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• W2132419558 abstract "Phospholipase Cγ isozymes (PLCγ1 and PLCγ2) have a crucial role in the regulation of a variety of cellular functions. Both enzymes have also been implicated in signaling events underlying aberrant cellular responses. Using N-ethyl-N-nitrosourea (ENU) mutagenesis, we have recently identified single point mutations in murine PLCγ2 that lead to spontaneous inflammation and autoimmunity. Here we describe further, mechanistic characterization of two gain-of-function mutations, D993G and Y495C, designated as ALI5 and ALI14. The residue Asp-993, mutated in ALI5, is a conserved residue in the catalytic domain of PLC enzymes. Analysis of PLCγ1 and PLCγ2 with point mutations of this residue showed that removal of the negative charge enhanced PLC activity in response to EGF stimulation or activation by Rac. Measurements of PLC activity in vitro and analysis of membrane binding have suggested that ALI5-type mutations facilitate membrane interactions without compromising substrate binding and hydrolysis. The residue mutated in ALI14 (Tyr-495) is within the spPH domain. Replacement of this residue had no effect on folding of the domain and enhanced Rac activation of PLCγ2 without increasing Rac binding. Importantly, the activation of the ALI14-PLCγ2 and corresponding PLCγ1 variants was enhanced in response to EGF stimulation and bypassed the requirement for phosphorylation of critical tyrosine residues. ALI5- and ALI14-type mutations affected basal activity only slightly; however, their combination resulted in a constitutively active PLC. Based on these data, we suggest that each mutation could compromise auto-inhibition in the inactive PLC, facilitating the activation process; in addition, ALI5-type mutations could enhance membrane interaction in the activated state. Phospholipase Cγ isozymes (PLCγ1 and PLCγ2) have a crucial role in the regulation of a variety of cellular functions. Both enzymes have also been implicated in signaling events underlying aberrant cellular responses. Using N-ethyl-N-nitrosourea (ENU) mutagenesis, we have recently identified single point mutations in murine PLCγ2 that lead to spontaneous inflammation and autoimmunity. Here we describe further, mechanistic characterization of two gain-of-function mutations, D993G and Y495C, designated as ALI5 and ALI14. The residue Asp-993, mutated in ALI5, is a conserved residue in the catalytic domain of PLC enzymes. Analysis of PLCγ1 and PLCγ2 with point mutations of this residue showed that removal of the negative charge enhanced PLC activity in response to EGF stimulation or activation by Rac. Measurements of PLC activity in vitro and analysis of membrane binding have suggested that ALI5-type mutations facilitate membrane interactions without compromising substrate binding and hydrolysis. The residue mutated in ALI14 (Tyr-495) is within the spPH domain. Replacement of this residue had no effect on folding of the domain and enhanced Rac activation of PLCγ2 without increasing Rac binding. Importantly, the activation of the ALI14-PLCγ2 and corresponding PLCγ1 variants was enhanced in response to EGF stimulation and bypassed the requirement for phosphorylation of critical tyrosine residues. ALI5- and ALI14-type mutations affected basal activity only slightly; however, their combination resulted in a constitutively active PLC. Based on these data, we suggest that each mutation could compromise auto-inhibition in the inactive PLC, facilitating the activation process; in addition, ALI5-type mutations could enhance membrane interaction in the activated state. Phosphoinositide-specific phospholipase C (PLC) 2The abbreviations used are: PLCγphospholipase CγENUN-ethyl-N-nitrosoureaDMEMDulbecco's modified Eagle's mediumBCRB-cell receptorEGFepidermal growth factorIP3inositol 1,4,5-trisphosphatePIP2phosphatidylinositol 4,5-bisphosphate. enzymes, comprising several families (PLCβ, γ, δ, ϵ, η, and ζ), have been established as crucial signaling molecules involved in regulation of a variety of cellular functions (1Suh P.G. Park J.I. Manzoli L. Cocco L. Peak J.C. Katan M. Fukami K. Kataoka T. Yun S. Ryu S.H. BMB Rep. 2008; 41: 415-434Crossref PubMed Google Scholar, 2Drin G. Scarlata S. Cell Signal. 