmethylxanthine (IBMX) and either cholera toxin or forskolin to cells, and persisted for at least 3 h. Treatment of cells with cyclic AMP agonists also enhanced, with similar kinetics, the phosphorylation of a 76 kDa protein co-precipitated by anti-PLC-tau monoclonal antibodies. Brief exposure of cells to cholera toxin/IBMX or forskolin/IBMX decreased inositol phosphate formation induced by the GTP-binding protein (G-protein) activator aluminium fluoride by approx. 50%, but was without effect on PDGF-stimulated inositol phosphate formation. These findings suggest that PLC-tau, and perhaps the 76 kDa co-precipitated protein, are substrates of cyclic AMP-dependent protein kinase in BALB/c-3T3 cells: however, the lack of effect of cyclic AMP elevation on PDGF-stimulated inositol phosphate formation indicates that the intrinsic activity of PLC-tau is unaltered by cyclic AMP-mediated phosphorylation.
1-Methyl-3-isobutylxanthine↗
Increase of the catalytic activity of phospholipase C-gamma 1 by tyrosine phosphorylation.
Phospholipase C-gamma 1 (PLC-gamma 1), an isozyme of the phosphoinositide-specific phospholipase C family, which occupies a central role in hormonal signal transduction pathways, is an excellent substrate for the epidermal growth factor (EGF) receptor tyrosine kinase. Epidermal growth factor elicits tyrosine phosphorylation of PLC-gamma 1 and phosphatidylinositol 4,5-bisphosphate hydrolysis in various cell lines. The ability of tyrosine phosphorylation to activate the catalytic activity of PLC-gamma 1 was tested. Tyrosine phosphorylation in intact cells or in vitro increased the catalytic activity of PLC-gamma 1. Also, treatment of EGF-activated PLC-gamma 1 with a tyrosine-specific phosphatase substantially decreased the catalytic activity of PLC-gamma 1. These results suggest that the EGF-stimulated formation of inositol 1,4,5-trisphosphate and diacylglycerol in intact cells results, at least in part, from catalytic activation of PLC-gamma 1 through tyrosine phosphorylation.
Catalysis↗
Feedback regulation of phospholipase C-beta by protein kinase C.
Treatment of a variety of cells and tissues with 12-O-tetradecanoylphorbol-13-acetate (TPA), an activator of protein kinase C (PKC) results in the inhibition of receptor-coupled inositol phospholipid-specific phospholipase C (PLC) activity. To determine whether or not the targets of TPA-activated PKC include one or more isozymes of PLC, studies were carried out with PC12, C6Bu1, and NIH 3T3 cells, which contain at least three PLC isozymes, PLC-beta, PLC-gamma, and PLC-delta. Treatment of the cells with TPA stimulated the phosphorylation of serine residues in PLC-beta, but the phosphorylation state of PLC-gamma and PLC-delta was not changed significantly. Phosphorylation of bovine brain PLC-beta by PKC in vitro resulted in a stoichiometric incorporation of phosphate at serine 887, without any concomitant effect on PLC-beta activity. We propose, therefore, that rather than having a direct effect on enzyme activity, the phosphorylation of PLC-beta by PKC may alter its interaction with a putative guanine nucleotide-binding regulatory protein and thereby prevent its activation.
Amino Acid Sequence↗
Epidermal growth factor and platelet-derived growth factor promote translocation of phospholipase C-gamma from cytosol to membrane.
