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• W1848900308 abstract "Phosphoinositide-specific phospholipase C-γ1 (PLC-γ1) has two pleckstrin homology (PH) domains, an N-terminal domain and a split PH domain. Here we show that pull down of NIH3T3 cell extracts with PLC-γ1 PH domain-glutathione S-transferase fusion proteins, followed by matrix-assisted laser desorption ionization-time of flight-mass spectrometry, identified β-tubulin as a binding protein of both PLC-γ1 PH domains. Tubulin is a main component of microtubules and mitotic spindle fibers, which are composed of α- and β-tubulin heterodimers in all eukaryotic cells. PLC-γ1 and β-tubulin colocalized in the perinuclear region in COS-7 cells and cotranslocated to the plasma membrane upon agonist stimulation. Membrane-targeted translocation of depolymerized tubulin by agonist stimulation was also supported by immunoprecipitation analyses. The phosphatidylinositol 4,5-bisphosphate (PIP2) hydrolyzing activity of PLC-γ1 was substantially increased in the presence of purified tubulin in vitro, whereas the activity was not promoted by bovine serum albumin, suggesting that β-tubulin activates PLC-γ1. Furthermore, indirect immunofluorescent microscopy showed that PLC-γ1 was highly concentrated in mitotic spindle fibers, suggesting that PLC-γ1 is involved in spindle fiber formation. The effect of PLC-γ1 in microtubule formation was assessed by overexpression and silencing PLC-γ1 in COS-7 cells, which resulted in altered microtubule dynamics in vivo. Cells overexpressing PLC-γ1 showed higher microtubule densities than controls, whereas PLC-γ1 silencing with small interfering RNAs led to decreased microtubule network densities as compared with control cells. Taken together, our results suggest that PLC-γ1 and β-tubulin transmodulate each other, i.e. that PLC-γ1 modulates microtubule assembly by β-tubulin, and β-tubulin promotes PLC-γ1 activity. Phosphoinositide-specific phospholipase C-γ1 (PLC-γ1) has two pleckstrin homology (PH) domains, an N-terminal domain and a split PH domain. Here we show that pull down of NIH3T3 cell extracts with PLC-γ1 PH domain-glutathione S-transferase fusion proteins, followed by matrix-assisted laser desorption ionization-time of flight-mass spectrometry, identified β-tubulin as a binding protein of both PLC-γ1 PH domains. Tubulin is a main component of microtubules and mitotic spindle fibers, which are composed of α- and β-tubulin heterodimers in all eukaryotic cells. PLC-γ1 and β-tubulin colocalized in the perinuclear region in COS-7 cells and cotranslocated to the plasma membrane upon agonist stimulation. Membrane-targeted translocation of depolymerized tubulin by agonist stimulation was also supported by immunoprecipitation analyses. The phosphatidylinositol 4,5-bisphosphate (PIP2) hydrolyzing activity of PLC-γ1 was substantially increased in the presence of purified tubulin in vitro, whereas the activity was not promoted by bovine serum albumin, suggesting that β-tubulin activates PLC-γ1. Furthermore, indirect immunofluorescent microscopy showed that PLC-γ1 was highly concentrated in mitotic spindle fibers, suggesting that PLC-γ1 is involved in spindle fiber formation. The effect of PLC-γ1 in microtubule formation was assessed by overexpression and silencing PLC-γ1 in COS-7 cells, which resulted in altered microtubule