FRINK Linked Data Fragments server

Matches in SemOpenAlex for { <https://semopenalex.org/work/W1507838062> ?p ?o ?g. }

• W1507838062 endingPage "1447" @default.
• W1507838062 startingPage "1438" @default.
• W1507838062 abstract "Phospholipase Cβ (PLCβ) isoforms, which are under the control of Gαq and Gβγ subunits, generate Ca2+ signals induced by a broad array of extracellular agonists, whereas PLCδ isoforms depend on a rise in cytosolic Ca2+ for their activation. Here we find that PLCβ2 binds strongly to PLCδ1 and inhibits its catalytic activity in vitro and in living cells. In vitro, this PLC complex can be disrupted by increasing concentrations of free Gβγ subunits. Such competition has consequences for signaling, because in HEK293 cells PLCβ2 suppresses elevated basal [Ca2+] and inositol phosphates levels and the sustained agonist-induced elevation of Ca2+ levels caused by PLCδ1. Also, expression of both PLCs results in a synergistic release of [Ca2+] upon stimulation in A10 cells. These results support a model in which PLCβ2 suppresses the basal catalytic activity of PLCδ1, which is relieved by binding of Gβγ subunits to PLCβ2 allowing for amplified calcium signals. Phospholipase Cβ (PLCβ) isoforms, which are under the control of Gαq and Gβγ subunits, generate Ca2+ signals induced by a broad array of extracellular agonists, whereas PLCδ isoforms depend on a rise in cytosolic Ca2+ for their activation. Here we find that PLCβ2 binds strongly to PLCδ1 and inhibits its catalytic activity in vitro and in living cells. In vitro, this PLC complex can be disrupted by increasing concentrations of free Gβγ subunits. Such competition has consequences for signaling, because in HEK293 cells PLCβ2 suppresses elevated basal [Ca2+] and inositol phosphates levels and the sustained agonist-induced elevation of Ca2+ levels caused by PLCδ1. Also, expression of both PLCs results in a synergistic release of [Ca2+] upon stimulation in A10 cells. These results support a model in which PLCβ2 suppresses the basal catalytic activity of PLCδ1, which is relieved by binding of Gβγ subunits to PLCβ2 allowing for amplified calcium signals. The binding of an agonist to its target G protein-coupled receptor stimulates heterotrimeric G proteins, which in turn can result in an increase in intracellular Ca2+ through the activation of phospholipase Cβ (PLCβ) 1The abbreviations used are: PLC, phospholipase C; PH, pleckstrin homology; PtdIns, phosphatidylinositol; PI(4,5)P2, phosphatidylinositol 4,5-bisphosphate; YFP, yellow fluorescent protein; BiFC, bimolecular fluorescent complex; HBSS, Hanks' balanced salt solution; DEPC, diethyl pyrocarbonate; PBS, phosphate-buffered saline; BSA, bovine serum albumin; PTX, pertussis toxin; HEK, human embryonic kidney; Ins(1,4,5)P3, inositol 1,4,5-trisphosphate; DABCS4L, 4-(dimethylamino)phenylazo-phenyl-4-sulfonyl chloride succinyl ester. (1Rebecchi M.J. Pentyala S.N. Physiol. Rev. 2000; 80: 1291-1335Crossref PubMed Scopus (831) Google Scholar, 2Rhee S.G. Annu. Rev. Biochem. 2001; 70: 281-312Crossref PubMed Scopus (1227) Google Scholar). PLCs catalyze the hydrolysis of a minor lipid component, phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2), to release the second messengers diacylglycerol and inositol 1,4,5-trisphosphate (Ins(1,4,5)P3). These messengers in turn activate protein kinase C and stimulate the release of Ca2+ from internal stores. All PLCβs (β1–β4) are regulated by Gαq subunits that are coupled to specific sets of G protein-coupled receptors. PLCβ2 and to a lesser extent PLCβ3 can also be stimulated by Gβγ subunits (1Rebecchi M.J. Pentyala S.N. Physiol. Rev. 2000; 80: 1291-1335Crossref PubMed Scopus (831) Google Scholar, 2Rhee S.G. Annu. Rev. Biochem. 