2007; 19: 1383-1392Crossref PubMed Scopus (50) Google Scholar, 3Bunney T.D. Katan M. Trends Cell Biol. 2006; 16: 640-648Abstract Full Text Full Text PDF PubMed Scopus (124) Google Scholar, 4Katan M. Biochem. J. 2005; 391: e7-9Crossref PubMed Scopus (62) Google Scholar). PLC-catalyzed formation of the second messengers, inositol 1,4,5-trisphosphate (IP3) and diacylglycerol, from phosphatidylinositol 4,5-bisphosphate (PIP2), constitutes one of the major cell signaling responses. These second messengers provide a common link from highly specific receptors for hormones, neurotransmitters, antigens, and growth factors to downstream, intracellular targets; thus, they contribute to regulation of biological functions as diverse as cell motility, fertilization, and sensory transduction. Despite this central role for PLC enzymes in signaling networks, the molecular details of their regulation and possible subversion of these regulatory mechanisms in disease remain poorly understood. phospholipase Cγ N-ethyl-N-nitrosourea Dulbecco's modified Eagle's medium B-cell receptor epidermal growth factor inositol 1,4,5-trisphosphate phosphatidylinositol 4,5-bisphosphate. Of two PLCγ enzymes, PLCγ1 is ubiquitously expressed and appears to regulate a multitude of cellular functions in many tissues. Plcg1-null mice die by embryonic day 9, highlighting the widespread importance of this enzyme (5Ji Q.S. Winnier G.E. Niswender K.D. Horstman D. Wisdom R. Magnuson M.A. Carpenter G. Proc. Natl. Acad. Sci. U.S.A. 1997; 94: 2999-3003Crossref PubMed Scopus (219) Google Scholar). PLCγ1 is activated in response to growth factor stimulation; in addition, its function in T-cell responses has been extensively documented (1Suh P.G. Park J.I. Manzoli L. Cocco L. Peak J.C. Katan M. Fukami K. Kataoka T. Yun S. Ryu S.H. BMB Rep. 2008; 41: 415-434Crossref PubMed Google Scholar). PLCγ2, in contrast, is most highly expressed in cells of the hematopoietic system and plays a key role in regulation of the immune response. Consistent with this, Plcg2-null mice display defects in the functioning of B cells, platelets, mast cells, and natural killer cells (6Wang D. Feng J. Wen R. Marine J.C. Sangster M.Y. Parganas E. Hoffmeyer A. Jackson C.W. Cleveland J.L. Murray P.J. Ihle J.N. Immunity. 2000; 13: 25-35Abstract Full Text Full Text PDF PubMed Scopus (408) Google Scholar). Both PLCγ enzymes have also been implicated in signaling events underlying aberrant cellular responses. PLCγ1 is critically involved in the regulation of cancer cell motility (7Wells A. Adv. Cancer Res. 2000; 78: 31-101Crossref PubMed Google Scholar, 8Jones N.P. Katan M. Mol. Cell. Biol. 2007; 27: 5790-5805Crossref PubMed Scopus (51) Google Scholar, 9Jones N.P. Peak J. Brader S. Eccles S.A. Katan M. J. Cell Sci. 2005; 118: 2695-2706Crossref PubMed Scopus (86) Google Scholar, 10van Rheenen J. Song X. van Roosmalen W. Cammer M. Chen X. Desmarais V. Yip S.C. Backer J.M. Eddy R.J. Condeelis J.S. J. Cell Biol. 2007; 179: 1247-1259Crossref PubMed Scopus (198) Google Scholar, 11Sala G. Dituri F. Raimondi C. Previdi S. Maffucci T. Mazzoletti M. Rossi C. Iezzi M. Lattanzio R. Piantelli M. Iacobelli S. Broggini M. Falasca M. Cancer Res. 2008; 68: 10187-10196Crossref PubMed Scopus (114) Google Scholar) while PLCγ2 has been implicated in deregulation