dynamics in vivo. Cells overexpressing PLC-γ1 showed higher microtubule densities than controls, whereas PLC-γ1 silencing with small interfering RNAs led to decreased microtubule network densities as compared with control cells. Taken together, our results suggest that PLC-γ1 and β-tubulin transmodulate each other, i.e. that PLC-γ1 modulates microtubule assembly by β-tubulin, and β-tubulin promotes PLC-γ1 activity. PLC-γ1 1The abbreviations used are: PLC-γ1, phospholipase C-γ1; PH, pleckstrin homology; GST, glutathione S-transferase; MALDI-TOF, matrix-assisted laser desorption ionization-time of flight; PIP2, phosphatidylinositol 4,5-bisphosphate; BSA, bovine serum albumin; siRNA, small interfering RNA; IP3, inositol 1,4,5-trisphosphate; SH, Src homology; HRP, horseradish peroxidase; FBS, fetal bovine serum; PVDF, polyvinylidene difluoride; PDGF, platelet-derived growth factor; EGF, epidermal growth factor; GFP, green fluorescent protein; mAb, monoclonal antibody; MES, 4-morpholineethanesulfonic acid; EF, elongation factor; GEF, guanine nucleotide exchange factor; GTPγS, guanosine 5′-3-O-(thio)triphosphate. 1The abbreviations used are: PLC-γ1, phospholipase C-γ1; PH, pleckstrin homology; GST, glutathione S-transferase; MALDI-TOF, matrix-assisted laser desorption ionization-time of flight; PIP2, phosphatidylinositol 4,5-bisphosphate; BSA, bovine serum albumin; siRNA, small interfering RNA; IP3, inositol 1,4,5-trisphosphate; SH, Src homology; HRP, horseradish peroxidase; FBS, fetal bovine serum; PVDF, polyvinylidene difluoride; PDGF, platelet-derived growth factor; EGF, epidermal growth factor; GFP, green fluorescent protein; mAb, monoclonal antibody; MES, 4-morpholineethanesulfonic acid; EF, elongation factor; GEF, guanine nucleotide exchange factor; GTPγS, guanosine 5′-3-O-(thio)triphosphate. is an important signaling molecule for cell proliferation and differentiation. Activated PLC-γ1 hydrolyzes PIP2 to produce inositol 1,4,5-trisphosphate (IP3) and diacylglycerol. These second messengers regulate the release of Ca2+ from intracellular stores and activate protein kinase C, respectively (1Berridge M.J. Nature. 1993; 361: 315-325Crossref PubMed Scopus (6147) Google Scholar, 2Nishizuka Y. FASEB J. 1995; 9: 484-496Crossref PubMed Scopus (2352) Google Scholar). PLC-γ1 has two Src homology (SH) 2 domains and an SH3 domain, both responsible for protein-protein interactions, and two pleckstrin homology (PH) domains, responsible for protein-protein and protein-lipid interactions. Of these, one PH domain is located in the 150 N-terminal amino acid residues, whereas the other is split by the SH2-SH2-SH3 domain (3Gibson T.J. Hyvonen M. Musacchio A. Saraste M. Birney E. Trends Biochem. Sci. 1994; 19: 349-353Abstract Full Text PDF PubMed Scopus (295) Google Scholar). PH domains bind with high specificity and affinity to phosphoinositides such as phosphatidylinositol phosphate, PIP2, phosphatidylinositol 1,4,5-trisphosphate, and IP3 (4Lemmon M.A. Falasca M. Ferguson K.M. Schlessinger J. Trends Cell Biol. 1997; 7: 237-242Abstract Full Text PDF PubMed Scopus (147) Google Scholar, 5Lemmon M.A. Ferguson K.M. Biochem. J. 2000; 350: 1-18Crossref PubMed Scopus (613) Google Scholar), and the PH domains of signaling molecules are often involved in targeted translocation of molecules to cell membranes (6Lemmon M.A. Ferguson