Treatment of HER 14 cells with epidermal growth factor (EGF) or platelet-derived growth factor (PDGF) induced a translocation of phospholipase C-gamma (PLC-gamma) from cytosol to membrane. In such growth factor-treated cells, cytosolic PLC-gamma was found to contain more phosphotyrosine than membrane-associated enzyme. Because these growth factors have been shown to promote both the physical association of PLC-gamma with their receptors and the subsequent phosphorylation of the enzyme directly by the membrane-bound receptor tyrosine kinases, the membrane association of PLC-gamma may simply be due to the formation of transient enzyme (receptor)-substrate (PLC-gamma) complexes. If this is the case, membrane-associated PLC-gamma would be expected to be released from membrane after undergoing tyrosine phosphorylation. However, tyrosine phosphorylation of membrane-associated PLC-gamma by the EGF receptor in vitro did not result in the release of PLC-gamma from membrane. Thus, the association of PLC-gamma with membrane would appear to involve more than enzyme-substrate complex.
Animals↗
Evidence that the epidermal growth factor receptor and non-tyrosine kinase hormone receptors stimulate phosphoinositide hydrolysis by independent pathways.
We have shown previously that exposure of a non-transformed continuous line of rat liver epithelial (WB) cells to epidermal growth factor (EGF), adrenaline, angiotensin II or [Arg8]vasopressin results in an accumulation of the inositol phosphates InsP1, InsP2 and InsP3 [Hepler, Earp & Harden (1988) J. Biol. Chem. 263, 7610-7619]. Studies were carried out with WB cells to determine whether the EGF receptor and other, non-tyrosine kinase, hormone receptors stimulate phosphoinositide hydrolysis by common, overlapping or separate pathways. The time courses for accumulation of inositol phosphates in response to angiotensin II and EGF were markedly different. Whereas angiotensin II stimulated a very rapid accumulation of inositol phosphates (maximal by 30 s), increases in the levels of inositol phosphates in response to EGF were measurable only following a 30 s lag period; maximal levels were attained by 7-8 min. Chelation of extracellular Ca2+ with EGTA did not modify this relative difference between angiotensin II and EGF in the time required to attain maximal phospholipase C activation. Under experimental conditions in which agonist-induced desensitization no longer occurred in these cells, the inositol phosphate responses to EGF and angiotensin II were additive, whereas those to angiotensin II and [Arg8]vasopressin were not additive. In crude WB lysates, angiotensin II, [Arg8]vasopressin and adrenaline each stimulated inositol phosphate formation in a guanine-nucleotide-dependent manner. In contrast, EGF failed to stimulate inositol phosphate formation in WB lysates in the presence or absence of guanosine 5'-[gamma-thio]triphosphate (GTP[S]), even though EGF retained the capacity to bind to and stimulate tyrosine phosphorylation of its own receptor. Pertussis toxin, at concentrations that fully ADP-ribosylate and functionally inactivate the inhibitory guanine-nucleotide regulatory protein of adenylate cyclase (Gi), had no effect on the capacity of EGF or hormones to stimulate inositol phosphate accumulation. In intact WB cells, the capacity of EGF, but not angiotensin II, to stimulate inositol phosphate accumulation was correlated with its capacity to stimulate tyrosine phosphorylation of the 148 kDa isoenzyme of phospholipase C. Taken together, these findings suggest that, whereas angiotensin II, [Arg8]vasopressin and alpha 1-adrenergic receptors are linked to activation of one or more phospholipase(s) C by an unidentified G-protein(s), the EGF receptor stimulates phosphoinositide hydrolysis by a different pathway, perhaps as a result of its capacity to stimulate tyrosine phosphorylation of phospholipase C-gamma.
Angiotensin II↗
Stimulation of phospholipase C-gamma 1 membrane association by epidermal growth factor.
Epidermal growth factor (EGF) treatment of A-431 epidermoid carcinoma cells elicited a redistribution of phospholipase C-gamma 1 (PLC-gamma 1) from a predominantly cytosolic localization to membrane fractions. The temporal coincidence of this redistribution with EGF stimulation of inositol phosphate formation and EGF increased phosphorylation of PLC-gamma 1 suggests that the membrane association of PLC-gamma 1 is a significant event in second messenger transduction.
Carcinoma, Squamous Cell↗
Regulation of D-myo-inositol 1,4,5-trisphosphate 3-kinase by cAMP-dependent protein kinase and protein kinase C.