of the immune responses resembling Btk-dependent X-linked agammaglobulinaemia and SLE disease in humans (12Guo S. Ferl G.Z. Deora R. Riedinger M. Yin S. Kerwin J.L. Loo J.A. Witte O.N. Proc. Natl. Acad. Sci. U.S.A. 2004; 101: 14180-14185Crossref PubMed Scopus (14) Google Scholar, 13Yu P. Constien R. Dear N. Katan M. Hanke P. Bunney T.D. Kunder S. Quintanilla-Martinez L. Huffstadt U. Schröder A. Jones N.P. Peters T. Fuchs H. Hrabé de Angelis M. Nehls M. Grosse J. Wabnitz P. Meyer T.P. Yasuda K. Schiemann M. Schneider-Fresenius C. Jagla W. Russ A. Popp A. Josephs M. Marquardt A. Laufs J. Schmittwolf C. Wagner H. Pfeffer K. Mudde G.C. Immunity. 2005; 22: 451-465Abstract Full Text Full Text PDF PubMed Scopus (125) Google Scholar, 14de Gorter D.J. Beuling E.A. Kersseboom R. Middendorp S. van Gils J.M. Hendriks R.W. Pals S.T. Spaargaren M. Immunity. 2007; 26: 93-104Abstract Full Text Full Text PDF PubMed Scopus (231) Google Scholar). It has been suggested that, in cancer cells, PLCγ1 could function as a key, rate-limiting, common component involved in cell motility triggered by several growth factors and integrins (7Wells A. Adv. Cancer Res. 2000; 78: 31-101Crossref PubMed Google Scholar). In some cancer cells, this increased motility could result from deregulation i.e. higher levels of expression of PLCγ1 (15Arteaga C.L. Johnson M.D. Todderud G. Coffey R.J. Carpenter G. Page D.L. Proc. Natl. Acad. Sci. U.S.A. 1991; 88: 10435-10439Crossref PubMed Scopus (191) Google Scholar, 16Nomoto K. Tomita N. Miyake M. Xhu D.B. LoGerfo P.R. Weinstein I.B. Mol. Carcinog. 1995; 12: 146-152Crossref PubMed Scopus (38) Google Scholar). The possibility that the activity of PLCγ could be up-regulated due to mutation has not yet been fully investigated in cancer. Previous studies of PLCγ2, however, have demonstrated the first gain-of-function mutation in a PLC molecule in the context of an organism, and shown that, in principle, PLC activity can be greatly enhanced by point mutations (13Yu P. Constien R. Dear N. Katan M. Hanke P. Bunney T.D. Kunder S. Quintanilla-Martinez L. Huffstadt U. Schröder A. Jones N.P. Peters T. Fuchs H. Hrabé de Angelis M. Nehls M. Grosse J. Wabnitz P. Meyer T.P. Yasuda K. Schiemann M. Schneider-Fresenius C. Jagla W. Russ A. Popp A. Josephs M. Marquardt A. Laufs J. Schmittwolf C. Wagner H. Pfeffer K. Mudde G.C. Immunity. 2005; 22: 451-465Abstract Full Text Full Text PDF PubMed Scopus (125) Google Scholar). Furthermore, this work has demonstrated that such a mutation is linked to a dramatic phenotypic disorder. By using a large scale ENU mutagenesis to discover new immune regulators, several mouse strains were generated with spontaneous autoimmune and inflammatory symptoms; two of these strains harbor a mutation in PLCγ2. In addition to the previously described ALI5 mutation (13Yu P. Constien R. Dear N. Katan M. Hanke P. Bunney T.D. Kunder S. Quintanilla-Martinez L. Huffstadt U. Schröder A. Jones N.P. Peters T. Fuchs H. Hrabé de Angelis M. Nehls M. Grosse J. Wabnitz P. Meyer T.P. Yasuda K. Schiemann M. Schneider-Fresenius C. Jagla W. Russ A. Popp A. Josephs M. Marquardt A. Laufs J. Schmittwolf C. Wagner H. Pfeffer K. Mudde G.C. Immunity. 2005; 22: 451-465Abstract Full Text Full Text PDF PubMed Scopus (125) Google Scholar) the ALI14 mutation has been identified very recently. 