K.M. Schlessinger J. Cell. 1996; 85: 621-624Abstract Full Text Full Text PDF PubMed Scopus (428) Google Scholar, 7Falasca M. Logan S.K. Lehto V.P. Baccante G. Lemmon M.A. Schlessinger J. EMBO J. 1998; 17: 414-422Crossref PubMed Scopus (481) Google Scholar). PH domains can also specifically bind cellular signaling proteins, such as the βγ-subunit of the heteromeric G-protein (8Touhara K. Inglese J. Pitcher J.A. Shaw G. Lefkowitz R.J. J. Biol. Chem. 1994; 269: 10217-10220Abstract Full Text PDF PubMed Google Scholar) and protein kinase C (9Yao L. Suzuki H. Ozawa K. Deng J. Lehe C. Fukamachi H. Anderson W.G. Kawakami Y. Kawakami T. J. Biol. Chem. 1997; 272: 13033-13039Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar). Although numerous studies have investigated protein-protein interactions via the SH domain of PLC-γ1, the PH domain-mediated signaling of PLC-γ1 has not yet been elucidated. Here we sought to identify proteins that specifically associate with the PLC-γ1 PH domains in cells, and we found that both PH domains of PLC-γ1 specifically bind to β-tubulin.Heterodimers of α- and β-tubulin are essential cytoskeletal components of the microtubules in all eukaryotes. Microtubules regulate cell division, cell shape, and cell motility via cycles of tubulin polymerization and depolymerization called microtubule instability (10Mitchison T.J. Kirschner M.W. Nature. 1984; 312: 237-242Crossref PubMed Scopus (2290) Google Scholar, 11Desai A. Mitchison T.J. Annu. Rev. Cell Dev. Biol. 1997; 13: 83-117Crossref PubMed Scopus (1923) Google Scholar). The mitotic spindle, which is a dynamic array of microtubules, is responsible for chromosome segregation into daughter cells during mitosis. Therefore, numerous studies have sought to identify proteins controlling microtubule instability. Two major groups of these proteins have been identified. The microtubule destabilizers, capable of depolymerizing microtubules into α- and β-tubulin heterodimers, include XKCM1 (12Walczak C.E. Mitchison T.J. Cell. 1996; 85: 943-946Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar), XKIF2 (13Desai A. Verma S. Mitchison T.J. Walczak C.E. Cell. 1999; 96: 69-78Abstract Full Text Full Text PDF PubMed Scopus (576) Google Scholar), stathmin/Op18 (14Belmont L.D. Mitchison T.J. Cell. 1996; 84: 623-631Abstract Full Text Full Text PDF PubMed Scopus (582) Google Scholar), and katanin (15McNally F.J. Okawa K. Iwamatsu A. Vale R.D. J. Cell Sci. 1996; 109: 561-567Crossref PubMed Google Scholar). The microtubule stabilizers, which polymerize tubulin heterodimers into microtubules, include Ran (16Wilde A. Lizarraga S.B. Zhang L. Wiese C. Gliksman N.R. Walczak C.E. Zheng Y. Nat. Cell Biol. 2001; 3: 221-227Crossref PubMed Scopus (184) Google Scholar), CRMP-2 (17Fukata Y. Itoh T.J. Kimura T. Menager C. Nishimura T. Shiromizu T. Watanabe H. Inagaki N. Iwamatsu A. Hotani H. Kaibuchi K. Nat. Cell Biol. 2002; 4: 583-591Crossref PubMed Scopus (619) Google Scholar), Tau (18Tseng H.C. Lu Q. Henderson E. Graves D.J. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 9503-9508Crossref PubMed Scopus (43) Google Scholar), and microtubule-associated protein (MAP) (19Trinczek B. Marx A. Mandelkow E.M. Murphy D.B. Mandelkow E. Mol. Biol. Cell. 1993; 4: 323-335Crossref PubMed Scopus (42) Google Scholar). Recently, a third group has been identified as regulating microtubule assembly. This group includes Hsp90 (20Garnier C. Barbier P. Gilli R. Lopez C. Peyrot V. Briand C. Biochem. Biophys. Res. Commun. 