The Ca2(+)-mobilizing second messenger D-myo-inositol 1,4,5-trisphosphate (Ins(1,4,5)P3) is converted to the putative messenger D-myo-inositol 1,3,4,5-tetrakisphosphate by Ins(1,4,5)P3 3-kinase. We found that cAMP-dependent protein kinase and protein kinase C phosphorylate, and thereby modulate, the activity of Ins(1,4,5)P3 3-kinase. cAMP-dependent kinase introduced a stoichiometric amount of phosphate at serine 109 of the 53-kDa polypeptide and caused a 1.8-fold increase in Vmax, whereas the protein kinase C-dependent phosphorylation reduced the Vmax to one-fourth of that of the unphosphorylated enzyme. Upon prolonged incubation, protein kinase C introduced phosphate at multiple sites in Ins(1,4,5)P3 3-kinase, and the resulting inactivation of the enzyme appeared to be well-correlated with the simultaneous phosphorylation of two major sites, serine 109 and serine 175. The Km for Ins(1,4,5)P3 was not affected significantly after phosphorylation by either protein kinase. We propose, therefore, that the phosphorylation of Ins(1,4,5)P3 3-kinase by cAMP-dependent kinase and protein kinase C constitutes mechanisms of cross-talk between cellular signaling pathways that use various second messengers such as inositol phosphates, diacylglycerol, Ca2+, and cAMP.
Amino Acid Sequence↗
Purification and properties of D-myo-inositol 1,4,5-trisphosphate 3-kinase from rat brain. Susceptibility to calpain.
A new, rapid method for purification of inositol(1,4,5)P3 3-kinase in high yield from rat brain is described. Purified enzyme exhibited a polypeptide of Mr = 53,000 on sodium dodecyl sulfate-polyacrylamide gel and a specific activity of 29 mumol/min/mg at 37 degrees C in the absence of calmodulin. Inclusion of calpain inhibitors was critical for obtaining the 53-kDa protein as the major product and 0.1% of the zwitterionic detergent, 3-[(3-cholamidopropyl)dimethylamino]-2-propanesulfonate, was necessary to stabilize enzyme activity. In the absence of calpain inhibitors, the 53-kDa protein degraded progressively during purification and yielded a mixture containing polypeptides of various sizes. Relative intensity of these degradation products on sodium dodecyl sulfate-polyacrylamide gel varied from one preparation to another. However, broad band(s) at the 42-45 kDa region and a band at 35 kDa were always weak, while bands of 53, 51, 40 (sometimes doublets), 33, and 32 KDa were usually strong. The fact that all of these polypeptides including the weak bands of 42-45 and 35 kDa were derived from the 53 kDa form was confirmed by their immunocross-reactivity with monoclonal antibodies to the 53 kDa form. When the 51, 40, and a mixture of the 33 and 32 kDa forms were obtained separately and nearly free from other forms, each of them exhibited catalytic activity. Nevertheless, calmodulin binds to polypeptides larger than 35,000 but not to the 33 and 32 kDa forms. Incubation of the purified 53 kDa form with calpain generated a fragmentation pattern nearly identical to that generated during purification in the absence of calpain inhibitors. Incubation with five other endoproteases produced proteolytic fragments slightly different from those by calpain. However, the general fragmentation patterns generated by the proteases were similar, suggesting that inositol(1,4,5)P3 3-kinase contains several motifs susceptible to a variety of proteases.
Animals↗
Effect of phospholipase C-gamma overexpression on PDGF-induced second messengers and mitogenesis.