3K. Abe, H. Fuchs, and M. Hrabé de Angelis, unpublished data. Strikingly, the well-characterized ALI5 phenotype has shown that the mutation affects many cellular functions deregulated in Plcg2-null mice. Notably, while in Plcg2-null mice such responses are lacking, the ALI5 mutation resulted in their enhancement. In particular, further analyses of the ALI5 mutation in the context of signaling in B-cells have demonstrated that calcium responses to the crosslinking of the B-cell receptor were enhanced and prolonged resulting in enhanced deletion of B cells and autoreactivity (13Yu P. Constien R. Dear N. Katan M. Hanke P. Bunney T.D. Kunder S. Quintanilla-Martinez L. Huffstadt U. Schröder A. Jones N.P. Peters T. Fuchs H. Hrabé de Angelis M. Nehls M. Grosse J. Wabnitz P. Meyer T.P. Yasuda K. Schiemann M. Schneider-Fresenius C. Jagla W. Russ A. Popp A. Josephs M. Marquardt A. Laufs J. Schmittwolf C. Wagner H. Pfeffer K. Mudde G.C. Immunity. 2005; 22: 451-465Abstract Full Text Full Text PDF PubMed Scopus (125) Google Scholar). The domain organization of PLCγ enzymes is characterized by the insertion of a highly structured region (PLCγ-specific array, γSA) between the two halves of the TIM-barrel catalytic domain common to all PLCs. The γSA comprises a split PH (spPH) domain flanking two tandem SH2 domains and a SH3 domain (1Suh P.G. Park J.I. Manzoli L. Cocco L. Peak J.C. Katan M. Fukami K. Kataoka T. Yun S. Ryu S.H. BMB Rep. 2008; 41: 415-434Crossref PubMed Google Scholar). A distinct regulatory feature of PLCγ enzymes is that their activation is linked to an increase in phosphorylation of specific tyrosine residues (most notably within the γSA) by receptor and non-receptor tyrosine kinases (17Poulin B. Sekiya F. Rhee S.G. Proc. Natl. Acad. Sci. U.S.A. 2005; 102: 4276-4281Crossref PubMed Scopus (88) Google Scholar, 18Sekiya F. Poulin B. Kim Y.J. Rhee S.G. J. Biol. Chem. 2004; 279: 32181-32190Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar). Furthermore, multiple protein-protein interactions (mainly mediated by SH2 domains) also contribute to activation and have an important role in localizing PLCγ into protein complexes with different binding partners, depending on cell type and specific cellular compartments. One mode of activation that is specific for the PLCγ2 isozyme is direct binding to and activation by Rac. The interaction involves the spPH domain, and this activation mechanism does not require tyrosine phosphorylation (19Piechulek T. Rehlen T. Walliser C. Vatter P. Moepps B. Gierschik P. J. Biol. Chem. 2005; 280: 38923-38931Abstract Full Text Full Text PDF PubMed Scopus (70) Google Scholar, 20Walliser C. Retlich M. Harris R. Everett K.L. Josephs M.B. Vatter P. Esposito D. Driscoll P.C. Katan M. Gierschik P. Bunney T.D. J. Biol. Chem. 2008; 283: 30351-30362Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar). In molecular terms, changes that lead to PLC activation in response to different input signals, or due to point mutations, are not well understood and require further studies. Here we describe further analysis of the two gain-of-function mutations, ALI5 and ALI14, obtained using ENU mutagenesis. These mutations map to different regions in PLCγ2, and we performed detailed analysis of these regions in both PLCγ isozymes. To characterize the molecular mechanism of gain-of-function, we combined studies in vitro and in different cellular signaling contexts. We have found that ALI5- and ALI14-type point mutations lead, by distinct mechanisms, to an enhancement of responses to a variety of input signals while their combination results in a constitutively active PLC enzyme. PLCγ1 antibody (P8104) was purchased from Sigma. PLCγ2 (sc-407) and Rac2 (sc-96) antibodies were purchased from Santa Cruz Biotechnology. GAPDH antibody (10R-G109a) was purchased from Fitzgerald. Penta-His antibody (38660) was purchased from Qiagen. Phosphotyrosine antibody (PY20) was purchased from BD Transduction Laboratories. 1-Palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (POPE), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoserine (POPS), and cholesterol were purchased from Avanti Polar Lipids. 