2001; 70: 281-312Crossref PubMed Scopus (1227) Google Scholar). Because Gβγ subunits have the potential to be released from all types of Gα-Gβγ heterotrimers, PLCβ2 and -β3 may be activated by a wider range of receptors. In contrast to other mammalian PLC enzymes, the cellular regulation of PLCδ enzymes is unclear. It is known that these enzymes are regulated by an increase in cellular Ca2+ levels because PLCδ is the only PLC family that is not active at basal Ca2+ levels but is strongly activated when cytoplasmic Ca2+ rises above basal levels (1Rebecchi M.J. Pentyala S.N. Physiol. Rev. 2000; 80: 1291-1335Crossref PubMed Scopus (831) Google Scholar, 3Ochocka A.M. Pawelczyk T. Acta Biochim. Pol. 2003; 50: 1097-1110Crossref PubMed Scopus (35) Google Scholar). This behavior suggests that PLCδs function to amplify, rather than initiate, calcium-mobilizing signals. At maximum Ca2+ concentrations, the specific catalytic activity of purified PLCδ1 is typically 50–100-fold greater than that of unstimulated PLCβ or PLCγ. When reconstituted into permeabilized PC12 and HL60 cells, PLCδ1, but not PLCβ1 or PLCγ1, shows substantial activation by physiologic calcium levels (4Allen V. Swigart P. Cheung R. Cockcroft S. Katan M. Biochem. J. 1997; 327: 545-552Crossref PubMed Scopus (175) Google Scholar). Overexpression of PLCδ1 in PC12 and Chinese hamster ovary cells enhances the increase in cellular Ca2+ and soluble inositol phosphate levels produced by bradykinin (5Kim Y-H. Park T.-J. Lee Y.H. Baek K.J. Suh P.-G. Ryu S.H. Kim K.-T. J. Biol. Chem. 1999; 274: 26127-26134Abstract Full Text Full Text PDF PubMed Scopus (116) Google Scholar) and thrombin (6Banno Y. Okano Y. Nozawa Y. J. Biol. Chem. 1994; 269: 15846-15852Abstract Full Text PDF PubMed Google Scholar). The Ca2+-amplification role of PLCδ1 has been clearly defined in keratinocytes derived from PLCδ1-null mice in which the sustained elevation of cytoplasmic Ca2+ that follows a PLCγ1-stimulated rapid rise does not occur but can be reconstituted when PLCδ1 is introduced (7Nakamura Y. Fukami K. Yu N. Takenaka K. Kataoka Y. Shirakata Y. Nishikawa S-I. Hashimoto K. Yoshida N. Takenawa T. EMBO J. 2003; 22: 2981-2991Crossref PubMed Scopus (89) Google Scholar). Several studies suggest that activation of PLCδ1 is under more complex control than the simple rise in cytoplasmic Ca2+. In frog oocytes expressing thrombin and platelet-derived growth factor receptors, microinjection of PLCδ1 antibody specifically inhibits thrombin but not platelet-derived growth factor-induced calcium mobilization (8Cho Y.S. Han M.K. Chae S.W. Park C.U. Kim U.H. FEBS Lett. 1993; 334: 257-260Crossref PubMed Scopus (9) Google Scholar). In Chinese hamster ovary cells, overexpression of PLCδ1 enhances the amount of inositol phosphates generated by ionomycin, but this increment is much smaller than the increase observed during thrombin stimulation (6Banno Y. Okano Y. Nozawa Y. J. Biol. Chem. 1994; 269: 15846-15852Abstract Full Text PDF PubMed Google Scholar). Similar results are obtained in bradykinin-stimulated PC12 cells expressing high levels of PLCδ1 (5Kim Y-H. Park T.-J. Lee Y.H. Baek K.J. Suh P.-G. Ryu S.H. Kim K.-T. J. Biol. Chem. 1999; 274: 26127-26134Abstract Full Text Full Text PDF PubMed Scopus (116) Google Scholar). Here, raising calcium with high extracellular potassium, thapsigargin, or ionomycin induces a measurable increase in inositol trisphosphate, yet this increment is substantially less than that observed with a maximum dose of bradykinin. Thus, although these observations support a generalized amplification hypothesis, they suggest that receptor-generated signals other than calcium also contribute to PLCδ1-dependent inositol phosphate generation. Whereas protein regulators of most mammalian PLCs have been identified, those for PLCδ1 have not been well established. A novel form of RhoGAP associates strongly with PLCδ1 in cell lysates (9Homma Y. Emori Y. EMBO J. 1995; 14: 286-291Crossref PubMed Scopus (189) Google Scholar), stimulating its catalytic activity at low levels of calcium (0.1 μm). There is compelling evidence that PLCδ1 is also regulated by an atypical GTP-binding protein, Gh, or transglutaminase (10Im M.J. Russell M.A. Feng J.F. Cell. Signal. 1997; 9: 477-482Crossref PubMed Scopus (96) Google Scholar). Gh is controlled by α1-adrenergic receptor α1b and α1d in heart and liver (11Das T. Baek K.J. Gray C.D. Im M. J. Biol. Chem. 1993; 268: 27398-27405Abstract Full Text PDF PubMed Google Scholar, 12Chen S. Lin F.G. Iismaa S. Lee K.N. Birckbichler P. Graham R. J. Biol. Chem. 1996; 271: 32385-32391Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar, 13Prasanna Murthy S.N. Lomasney J.W. Mak E.C. Lorand L. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 111815-111819Google Scholar), as well as oxytocin receptors in myometrium (14Baek K.J. Kwon N.S. Lee H.S. Kim M.S. Muralidhar P. Im M. Biochem. J. 1996; 315: 739-744Crossref PubMed Scopus (63) Google Scholar). PLCδ1 stimulates GDP/GTP exchange on TGII/Gh (11Das T. Baek K.J. Gray C.D. Im M. J. Biol. Chem. 1993; 268: 27398-27405Abstract Full Text PDF PubMed Google Scholar, 15Baek K.J. Kang S. Damron D. Im M. J. Biol. Chem. 2001; 276: 5591-5597Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar), and reciprocally, Gh allows PLCδ1 to be stimulated at lower Ca2+ concentrations. Although these studies show that TGII/Gh couples heptahelical receptors to PLCδ1, the fraction of the total cellular response that this represents is unclear. Other regulators of PLCδ1 have been suggested. Association of PLCδ1 to GAP43 results in a rise in cytoplasmic Ca2+, although it is uncertain whether this is because of increased membrane association of PLCδ1 (16Caprini M. Gomis A. Cabedo H. Planells-Cases R. Belmonte C. Viana F. Ferrer-Montiel A. EMBO J. 2003; 22: 3004-3014Crossref PubMed Scopus (31) Google Scholar). The activity of PLCδ4 is suppressed by a inactive PLCδ4-Alt3 variant (17Nagano K. Fukami K. Minagawa T. Watanabe Y. Ozaki C. Takenawa T. J. Biol. Chem. 1999; 274: 2872-2879Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar), but it is unclear whether comparable regulators of other PLCδ isozymes exist. Most PLCβ and PLCδ isozymes are widely expressed with varying tissue distribution. PLCβ2, which is the focus of this study, is expressed at high levels in cells of hematopoietic origin. Mice lacking this isozyme have abnormal chemokine signaling, possibly through Gβγ released from Gαi/o-coupled receptors (18Lee S.B. Rao A.K. Lee K.H. Yang X. Bae Y.S. Rhee S.G. Blood. 1996; 88: 1684-1691Crossref PubMed Google Scholar). PLCδ1 is the most widely distributed PLCδ isotype and is most strongly expressed in skeletal muscle, some layers of the skin, the spleen, testis, and lung (1Rebecchi M.J. Pentyala S.N. Physiol. Rev. 2000; 80: 1291-1335Crossref PubMed Scopus (831) Google Scholar, 3Ochocka A.M. Pawelczyk T. Acta Biochim. Pol. 2003; 50: 1097-1110Crossref PubMed Scopus (35) Google Scholar, 19Homma Y. Takenawa T. Emori Y. Sorimachi H. Suzuki K. Biochem. Biophys. Res. Commun. 1989; 164: 406-412Crossref PubMed Scopus (97) Google Scholar). In the adult brain, PLCδ1 is found primarily in glial cells (20Yamada H. Mizuguchi M. Rhee S.G. Kim S.U. Brain Res. 1991; 59: 7-16Crossref Scopus (22) Google Scholar, 21Yamada M. Kakita A. Mizuguchi M. Rhee S.G. Kim S.U. Ikuta F. Brain Res. 1993; 606: 335-340Crossref PubMed Scopus (25) Google Scholar); however, like PLCβ2, its presence is low in most neurons. Previous work in our laboratories has focused on characterizing the regulation of PLCδ1 and PLCβ2. Here we report that PLCβ2 inhibits PLCδ and that this inhibition is relieved upon binding of Gβγ to PLCβ2. Our results suggest a novel mechanism in which G protein stimulation has the ability to amplify PLCβ2-generated Ca2+ signals through simultaneous activation of PLCδ1 under some cellular conditions. In Vitro Fluorescence Studies—Gβ1γ2 subunits and PLCβ2 were expressed in Sf9 cells by using a baculovirus system (22Kozasa T. Gilman A.G. J. Biol. Chem. 1995; 270: 1734-1741Abstract Full Text Full Text PDF PubMed Scopus (298) Google Scholar). Gβ1γ2 subunits were reconstituted into preformed lipid bilayers by simple addition (23Runnels L.W. Scarlata S.F. Biochemistry. 1998; 37: 15563-15574Crossref PubMed Scopus (29) Google Scholar). PLCδ1 was bacterially expressed and purified (24Tall E. Dorman G. Garcia P. Runnels L. Shah S. Chen J. Profit A. Gu Q. Chaudhary A. Prestwich G.D. Rebecchi M.J. Biochemistry. 1997; 36: 7239-7248Crossref PubMed Scopus (65) Google Scholar). Association measurements between membrane-bound proteins were carried out in large unilamellar vesicles composed of 67% anionic 1-palmitoyl-2-oleoyl-l-phosphatidylserine and 33% 1-palmitoyl-2-oleoyl-l-phosphatidylcholine, where both PLC enzymes are membrane-bound above 250 μm total lipid (data not shown) (25Garcia P. Gupta R. Shah S. Morris A.J. Rudge S. Scarlata S. Petrova V. McLaughlin S. Rebecchi M. Biochemistry. 1995; 34: 16228-16234Crossref PubMed Scopus (255) Google Scholar, 26Runnels L.W. Jenco J. Morris A. Scarlata S. Biochemistry. 1996; 35: 16824-16832Crossref PubMed Scopus (70) Google Scholar). Protein-protein association was assessed using the fluorescence methods described previously (27Guo Y. Philip F. Scarlata S. J. Biol. Chem. 2003; 278: 29995-30004Abstract Full Text Full Text PDF PubMed Scopus (17) Google Scholar). Briefly, proteins were covalently labeled with either acrylodan or coumarin (26Runnels L.W. Jenco J. Morris A. Scarlata S. Biochemistry. 