1998; 250: 414-419Crossref PubMed Scopus (76) Google Scholar), Gβγ subunit (21Roychowdhury S. Panda D. Wilson L. Rasenick M.M. J. Biol. Chem. 1999; 274: 13485-13490Abstract Full Text Full Text PDF PubMed Scopus (97) Google Scholar), G-protein coupled receptor kinase-2 (22Pitcher J.A. Hall R.A. Daaka Y. Zhang J. Ferguson S.S. Hester S. Miller S. Caron M.G. Lefkowitz R.J. Barak L.S. J. Biol. Chem. 1998; 273: 12316-12324Abstract Full Text Full Text PDF PubMed Scopus (133) Google Scholar), and pyruvate kinase (23Vertessy B.G. Bankfalvi D. Kovacs J. Low P. Lehotzky A. Ovadi J. Biochem. Biophys. Res. Commun. 1999; 254: 430-435Crossref PubMed Scopus (28) Google Scholar).Because PLC-γ1 plays pivotal roles in the cellular signaling pathways responsible for triggering the production of second messengers including Ca2+ and IP3, and because overexpression of PLC-γ1 results in cellular transformation (24Chang J.-S. Noh D.Y. Park I.A. Kim M.J. Song H. Ryu S.H. Suh P.-G. Cancer Res. 1997; 57: 5465-5468PubMed Google Scholar), it is critical to identify the proteins that regulate activity of PLC-γ1. Here we sought to identify proteins that specifically interact with the PH domains of PLC-γ1, and we showed for the first time that β-tubulin can bind both PH domains of PLC-γ1. In addition, we determined that PLC-γ1 and β-tubulin transmodulate each other in vivo.EXPERIMENTAL PROCEDURESAntibodies—Monoclonal anti-β-tubulin and anti-α-tubulin were purchased from Chemicon (Temecula, CA) and Sigma, and polyclonal anti-β-tubulin was purchased from Santa Cruz Biotechnology (Santa Cruz, CA). The horseradish peroxidase (HRP)-conjugated goat anti-mouse and goat anti-rabbit antibodies were purchased from Upstate Biotechnology, Inc. (Lake Placid, NY). Monoclonal anti-FLAG M5, polyclonal anti-PLC-γ1, and highly purified tubulin from bovine brain were obtained from Sigma. Fluorescein-conjugated Affinipure goat anti-rabbit IgG and rhodamine-conjugated Affinipure goat anti-mouse IgG were from Jackson ImmunoResearch (West Grove, PA).In Vitro Binding Assay with GST Fusion Proteins—By using rat PLC-γ1 cDNA (25Suh P.-G. Ryu S.H. Moon K.H. Suh H.W. Rhee S.G. Proc. Natl. Acad. Sci. U. S. A. 1988; 85: 5419-5423Crossref PubMed Scopus (126) Google Scholar) as the template, GST constructs for fusion proteins were generated by PCR as described previously (26Chang J.-S. Seok H. Kwon T.K. Min D.S. Ahn B.H. Lee Y.H. Suh J.W. Kim J.W. Iwashita S. Omori A. Ichinose S. Numata O. Seo J.K. Oh Y.S. Suh P.-G. J. Biol. Chem. 2002; 277: 19697-19702Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar). GST fusion proteins were expressed in Escherichia coli, and the lysates were incubated with glutathione-Sepharose beads, then washed extensively with Igepal buffer (20 mm Tris-Cl, pH 7.5, 1% Igepal CA-630, 300 mm NaCl, 2 mm MgCl2, 1 mm EDTA, 10 μg/ml aprotinin, 10 μg/ml leupeptin, 1 mm phenylmethylsulfonyl fluoride, and 1 mm sodium orthovanadate), resolved by 10% SDS-PAGE, and then transferred to polyvinylidene difluoride (PVDF) membranes. The membrane-bound proteins were detected with an ECL detection system (Amersham Biosciences) using monoclonal anti-FLAG and HRP-conjugated goat anti-mouse antibodies.DNA Construction and