1,2-dipalmitoyl derivatives of phosphatidylinositol-(4,5)-bisphosphate (PIP2), phosphatidylinositol-(3,4,5)-trisphosphate (PIP3), and phosphatidylinositol were from Cayman Chemical. For the activity measurements l-α-PIP2 was purchased from Sigma and PIP2 [inositol 2-3H (N)] from PerkinElmer Life Sciences. The Ali14 mutation was generated in a large scale N-ethyl-N-nitrosourea (ENU) mouse mutagenesis program (21Hrabé de Angelis M. Flaswinkel H. Fuchs H. Rathkolb B. Soewarto D. Marschall S. Heffner S. Pargent W. Wuensch K. Jung M. Reis A. Richter T. Alessandrini F. Jakob T. Fuchs E. Kolb H. Kremmer E. Schaeble K. Rollinski B. Roscher A. Peters C. Meitinger T. Strom T. Steckler T. Holsboer F. Klopstock T. Gekeler F. Schindewolf C. Jung T. Avraham K. Behrendt H. Ring J. Zimmer A. Schughart K. Pfeffer K. Wolf E. Balling R. Nat. Genet. 2000; 25: 444-447Crossref PubMed Scopus (581) Google Scholar). The first ALI14 mutant mouse was identified in the dominant dysmorphology screen (22Fuchs H. Schughart K. Wolf E. Balling R. Hrabé de Angelis M. Mamm. Genome. 2000; 11: 528-530Crossref PubMed Scopus (35) Google Scholar) based on a phenotype marked by swollen footpads due to inflammation. The mutation has been stably maintained on the original C3HeB/FeJ genetic background through more than 20 backcrosses to the wild type. Detailed analysis of the phenotype, genetic mapping, and mutation detection of Ali14 have been performed using the same methodology as for ALI5 mice (13Yu P. Constien R. Dear N. Katan M. Hanke P. Bunney T.D. Kunder S. Quintanilla-Martinez L. Huffstadt U. Schröder A. Jones N.P. Peters T. Fuchs H. Hrabé de Angelis M. Nehls M. Grosse J. Wabnitz P. Meyer T.P. Yasuda K. Schiemann M. Schneider-Fresenius C. Jagla W. Russ A. Popp A. Josephs M. Marquardt A. Laufs J. Schmittwolf C. Wagner H. Pfeffer K. Mudde G.C. Immunity. 2005; 22: 451-465Abstract Full Text Full Text PDF PubMed Scopus (125) Google Scholar) and is being described elsewhere. 4K. Abe, H. Fuchs, and M. Hrabé de Angelis, manuscript in preparation. cDNA encoding bovine PLCγ1 and human PLCγ2 were inserted into pTriEx-4 (Novagen). pcDNA3.1(+) Rac2G12V construct was described in (20Walliser C. Retlich M. Harris R. Everett K.L. Josephs M.B. Vatter P. Esposito D. Driscoll P.C. Katan M. Gierschik P. Bunney T.D. J. Biol. Chem. 2008; 283: 30351-30362Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar). Human PLCγ1 encoding residues 3–1224 was inserted into pDest10 (Invitrogen) using Gateway® Technology. This construct was used to produce baculoviruses. QuikChange PCR mutagenesis (Stratagene) was used to introduce point mutations. All mutants were fully sequenced to verify the fidelity of PCR. COS-7 cells were maintained at 37 °C in a humidified atmosphere of 95% air and 5% CO2 in Dulbecco's modified Eagle's medium (DMEM) (Invitrogen) supplemented with 10% (v/v) fetal bovine serum (Invitrogen) and 2.5 mm glutamine. Prior to transfection cells were seeded into 6-well plates at a density of 2.5 × 105 cells/well and grown for 16 h in 2 ml/well of the same medium. For transfection, 1.0 μg of PLCγ DNA was mixed with 1 μl of PlusReagentTM and 7 μl of LipofectamineTM (Invitrogen) and added to the cells in 0.8 ml of DMEM without serum. The cells were incubated for 3.5 h at 37 °C, 5% CO2 before the transfection mixture was removed and replaced with DMEM-containing serum. 