1996; 35: 16824-16832Crossref PubMed Scopus (70) Google Scholar, 28Runnels L.W. Scarlata S. Biochemistry. 1999; 38: 1488-1496Crossref PubMed Scopus (85) Google Scholar). The labeled proteins were excited at 340 nm and scanned from 380 to 500 nm. The emission intensity was taken from the integrated area of the spectrum. We found that the emission intensity of labeled PLCβ2 showed a substantial and reproducible increase upon the addition of unlabeled PLCδ1 and gave a titration curve that showed the appropriate shift in midpoint when the initial concentration of acrylodan-PLCβ2 was changed, thereby reflecting protein-protein association. Protein-protein associations were also assessed by fluorescence resonance energy transfer using coumarinand DABCSYL-labeled proteins and were carried out similarly by using the methods and analysis described previously (28Runnels L.W. Scarlata S. Biochemistry. 1999; 38: 1488-1496Crossref PubMed Scopus (85) Google Scholar). PLC Activity Measurements—PLC activity measurements were conducted by using purified proteins and detergent-mixed micelles substrates as described previously (26Runnels L.W. Jenco J. Morris A. Scarlata S. Biochemistry. 1996; 35: 16824-16832Crossref PubMed Scopus (70) Google Scholar, 29Cifuentes M.E. Delaney T. Rebecchi M.J. J. Biol. Chem. 1994; 269: 1945-1948Abstract Full Text PDF PubMed Google Scholar). Construction of BiFC Plasmid—The vectors used to construct YFP fusion proteins were generously provided by Dr. Thomas Kerppola (University of Michigan). These vectors contained YFP fragments (FLAG/YN-Smad3 or FLAG/YC-Smad4) that can associate to form a bimolecular fluorescent complex (BiFC). The coding sequence of human PLCβ2 was amplified by using PCR with PLCβ2.pVL1392 plasmid as template and was subcloned through EcoRI/XbaI sites into the FLAG/YN-Smad3 vector. The primer set was 5′-CGGCGGCCGCGGAATTCTGCAAAGAGGAACG-3′ and 3′-CCCACAGCTCTAGAAGGCTGGGGCTCCTTTTTCCTGGGGG 5′, which contained restriction enzyme sites EcoRI and XbaI (underlined), respectively. PLCβ2 was subcloned through an ECOR/XbaI site into FLAG/YN-SMAD3 vector. Similarly, human PLCδ1 was amplified by using PCR with PLCδ1.pet3a plasmid as template (the primer set was 5′-GTTTAACTGGTACCAGGAGATATACATATGGACTC-3′ and 3′-CTCAGGGGGGACCCCTCTAGATTCCTCCAGCCTAGTCC-5′, which contained restriction enzyme sites KpnI and XbaI (underlined), respectively) and was subcloned through KpnI/XbaI sites into FLAG/YC-Smad4 vector. The PLCβ2-FLAG/YN-Smad3 and PLCδ1-FLAG/YC-Smad4 plasmids were sequenced by checking their reading frames. Cell Culture and Overexpression of PLCs—HEK293 cells and A10 cells were maintained in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum and antibiotics. For A10 cells, the medium was supplemented with 1 mm sodium pyruvate at 37 °C in 5% CO2. For fluorescence studies, HEK293 cells were cotransfected with BiFC-PLCβ2/PLCδ1 vectors (15 μg/106 cells in 60-mm dish) by calcium phosphate coprecipitation. HEK293 cells transfected with either BiFC-PLCβ2/PLCδ1 vectors or vectors lacking the BiFC tags were used in the Ca2+ and inositol phosphate studies as both produced identical results. A Western blot showing the levels of expression is given in Fig. 3. In the pertussis toxin (PTX)-treated cells, HEK293 cells were incubated with 100 ng/ml for 16–20 h. Imaging of Fluorescence in Living Cells—24 h after transfection, cells from 60-mm dishes were plated into Labtek chambered glass coverslips (Bio-Rad). Fluorescence emission from living cells was measured 48–72 h after transfection on a Zeiss Axiovert microscope using 63× 1.4 numerical aperature oil objective. Emission spectra of BiFC complexes from a 100-mm dish of living cells were taken on an ISS spectrofluorometer (Champaign, IL). Cells expressing BiFC-PLCβ2/PLCδ1 were placed in a 1-cm cuvette with stirring at an adjusted concentration of 1 × 106 cells/ml. Spectra were taken at λexc = 500 nm and scanning from 535 to 600 nm. Cut-off filters (525 nm, Corion Optical) were placed before the monochromators to help reduce the amount of scattered light. Background samples were obtained from cultures transfected with only