Expression—PCR-amplified mouse cDNAs encoding various tubulin isotypes (Mβ1, Mβ2, Mβ3, Mβ6, and Mα1, kindly provided by Dr. Sally Lewis, New York University) (27Wang D. Villasante A. Lewis S.A. Cowan N.J. J. Cell Biol. 1986; 103: 1903-1910Crossref PubMed Scopus (211) Google Scholar) were ligated into the EcoRI/SalI restriction sites of the N-terminal epitope tagging vector, pFLAG-CMV-2 (Sigma). When expressed, all α- and β-tubulin cDNAs encoded an N-terminal 9-amino acid FLAG epitope tag. Similarly, the rat cDNA encoding PLC-γ1 was inserted into the HindIII/XbaI sites of the pFLAG-CMV-2 vector for mammalian expression. For localization studies in COS-7 cells, we constructed vectors in which the enhanced green fluorescent protein (GFP) was fused with the PH domains of PLC-γ1. cDNAs encoding the PH domains of PLC-γ1 were subcloned into the EcoRI/XbaI sites of pEGFP-C2 (Clontech). Expression of GFP-PH1 (amino acids 25–145 of PLC-γ1) and GFP-nPH2 (amino acids 477–547 of PLC-γ1) was assessed by immunofluorescent microscopy and immunoblotting using an anti-GFP antibody (Zymed Laboratories Inc.). All constructs were prepared using the plasmid maxi kit (Qiagen, Santa Clarita, CA) and confirmed by DNA sequencing of the ligation sites. COS-7 cells were transfected with the various constructs using the Lipofectamine reagent (Invitrogen). Forty eight hours after transfection, the cells were either harvested for immunoblotting/immunoprecipitation or fixed for indirect immunofluorescent microscopy.Immunoprecipitation and Immunoblotting—Cells transfected with plasmids encoding the various FLAG-tagged mouse tubulin constructs were washed twice with phosphate-buffered saline and lysed with radioimmunoprecipitation assay buffer (20 mm HEPES, pH 7.2, 1% Triton X-100, 1% sodium deoxycholate, 0.1% SDS, 150 mm NaCl, 1 mm sodium orthovanadate, 10 μg/ml leupeptin, 10 μg/ml aprotinin, and 1 mm phenylmethylsulfonyl fluoride). For agonist stimulation, cells were serum-starved for 30 h and then stimulated with either 20% fetal bovine serum (FBS) or 0.2 μg/ml epidermal growth factor (EGF) for 10–20 min. The lysate supernatants were precleared by incubation with anti-FLAG M2 affinity gel (Sigma) for 30 min. Precleared cell lysates were then incubated for 2 h with anti-FLAG antibodies conjugated with 50 μl of a 50% slurry of anti-FLAG M2 affinity gel. The immune complexes were collected by centrifugation, washed three times with ice-cold radioimmunoprecipitation assay buffer, then resolved by 10% SDS-PAGE, and blotted to a PVDF membrane. The blot was probed with either anti-PLC-γ1 or anti-FLAG antibodies, and the immunoreactive bands were visualized by ECL detection using HRP-conjugated goat anti-mouse IgG.Far Western Blot Analysis—Purified bovine tubulin (0.2 μg per lane) was resolved by 10% SDS-PAGE and transferred onto a PVDF membrane. Nonspecific binding was blocked by incubation of membranes in 2% skim milk in Tris-buffered Tween 20 (TBT) for 1 h at room temperature. The membranes were then incubated with the GST, GST-PH1, GST-nPH2, or GST-SH3 fusion proteins (0.5 μg/ml) in blocking buffer for 14 h at 4 °C. After washing in TBT buffer, the membranes were incubated with anti-GST antibody for 2 h at room temperature. The membranes were then washed in TBT buffer, and bound proteins were detected by incubation with a secondary HRP-conjugated