24 h after transfection, the cells were washed twice with inositol-free DMEM without serum and incubated for 24 h in 1.5 ml of the same medium supplemented with 0.25% fatty acid free bovine serum albumin (Sigma) and 1.5 μCi/ml myo-[2-3H]inositol (MP Biomedicals). After a further 24 h, the cells were incubated in 1.2 ml of inositol-free DMEM without serum containing 20 mm LiCl with or without stimulation with 100 ng/ml EGF (Calbiochem). The cells were lysed by addition of 1.2 ml of 4.5% perchloric acid. After incubating the samples on ice for 30 min, they were centrifuged for 20 min at 3700 × g. Supernatants and pellets were separated. The supernatants were neutralized by addition of 3 ml of 0.5 m potassium hydroxide/9 mm sodium tetraborate and centrifuged for a further 20 min at 3700 × g. Supernatants were loaded onto AG1-X8 200–400 columns (Bio-Rad) that had been converted to the formate form by addition of 2 m ammonium formate/0.1 m formic acid and equilibrated with water. The columns were washed three times with 5 ml of 60 mm ammonium formate/5 mm sodium tetraborate, and inositol phosphates were eluted with 5 ml of 1.2 m ammonium formate/0.1 m formic acid. 5 ml Ultima-Flo scintillation fluid (PerkinElmer Life Sciences) was added to the eluates and the radioactivity quantified by liquid scintillation counting. The values represent total inositol phosphates. The pellets from the first centrifugation were resuspended in 100 μl of water and 375 μl of chloroform/methanol/HCl (200:100:15) was added. The samples were vortexed, and an additional 125 μl of chloroform and 125 μl of 0.1 m HCl were added. After further vortexing, the samples were centrifuged at 700 × g for 10 min. 10 μl of the lower phase were placed in a scintillation vial with 3 ml of Ultima-Flo scintillation fluid and the radioactivity quantified by liquid scintillation counting. The obtained values correspond to radioactivity in inositol lipids. PLC activity is expressed as the total inositol phosphates formed relative to the amount of [3H]myo-inositol in the phospholipid pool. Because the differences in steady state labeling of inositol lipids are small (within 20%), this normalized PLC activity corresponds closely to PLC values expressed as total inositol phosphates; however, the error bars between the duplicates are generally smaller. For production of recombinant PLCγ1 (3–1224) (wild type and mutants), baculovirus-infected Sf9 cells were grown in suspension culture in Sf-900 II SFM medium (Invitrogen) in 2000-ml roller bottles. Cells were infected with baculovirus at 1.8 × 106 cell/ml in a 400-ml culture and incubated for 72 h at 27 °C on a rotary shaker at 140 rpm. Cells were harvested by centrifugation at 2000 × g for 15 min and then snap-frozen in liquid nitrogen. Pellets were stored at −80 °C until required. Frozen pellets were lysed with 20 ml of lysis buffer (50 mm Tris/Cl, 400 mm NaCl, 1 mm MgCl2 1% Triton X-100, 1 mm TCEP, 1 mm AEBSF, 1× Complete EDTA-free protease inhibitor mixture (Roche Applied Science), 400 units of DNaseI, pH 8.0) at 4 °C on a rotating wheel for 1 h. The lysate was centrifuged at 17,500 × g for 1 h, and the recombinant protein was then purified by a five step process. First, the supernatant was loaded onto a 5 ml of HisTrap column (GE Healthcare) with wash buffer A (25 mm Tris/Cl, 500 mm NaCl, 40 mm imidazole, and 1 mm tris(2-carboxyethyl)phosphine (TCEP), pH 8.0) and eluted with elution buffer B (25 mm Tris/Cl, 500 mm NaCl, 500 mm imidazole, and 1 mm TCEP, pH 8.0). Second, the purification tags were proteolytically cleaved overnight by TeV protease in cleavage and dialysis buffer C (25 mm Tris/Cl, 150 mm NaCl, 10 mm imidazole, and 1 mm TCEP, pH 8.0) at 4 °C. Third, the cleaved protein was passed over a Ni2+-loaded HiTrap chelating column (GE Healthcare) in buffer C and the flow-through collected. Fourth, the flow-through fraction was loaded on a Superdex 200 26/60 gel filtration column (GE Healthcare) in gel filtration buffer D (25 mm Tris/Cl, 50 mm NaCl, and 1 mm TCEP, pH 8.0) and fractions of monomeric protein collected. Finally, these fractions were concentrated by loading on a 1-ml Resource Q column (GE Healthcare) in wash buffer E (20 mm Tris/Cl and 1 mm TCEP, pH 8.0) and elution on a NaCl gradient with elution buffer F (20 mm Tris/Cl, 1 