one of the BiFC constructs. Background spectra, which contributed 12–22% of the signal, were subtracted from the corresponding sample spectra. For immunostaining, cells were grown on Labtek chambers, fixed in 4% formaldehyde solution in PBS, and permeabilized in 0.1% Triton X-100. Cells were blocked in PBS containing 5% goat serum, 1% BSA, and 50 mm glycine overnight. The monoclonal antibody against PLCδ1 (Upstate Biotechnology, Inc.) was used as the primary antibody. Polyclonal antibody against PLCβ2 (Santa Cruz Biotechnology) was also used as the primary antibody. Primary antibodies were diluted 1:200 in PBS containing 0.5% BSA. Cells were incubated in primary antibody at 37 °C for 1 h. This was followed by three washes of 7 min each in PBS. Secondary antibodies were diluted at 1:2000 in PBS, 0.5% BSA. Fluorescein isothiocyanate-conjugated rabbit secondary antibody was to detect PLCδ1 antibody, and DSRed-conjugated anti-mouse secondary antibody was used to detect antibody against PLCβ2. After 1 h of incubation at 37 °C, the cells were washed by PBS (three times at 7 min each). Finally, PBS was added to the cells, and the specimens were viewed under the Zeiss Axiovert fluorescence microscope. Preparation of BiFC-PLCβ2/PLCδ1 Membranes—Cells expressing BiFC-PLCβ2/PLCδ1 were harvested and centrifuged at 500 × g for 5 min. The pellet was washed with PBS, resuspended in lysis buffer (10 mm Tris, pH 7.4, 1 mm MgCl2, 1 mm dithiothreitol, 1 mm phenylmethylsulfonyl fluoride, 10 μg/ml pepstatin, and 10 μg/ml leupeptin), and homogenized. The lysate was then centrifuged at 500 × g for 5 min, and the supernatant was further centrifuged at 50,000 × g for 30 min at 4 °C. The pellet containing the cell membranes was resuspended, and the protein concentration was adjusted to 0.5 mg/ml. Both purified Gβγ subunits and PLCβ2 were briefly dialyzed in 20 mm Hepes, 0.16 m KCl, 1mm dithiothreitol, and 1 mm EGTA, pH 7.4, to remove detergent in the Gβγ storage buffer. Measurement of Cellular [Ca2+]i—Cellular [Ca2+]i was determined with the fluorescent calcium indicator dye fura-2/AM in an ISS spectrofluorometer. Briefly, cell monolayers were washed with Hanks' balanced salt solution (HBSS) (118 mm NaCl, 5 mm KCl, 1 mm CaCl2,1mm MgCl2,5mm glucose, 15 mm Hepes, 1% BSA, pH 7.4), and the cells were detached by a buffer stream. Suspended cells were labeled with 1 μm fura-2/AM for 45 min at 37 °C in HBSS with rotation. Thereafter, cells were washed twice and incubated in HBSS for another 20 min. After centrifugation, the cells were resuspended at a density of 1 × 106 cells/ml and were measured in a continuously stirred cell suspension at room temperature. The ratio of fluorescence emitted at 340 and 380 nm (340:380 ratio) of cells was converted to Ca2+ concentration by the method of Tsien and co-workers (30Grynkiewicz G. Poenie M. Tsien R.Y. J. Biol. Chem. 1985; 260: 3440-3450Abstract Full Text PDF PubMed Scopus (80) Google Scholar) using the relation: [Ca2+]i free (nm) = ((R - Rmin)/(Rmax - R)) ·(Fmax/Fmin) ·Kd (nm). The Fura-2/AM Kd value is ∼225 nm. Fmax is the fluorescence at that wavelength in EDTA buffer, and Fmin is the fluorescence in the presence of detergent and excess calcium. R is the measured ratio, and Rmin and Rmax are the ratios corresponding to the EDTA and detergent/excess calcium conditions, respectively. In some experiments, [Ca2+]i was determined in the absence of extracellular Ca2+ by treatment cell with 1 mm EGTA. Measurements of Inositol Phosphate Formation and Phosphoinositide Analysis in Intact Cells—Cells were prelabeled with myo-[3H]inositol (1 μCi/ml) for 2 days in inositol-free medium. The cells were harvested, kept in suspension, and incubated for 10 min at 37 °C with HBSS/LiCl (118 mm NaCl, 5 mm KCl, 1 mm CaCl2,1mm MgCl2,5mm d-glucose, and 15 mm Hepes, pH 7.4, supplemented with 10 mm LiCl), before challenging with agonist. For agonist stimulation, the cells were incubated with agonist (5 μm carbachol) in the presence of LiCl for 30 min at 37 °C. All reactions were stopped by removing the incubation medium and lysing the cells in 1 ml of ice-cold methanol. After the addition of 1 ml of chloroform and 0.5 ml of H2O, phase separation was performed by centrifugation at 2000 × g for 10 min at 4 °C. The aqueous upper phase was applied to AG 1-X8 anion exchange columns to isolate