anti-goat antibody and visualized with an ECL detection system.Expression and Purification of PLC-γ1—The cDNAs for the wild type PLC-γ1 was introduced into the HindIII/XbaI sites of the pFLAG-CMV-2 vector. The recombinant PLC-γ1 proteins contained a 9-amino acid FLAG epitope at their N termini. COS-7 cells grown on 100-mm culture dishes were transfected with 5 μg of recombinant plasmid and 10 μl of Lipofectamine (Invitrogen) according to the manufacturer's specifications. Two days after transfection, cells were harvested, and the recombinant PLC-γ1 proteins were recovered using a pFLAG-CMV-2 purification kit (Sigma).Sedimentation Experiments—Purified tubulin (10 μm) was incubated with 0.5 μm of PLC-γ1 or BSA (control) in tubulin polymerizing buffer (0.1 m MES, 1 mm EGTA, 0.5 mm MgCl2, 0.1 mm EDTA, 2.5 m glycerol and 0.1 mm GTP, pH 6.5) for 30 min at 37 °C. The samples were then centrifuged at 15,000 × g for 1 h at room temperature. The supernatant and pellet fractions were resolved by 10% SDS-PAGE, and the gel was stained with Coomassie Brilliant Blue for visualization. A separate fractionation was performed; the resulting supernatant was mixed with 20% glycerol and 1 mm GTP, and the sample was incubated at 37 °C for polymerization for 30 min and then centrifuged at 100,000 × g for 45 min. The pellet was resuspended in ice-cold tubulin polymerizing buffer, homogenized, incubated on ice for depolymerization, and centrifuged at 100,000 × g. These polymerization/depolymerization cycles were repeated two times, and the supernatants and pellets from each step were resolved by 10% SDS-PAGE and transferred to PVDF membranes for Western immunoblotting using an anti-PLC-γ1 antibody.Immunofluorescent Microscopy—COS-7 cells were seeded on glass coverslips in 6-well plates and transfected with 5 μl of Lipofectamine and one of the following: 2 μg of the pFLAG-CMV-2-PLC-γ1 vector, small interfering RNA (siRNA) for PLC-γ1, or empty pFLAG-CMV-2 vector. The siRNAs for human PLC-γ1 were purchased from Dharmacon Inc. (Lafayette, CO); the SMARTpool siRNAs consisted of combinations of GGAAGAAGCAGCTGTGGTT, CCAACCAGCTTAAGAGGAA, GAAGTGAACATGTGGATCA, and GAGCAGTGCCTTTGAAGAA. Following transfection, the cells were grown for 2 days in Dulbecco's modified Eagle's medium with 0.5% FBS. For agonist stimulation, the cells were serum-starved for at least 30 h and then stimulated with either 20% FBS (Invitrogen) or 0.2 μg/ml EGF (Sigma) for 10–20 min. The cells were fixed at 37 °C for 10 min in 4% paraformaldehyde and then incubated with affinity-purified monoclonal anti-FLAG or polyclonal anti-PLC-γ1 antibodies for 1 h at room temperature in a humidity chamber. Following complete washing with phosphate-buffered saline, the cells were incubated with fluorescein-conjugated Affinipure goat anti-rabbit IgG or rhodamine-conjugated Affinipure goat anti-mouse IgG. Immunostained cells were observed with a fluorescent microscope (Nikon Eclipse E600 Epifluorescence Microscope), and the images were captured with a digital image microscope camera.Mass Spectrometry—Gel slices corresponding to the appropriate protein bands were crushed and destained by washing with 50% acetonitrile in 25 mm NH4HCO3. The gel slices were then incubated overnight with trypsin (Promega) in 25 mm NH4HCO3 at 37 °C. The resulting peptides were eluted with matrix solution (5 