m NaCl and 1 mm TCEP, pH 8.0). Fractions containing concentrated protein were snap-frozen and stored at −80 °C. The assay was based on the method described in Ellis et al. (23Ellis M.V. James S.R. Perisic O. Downes C.P. Williams R.L. Katan M. J. Biol. Chem. 1998; 273: 11650-11659Abstract Full Text Full Text PDF PubMed Scopus (137) Google Scholar). The reaction contained 20 mm Tris/Cl pH 6.8, 0.4 mg/ml bovine serum albumin, 5 mm 2-mercaptoethanol, 4 mm EGTA, 2 mm CaCl2, 100 mm NaCl, 0.4% sodium cholate, 200 μm PIP2, and 25 ng of protein in 50 μl. Reactions were incubated at 37 °C for 20 min. The one-dimensional 1H NMR spectrum of spPH proteins was obtained at 25 °C on a Varian UnityPLUS spectrometer (500 MHz) using pulse field gradient-based water suppression. Resonances were obtained from the backbone NH and aromatic side chain CH protons (20Walliser C. Retlich M. Harris R. Everett K.L. Josephs M.B. Vatter P. Esposito D. Driscoll P.C. Katan M. Gierschik P. Bunney T.D. J. Biol. Chem. 2008; 283: 30351-30362Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar). Heats of interaction were measured as described in Bunney et al. (24Bunney T.D. Opaleye O. Roe S.M. Vatter P. Baxendale R.W. Walliser C. Everett K.L. Josephs M.B. Christow C. Rodrigues-Lima F. Gierschik P. Pearl L.H. Katan M. Mol. Cell. 2009; 34: 223-233Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar). Briefly, a MSC system (Microcal) with a cell volume of 1,458 ml was used. Proteins were loaded in the sample cell at 300 mm and titrated with the binding partner in the syringe (2 mm). The titrations were performed with stirring at 260 rpm, at 25 °C. The data were fitted with a single site model using Origin software (Microcal) (24Bunney T.D. Opaleye O. Roe S.M. Vatter P. Baxendale R.W. Walliser C. Everett K.L. Josephs M.B. Christow C. Rodrigues-Lima F. Gierschik P. Pearl L.H. Katan M. Mol. Cell. 2009; 34: 223-233Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar). Alignments of PLCδ1 with PLCγ1 and PLCγ2 were made with ClustalW and used in combination with the PLCδ1 structure (PDB:2ISD) to identify the X and Y region in the catalytic domains. The raw sequences of these regions from PLCγ1 and PLCγ2 were loaded in DeapView/Swiss-PDBViewer and three-dimensional models constructed using the SwissModel server (25Bordoli L. Kiefer F. Arnold K. Benkert P. Battey J. Schwede T. Nat. Protoc. 2009; 4: 1-13Crossref PubMed Scopus (990) Google Scholar). Side chains were repositioned in optimized conformations using SCWRL (26Dunbrack Jr., R.L. Proteins Suppl. 1999; 3: 81-87Crossref PubMed Scopus (66) Google Scholar). Stereochemical verifications of the models were carried out using Procheck (27Laskowski R.A. Rullmannn J.A. MacArthur M.W. Kaptein R. Thornton J.M. J. Biomol. NMR. 1996; 8: 477-486Crossref PubMed Scopus (4430) Google Scholar) and ProSA (28Wiederstein M. Sippl M.J. Nucleic Acids Res. 2007; 35: W407-410Crossref PubMed Scopus (3494) Google Scholar). The models were analyzed using DeapView/Swiss-PDBViewer and Pymol. All surface plasmon resonance measurements were performed at 23 °C using a lipid-coated L1 chip in the BIACORE X system as described previously (29Stahelin R.V. Cho W. Biochemistry. 2001; 40: 4672-4678Crossref PubMed Scopus (145) Google Scholar). Briefly, after washing the sensor chip surface with the running buffer (50 mm Tris/Cl, pH 7.4, containing 0.16 m KCl), POPC/POPE/POPS/cholesterol/PIP3 (12: 33: 22: 8: 22: 3%) and POPC (100%) vesicles were injected at 5 ml/min to the active surface and the control surface, respectively, to give the same resonance unit (RU) values. The level of lipid coating for both surfaces was kept at the minimum that is necessary for preventing the nonspecific adsorption to the sensor chips. This low surface coverage minimized