myo-[3H]-inositol phosphate formation. For determination of phosphoinositide levels, the lower phase was collected and evaporated by vacuum drying, and the lipids were resuspended in 50 μl of chloroform and spotted onto LK5D linear-k silica gel TLC plates (Whatman). The plates were developed in chloroform, methanol, 2.5 m ammonium hydroxide (9:7:2, v/v). The areas corresponding to authentic PtdIns (Rf = 0.64), PtdIns(4)P (Rf = 0.45), and PtdIns(4,5)P2 (Rf = 0.25) were scraped into vials, and the radioactivity was measured by liquid scintillation counting. The amount of disintegrations/min was normalized for protein content. PLCβ2 and PLCδ1 Strongly Interact on Model Membrane Surfaces—This study began with the unexpected finding that PLCβ2 and PLCδ1 form heteromeric complexes in solution and on membrane surfaces. We directly measured the formation of PLCβ2 ·PLCδ1 complexes by using fluorescence methods to quantify the association energy between proteins. Protein-protein association was monitored by the change in the fluorescence emission of the probe acrylodan covalently linked to PLCβ2 (see “Materials and Methods”). This probe undergoes an increase in emission intensity and energy upon the association of Gβγ subunits with PLCβ2 and yields a dissociation constant identical to that obtained by using fluorescence resonance energy transfer (27Guo Y. Philip F. Scarlata S. J. Biol. Chem. 2003; 278: 29995-30004Abstract Full Text Full Text PDF PubMed Scopus (17) Google Scholar). In Fig. 1A, we show the normalized increase in acrylodan-PLCβ2 fluorescence when PLCδ1 is added in solution. This increase was not observed when buffer or unlabeled PLCβ2 was substituted for PLCδ1. These data can be fit to a bimolecular association constant that gives an apparent affinity of Kapp ∼200 nm. Both enzymes are capable of membrane binding, and it is expected that membrane binding would increase their affinity because of a reduction in dimensionality. PLCδ1 was added to acrylodan-PLCβ2 under conditions where both enzymes were completely membrane-bound (49Hepler J.R. Gilman A.G. Trends Biochem. Sci. 1992; 17: 383-387Abstract Full Text PDF PubMed Scopus (928) Google Scholar). Under these conditions, a stronger apparent affinity of Kapp = 9.2 ± 4 nm was observed due to the concentrating effect of being bound to a membrane surface (see “Discussion”). The affinity of PLCδ1 for PLCβ2 on membranes was unchanged when the free Ca2+ concentration was raised from 0 to 12 μm using Ca2+/EGTA buffers. Similar studies monitoring the self-association of the two isolated proteins under membrane-bound conditions, which would promote associations, showed that PLCβ2, which appears monomeric by chromatography under our purification conditions (31Runnels L.W. Regulation of Phospholipase C-β Isozymes by Heterotrimeric GTP-binding Proteins. Ph.D. thesis, State University of New York, Stony Brook1997Google Scholar), does not self-associate at concentrations up to 300 nm. Similarly, PLCδ1 does not self-associate at concentrations up to 320 nm (equivalent to the highest concentration achieved in the PLC binding measurements). Thus, the association between membrane-bound PLCβ2 and PLCδ1 is not explained by a tendency of the PLCs to form homomeric complexes. PLCβ2 Inhibits the Activity of PLCδ1—We tested whether the association between PLCβ2 and PLCδ1 affected their enzymatic activities. These studies utilized the substrate [3H]PI(4)P rather than PI(4,5)P2. The rationale for using [3H]PI(4)P is that the reduced Km value of PLCδ1 toward this substrate as compared with PI(4,5)P2 allowed us to carry out activity assays at enzyme concentrations above the apparent dissociation constant of PLCβ2-PLCδ1. We found that the catalytic activity of a mixture of PLCβ2 and PLCδ1 was much less than the additive values obtained from the isolated enzymes (Fig. 2A), suggesting that the PLCβ2-PLCδ1 association inhibits one or both of the enzymes. We thus began a series of studies to determine whether one of the enzymes is inhibited by the other. PLCδ1 binds to membranes primarily through a high affinity site for PI(4,5)P2/Ins(1,4,5)P3 in its N-terminal pleckstrin homology (PH) domain in addition to the low affinity catalytic binding site for substrate/product (32Rebecchi M.J. Scarlata S. Annu. Rev. Biophys. Biomol. Struct. 