Platelet-derived growth factor (PDGF) stimulates phospholipase C (PLC) activity and the phosphorylation of the gamma isozyme of PLC (PLC-gamma) in vitro and in living cells. The role of PLC-gamma in the phosphoinositide signaling pathway was addressed by examining the effect of overexpression of PLC-gamma on cellular responses to PDGF. Overexpression of PLC-gamma correlated with PDGF-induced tyrosine phosphorylation of PLC-gamma and with PDGF-induced breakdown of phosphatidylinositol 4,5-bisphosphate (PIP2). However, neither bradykinin- nor lysophosphatidic acid-induced phosphoinositide metabolism was enhanced in the transfected cells, suggesting that the G protein-coupled phosphoinositide responses to these ligands are mediated by other PLC isozymes. The enhanced PDGF-induced generation of inositol trisphosphate (IP3) did not enhance intracellular calcium signaling or influence PDGF-induced DNA synthesis. Thus, enzymes other than PLC-gamma may limit PDGF-induced calcium signaling and DNA synthesis. Alternatively, PDGF-induced calcium signaling and DNA synthesis may use biochemical pathways other than phosphoinositide metabolism for signal transduction.
Animals↗
Molecular cloning and expression of a complementary DNA for inositol 1,4,5-trisphosphate 3-kinase.
A complementary DNA (cDNA) clone that encodes inositol 1,4,5-trisphosphate 3-kinase was isolated from a rat brain cDNA expression library with the use of monoclonal antibodies. This clone had an open reading frame that would direct the synthesis of a protein consisting of 449 amino acids and with a molecular mass of 49,853 daltons. The putative protein revealed a potential calmodulin-binding site and six regions with amino acid compositions (PEST regions) common to proteins that are susceptible to calpain. Expression of the cDNA in COS cells resulted in an approximately 150-fold increase in inositol 1,4,5-trisphosphate 3-kinase activity of these cells.
Amino Acid Sequence↗
Tyrosine residues in bovine phospholipase C-gamma phosphorylated by the epidermal growth factor receptor in vitro.
We have identified the sites phosphorylated in vitro by epidermal growth factor (EGF) receptor kinase in bovine brain phospholipase C-gamma (PLC-gamma). They are tyrosine residues 472, 771, 783, and 1254. The rate of phosphorylation was fastest with the sites at 771 and 783, then at 1254, and slowest at 472. PLC-gamma isolated from cells treated with EGF is known to contain at least four tyrosine phosphate-containing peptides and two of them are identified to be residues 771 and 1254 in the accompanying paper (Wahl, M. I., Nishibe, S., Kim, J. W., Kim, H., Rhee, S. G., and Carpenter, G. (1990) J. Biol. Chem. 265, 3944-3948). The 3 residues 472, 771, and 783 are located closely to the regions of PLC-gamma which exhibit a high sequence similarity to the regulatory domain of the src family tyrosine kinases. Nevertheless, the tyrosine phosphorylation did not affect the catalytic activity of PLC-gamma in vitro. We propose, therefore, that the phosphorylation of PLC-gamma by EGF receptor kinase alters its interaction with putative inhibitory proteins and leads to its activation.
Amino Acid Sequence↗
Identification of two epidermal growth factor-sensitive tyrosine phosphorylation sites of phospholipase C-gamma in intact HSC-1 cells.
The 145-kDa phospholipase C isozyme, PLC-gamma, is an excellent substrate for the epidermal growth factor (EGF) receptor both in vivo and in vitro. We now demonstrate that EGF treatment of HSC-1 cells, a human squamous cell carcinoma-derived cell line that expresses high levels of the EGF receptor, rapidly induces tyrosine phosphorylation of two-thirds of the total cellular PLC-gamma pool. A two-step immunoaffinity protocol was used for large-scale isolation of phosphorylated PLC-gamma from the cytosol of EGF-treated HSC-1 cells. Phosphorylated PLC-gamma was digested with trypsin, then phosphotyrosine-containing peptides were purified by phosphotyrosine affinity chromatography and reverse-phase high performance liquid chromatography. The two major phosphotyrosine-containing tryptic peptides were sequenced. Comparison of the sequence data with the bovine brain PLC-gamma amino acid sequence indicated that the major, EGF-sensitive tyrosine phosphorylation sites of human PLC-gamma correspond to the bovine brain PLC-gamma tyrosine residues 771 and 1254. The former residue is adjacent to regions of PLC-gamma that contain high homology to the non-catalytic, amino-terminal region of the src tyrosine kinase. The latter residue lies near the carboxyl terminus of the PLC-gamma molecule. The accompanying manuscript (Kim J.W., Sim, S.S., Kim, U-H., Nishibe, S., Wahl, M. I., Carpenter, G., and Rhe, S. G. (1990) J. Biol. Chem. 265, 3940-3943) identifies these same 2 residues plus 2 additional tyrosine phosphorylation sites through large-scale in vitro phosphorylation of purified bovine brain PLC-gamma by the EGF receptor.