the mass transport effect and kept the total protein concentration above the total concentration of protein binding sites on vesicles. For PLCγ1 lipid binding measurements, the flow rate was maintained at 15 ml/min for both association and dissociation phases. The PLCγ2 residue altered in ALI5 mice (Asp-993) has been mapped to the structurally defined catalytic domain (Fig. 1A) that has high sequence similarity between different PLC families (Fig. 1B and supplemental Fig. S1). The residue corresponding to Asp-993 in PLCγ2 is present in all PLC families (Fig. 1B and supplemental, Fig. S1). The two available structures of the catalytic domain from PLCδ1 and PLCβ2 superimpose well and provided a good template for modeling the catalytic domain from PLCγ enzymes. These models are shown in Fig. 1C. Residue Asp-993 and the corresponding Asp-1019 in PLCγ1 are present within one of 3 loops that form a ridge around the active site. The ridge region contains several negatively charged and hydrophobic residues. Residues Asp-1019, Asp-342, Phe-344, and Glu-347 in PLCγ1 correspond to Asp-993, Asp-334, Leu-336, and Glu-339 in PLCγ2, respectively (Fig. 1D). The ALI5 mutation in PLCγ2, D993G, has been analyzed in the context of B-cell signaling, and the data showed an increase in IP3 and calcium generation in stimulated cells in response to BCR activation (13Yu P. Constien R. Dear N. Katan M. Hanke P. Bunney T.D. Kunder S. Quintanilla-Martinez L. Huffstadt U. Schröder A. Jones N.P. Peters T. Fuchs H. Hrabé de Angelis M. Nehls M. Grosse J. Wabnitz P. Meyer T.P. Yasuda K. Schiemann M. Schneider-Fresenius C. Jagla W. Russ A. Popp A. Josephs M. Marquardt A. Laufs J. Schmittwolf C. Wagner H. Pfeffer K. Mudde G.C. Immunity. 2005; 22: 451-465Abstract Full Text Full Text PDF PubMed Scopus (125) Google Scholar). We first tested whether the impact of this mutation is restricted to B-cell components or has a more general impact on PLCγ2 activity. After transfection in COS cells, the PLCγ2 D993G variant has enhanced activation (about 6-fold) following EGF stimulation or co-transfection with the activated variant of Rac (Fig. 2, A and B). Subsequently, we analyzed the corresponding PLCγ1 mutation with respect to EGF-stimulated activation; this signaling context has been particularly well defined for the PLCγ1 isozyme. The effect of D1019G replacement was similar to the D993G mutation in PLCγ2, giving a severalfold enhancement of activity over the wild type (Fig. 2D). We further tested whether enhanced PLC activation following replacement of Asp-993 or the corresponding Asp-1019 was a consequence of the removal of negative charge in this region or was due to a change in local conformation. As shown in Fig. 2D, the replacement of Asp-1019 by alanine, leucine or asparagine, like the ALI5 mutation (replacement by glycine), resulted in an enhanced activation in EGF-stimulated cells. The most striking activation was observed for the D1019L variant; here the leucine side chain has a similar size to aspartic acid but lacks the negative charge. These data suggest that in the wild-type PLC enzyme, the negative charge of the conserved Asp-1019 residue has a role in limiting PLC activity toward the substrate, presented within the overall negatively charged plasma membrane, and that the removal of this negative charge leads to gain-of-func" @default.
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• W2132419558 date "2009-08-01" @default.
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• W2132419558 title "Characterization of Phospholipase Cγ Enzymes with Gain-of-Function Mutations" @default.
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• W2132419558 doi "https://doi.org/10.1074/jbc.m109.019265" @default.
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