1998; 27: 503-528Crossref PubMed Scopus (250) Google Scholar). This high affinity site keeps PLCδ1 membrane-bound as long as substrate is present in the membrane and the amount of product in the aqueous phase is low. In contrast, the corresponding pleckstrin homology of PLCβ2 binds to membranes with little specificity (33Wang T. Pentyala S. Rebecchi M. Scarlata S. Biochemistry. 1999; 38: 1517-1527Crossref PubMed Scopus (95) Google Scholar). Addition of Ins(1,4,5)P3 to PLCδ1 should greatly reduce its activity because it will inhibit the binding of the PH domain to substrate-containing membranes. Additionally, the presence of product will compete for substrate in the catalytic site, although very high levels of product are needed for inhibition by this latter process. Conversely, product inhibition of PLCβ2 will only occur at high levels of product. We measured the ability of Ins(1,4,5)P3 to inhibit the PLCβ2/PLCδ1 mixture, and we found that the activity of the mixtures was not significantly changed. This result suggests that the activity of PLCδ1 may be inhibited when bound to PLCβ2. To better isolate which enzyme is inhibited in the PLCβ2 ·PLCδ1 complex, we chemically treated PLCδ1 with diethyl pyrocarbonate (DEPC). This agent forms a covalent adduct with one or both of the catalytic His residues, thereby inactivating the histidine-dependent enzyme (34Vik S.B. Hatefi Y. Proc. Natl. Acad. Sci. U.S. A. 1981; 78: 6749-6753Crossref PubMed Scopus (38) Google Scholar). Treatment of PLCδ1 resulted in complete loss of PLCδ1 activity but did not affect its ability to bind substrate membrane, and the apparent affinity with PLCβ2 on membrane surfaces fell within the error of its unmodified enzyme (Kapp ∼10 nm on 200 μm lipid). Most importantly, addition of DEPC-treated PLCδ1 to wild type PLCβ2 up to 20 nm did not change its rate of PI(4,5)P2 hydrolysis (Fig. 2B). We further tested whether PLCβ2 inhibits PLCδ1 activity by using a point mutant of PLCβ2, H327N. This residue is necessary to stabilize the transition state charge of the phosphate group, and mutation of the corresponding residue on PLCδ profoundly reduces activity (35Cheng H.F. Jiang M.J. Chen C.L. Liu S.M. Wong L.P. Lomasney J.W. King K. J. Biol. Chem. 1995; 270: 5495-5505Abstract Full Text Full Text PDF PubMed Scopus (80) Google Scholar, 36Ellis M.V. Katan M. Biochem. J. 1995; 307: 69-75Crossref PubMed Scopus (54) Google Scholar). We find that the enzymatic activity is reduced 50–100-fold, and as expected, its binding to membranes and Gβγ subunits is within error to the wild type enzyme (Kapp ∼180 mm in solution). At activating Ca2+ levels, PLCδ1 activity decreased ∼4-fold with increasing amounts of H327N-PLCβ2, with an EC50 similar to the Kd for PLCδ1 association (Fig. 2B). These results demonstrate that PLCβ2 suppresses the catalytic activity of PLCδ1 at activating levels of Ca2+. Gβγ Subunits Disrupt PLCβ2-PLCδ1 Association in Vitro— PLCβ2 and PLCδ1 both bind to Gβγ subunits, although binding to the former enzyme is of higher affinity and results in enzyme activation (33Wang T. Pentyala S. Rebecchi M. Scarlata S. Biochemistry. 1999; 38: 1517-1527Crossref PubMed Scopus (95) Google Scholar). We tested whether Gβγ subunits could interfere with PLCβ2-PLCδ1 association by measuring complex formation on membranes in the absence or presence of Gβγ subunits by fluorescence resonance energy transfer using the coumarin-DABCSYL donor-acceptor pair. Addition of DABC-SYL-PLCβ2 to coumarin-PLCδ1 resulted in a decrease in donor fluorescence as the two proteins associate (Fig. 1C), but this decrease was largely prevented by the addition of 10 nm Gβγ. Association of PLCβ2 and PLCδ1 in Living Cells—To determine whether the two PLC subtypes associate in living cells, we initially used indirect immunofluorescence to view endogenous PLC in fixed rat aorta smooth muscle cells (A10) because these cells express both PLCs (see Ref. 37Di Salvo J. R. Nelson S. FEBS Lett. 1998; 422: 85-88Crossref PubMed Scopus (20) Google Scholar and Sup" @default.
• W1507838062 created "2016-06-24" @default.
• W1507838062 creator A5032136628 @default.
• W1507838062 creator A5080995402 @default.
• W1507838062 creator A5083183210 @default.
• W1507838062 date "2005-01-01" @default.
• W1507838062 modified "2023-09-28" @default.
• W1507838062 title "Phospholipase Cβ2 Binds to and Inhibits Phospholipase Cδ1" @default.