Amino Acid Sequence↗
Inhibition of serum- and ras-stimulated DNA synthesis by antibodies to phospholipase C.
Several immunologically distinct isozymes of inositol phospholipid-specific phospholipase C (PLC) have been purified from bovine brain. Murine NIH 3T3 fibroblasts were found to express PLC-gamma, but the expression of PLC-beta was barely detectable by radioimmunoassay or protein immunoblot. A mixture of monoclonal antibodies was identified that neutralizes the biological activity of both endogenous and injected purified PLC-gamma. When co-injected with oncogenic Ras protein or PLC-gamma, this mixture of antibodies inhibited the induction of DNA synthesis that characteristically results from the injection of these proteins into quiescent 3T3 cells. However, when oncogenic Ras protein or PLC-gamma was co-injected with a neutralizing monoclonal antibody to Ras, only the DNA synthesis induced by the Ras protein was inhibited--that induced by PLC was unaffected. These results suggest that the Ras protein is an upstream effector of PLC activity in phosphoinositide-specific signal transduction and that PLC-gamma activity is necessary for Ras-mediated induction of DNA synthesis.
Animals↗
Characterization of phosphatidylinositol-specific phospholipase C defects associated with thrombin-induced mitogenesis.
In a previous paper (Rath, H. M., Doyle, G. A. R., and Silbert, D. F. (1989) J. Biol. Chem. 264, 13387-13390), we reported a selection for the isolation of Chinese hamster lung fibroblasts (CCL39) defective in thrombin-induced mitogenesis. One mutant, D1-6b, had decreased production of inositol phosphates when challenged with activators of phosphatidylinositol turnover and extracts of this mutant showed a marked decrease in phospholipase C (PLC) activity toward phosphatidylinositol. In the current studies, the PLC activities of wild type CCL39 and D1-6b cytosolic extracts are further characterized. Wild type cytosol had at least two phosphatidylinositol-specific PLC isoenzymes, which could be separated by anion exchange chromatography and behaved differently in thermal inactivation studies. Since gel filtration of PLC activity in wild type extracts gave Mr values similar to that of previously characterized PLCs (140,000-200,000), immunoblots with antibodies to bovine brain isoenzymes were used to show that the PLC activities obtained by anion exchange chromatography were PLC-delta and PLC-gamma. Immunoblots with mutant D1-6b cytosol confirmed the presence of the PLC-gamma but showed no detectable PLC-delta. This activity in the mutant extracts eluted at the same conductivity on anion exchange columns and had the same kinetics of thermal inactivation as the PLC-gamma found in the wild type extracts. PLC-gamma from mutant extracts was active in assays containing phospholipid detergent mixed micelles but not in assays utilizing phospholipid vesicles, in sharp contrast to PLC-gamma from CCL39 extracts, which was active under either condition. Thus, the phosphatidylinositol-specific phospholipase C activity of mutant D1-6b is diminished both by the loss of PLC-delta and by the compromised behavior of PLC-gamma.
Animals↗
Protein that prevents mercaptan-mediated protein oxidation.
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Aerobiosis↗