• W1507838062 cites W1503425505 @default.
• W1507838062 cites W1582659466 @default.
• W1507838062 cites W1604907458 @default.
• W1507838062 cites W1617760412 @default.
• W1507838062 cites W1958742527 @default.
• W1507838062 cites W1965169361 @default.
• W1507838062 cites W1971420697 @default.
• W1507838062 cites W1983021456 @default.
• W1507838062 cites W1984212762 @default.
• W1507838062 cites W1985916633 @default.
• W1507838062 cites W1986861517 @default.
• W1507838062 cites W1988408094 @default.
• W1507838062 cites W1991892690 @default.
• W1507838062 cites W1999453187 @default.
• W1507838062 cites W2011653196 @default.
• W1507838062 cites W2011915496 @default.
• W1507838062 cites W2017196830 @default.
• W1507838062 cites W2032674795 @default.
• W1507838062 cites W2034983225 @default.
• W1507838062 cites W2036239457 @default.
• W1507838062 cites W2037913601 @default.
• W1507838062 cites W2042970033 @default.
• W1507838062 cites W2049344504 @default.
• W1507838062 cites W2052977964 @default.
• W1507838062 cites W2059154200 @default.
• W1507838062 cites W2068131808 @default.
• W1507838062 cites W2071833308 @default.
• W1507838062 cites W2072644328 @default.
• W1507838062 cites W2072778082 @default.
• W1507838062 cites W2079853404 @default.
• W1507838062 cites W2082688327 @default.
• W1507838062 cites W2096549360 @default.
• W1507838062 cites W2102436071 @default.
• W1507838062 cites W2117983839 @default.
• W1507838062 cites W2118224224 @default.
• W1507838062 cites W2141260882 @default.
• W1507838062 cites W2148310842 @default.
• W1507838062 cites W2152571111 @default.
• W1507838062 cites W2189033227 @default.
• W1507838062 cites W2223734153 @default.
• W1507838062 cites W2265371507 @default.
• W1507838062 cites W2399849921 @default.
• W1507838062 cites W2416727200 @default.
• W1507838062 cites W2425190918 @default.
• W1507838062 cites W35097119 @default.
• W1507838062 cites W4248350370 @default.
• W1507838062 cites W4300803924 @default.
• W1507838062 doi "https://doi.org/10.1074/jbc.m407593200" @default.
• W1507838062 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/15509571" @default.
• W1507838062 hasPublicationYear "2005" @default.
• W1507838062 type Work @default.
• W1507838062 sameAs 1507838062 @default.
• W1507838062 citedByCount "62" @default.
• W1507838062 countsByYear W15078380622012 @default.
• W1507838062 countsByYear W15078380622013 @default.
• W1507838062 countsByYear W15078380622014 @default.
• W1507838062 countsByYear W15078380622015 @default.
• W1507838062 countsByYear W15078380622016 @default.
• W1507838062 countsByYear W15078380622018 @default.
• W1507838062 countsByYear W15078380622019 @default.
• W1507838062 countsByYear W15078380622020 @default.
• W1507838062 countsByYear W15078380622022 @default.
• W1507838062 crossrefType "journal-article" @default.
• W1507838062 hasAuthorship W1507838062A5032136628 @default.
• W1507838062 hasAuthorship W1507838062A5080995402 @default.
• W1507838062 hasAuthorship W1507838062A5083183210 @default.
• W1507838062 hasBestOaLocation W15078380621 @default.
• W1507838062 hasConcept C12927208 @default.
• W1507838062 hasConcept C181199279 @default.
• W1507838062 hasConcept C185592680 @default.
• W1507838062 hasConcept C195794163 @default.
• W1507838062 hasConcept C2778597717 @default.
• W1507838062 hasConcept C55493867 @default.
• W1507838062 hasConcept C6182249 @default.
• W1507838062 hasConcept C78297661 @default.
• W1507838062 hasConceptScore W1507838062C12927208 @default.
• W1507838062 hasConceptScore W1507838062C181199279 @default.
• W1507838062 hasConceptScore W1507838062C185592680 @default.
• W1507838062 hasConceptScore W1507838062C195794163 @default.
• W1507838062 hasConceptScore W1507838062C2778597717 @default.
• W1507838062 hasConceptScore W1507838062C55493867 @default.
• W1507838062 hasConceptScore W1507838062C6182249 @default.
• W1507838062 hasConceptScore W1507838062C78297661 @default.
• W1507838062 hasIssue "2" @default.
• W1507838062 hasLocation W15078380621 @default.
• W1507838062 hasOpenAccess W1507838062 @default.
• W1507838062 hasPrimaryLocation W15078380621 @default.
• W1507838062 hasRelatedWork W141142636 @default.
• W1507838062 hasRelatedWork W1972664132 @default.
• W1507838062 hasRelatedWork W1978004062 @default.

• next