mg/ml α-cyano-4-hydroxycinnamic acid, 0.1% trifluoroacetic acid, and 50% acetonitrile) and applied to the MALDI target plate. Peptide molecular weights were measured on a MALDI-TOF mass spectrometer (Voyager-DE STR, Applied Biosystems, Inc.). Peptide mass maps were searched against theoretically derived maps from proteins found in the nonredundant protein data base (NCBI) using the ProFound online program (www.proteometrics.com).Fractionation of Monomeric and Polymeric Tubulin—Polymerized (polymeric) and depolymerized (monomeric) tubulins were differentially fractionated from COS-7 cells transfected with vector alone, pFLAG-CMV-2-PLC-γ1, and siRNA for PLC-γ1 according to procedures described previously (28Breitfeld P.P. McKinnon W.C. Mostov K.E. J. Cell Biol. 1990; 111: 2365-2373Crossref PubMed Scopus (130) Google Scholar). Briefly, the transfected cells on a 6-well plate were washed two times with phosphate-buffered saline and incubated with 0.3 ml of tubulin polymerizing buffer plus 10 μg/ml leupeptin, 10 μg/ml aprotinin, 1 mm phenylmethylsulfonyl fluoride, 1 mm MgSO4, and 0.1% Triton X-100 for 20 min at 37 °C. The supernatant from a 6-well plate was removed carefully as monomeric tubulin fractions. Polymeric tubulin was extracted from the remaining Triton X-100-insoluble fraction by extracting in SDS lysis buffer (25 mm Tris, pH 7.4, 0.4 m NaCl, 0.5% SDS) for 10 min at 37 °C. Aliquots from each fraction were resolved by 10% SDS-PAGE and Western immunoblotted with anti-tubulin and anti-PLC-γ1 antibodies.PLC-γ1 Activity Assay—PLC-γ1 activity was measured as described previously (29Hepler J.R. Kozasa T. Smrcka A.V. Simon M.I. Rhee S.G. Sternweis P.C. Gilman A.G. J. Biol. Chem. 1993; 268: 14367-14375Abstract Full Text PDF PubMed Google Scholar). Briefly, the substrate was prepared as sonicated vesicles of 75 mm PIP2, 75 mm [3H]PIP2 (9,000–10,000 cpm/assay), and 750 mm phosphatidylethanolamine in 50 mm HEPES buffer, pH 7.0. Reactions were performed for 20 min at 30 °C in a 100-μl final volume containing 10 or 100 ng of PLC-γ1 and 2 mm Ca2+, and terminated by addition of 1 ml of chloroform/methanol/HCl (50:50:0.5) and 400 μl of 1 n HCl. The mixtures were vortexed and centrifuged for 10 min at 2,000 rpm. The aqueous phase containing [3H]IP3 was collected and subjected to scintillation counting. The effect of β-tubulin was examined by adding the indicated amounts of α- and β-tubulin to the PLC-γ1 assay mixture.RESULTSThe PH Domains of PLC-γ1 Directly Bind to β-Tubulin—To identify new PLC-γ1-associated proteins, the two PH domains of PLC-γ1 were fused to GST and used for pull-down assays. GSH-Sepharose-coupled GST-PH1 and GST-nPH2 proteins were incubated with NIH3T3 cell lysates (Fig. 1A), using the technique that had been used previously to isolate eukaryotic translational elongation factor (EF)-1α as a PH domain-associated protein (26Chang J.-S. Seok H. Kwon T.K. Min D.S. Ahn B.H. Lee Y.H. Suh J.W. Kim J.W. Iwashita S. Omori A. Ichinose S. Numata O. Seo J.K. Oh Y.S. Suh P.-G. J. Biol. Chem. 2002; 277: 19697-19702Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar). Because the GST-cPH2 fusion protein did not show any visible protein band on our SDS-polyacrylamide gel (26Chang J.-S. Seok H. Kwon T.K. Min D.S. Ahn B.H. Lee Y.H. Suh J.W. Kim J.W. Iwashita S. Omori A. Ichinose S. Numata O. Seo J.K. Oh Y.S. Suh P.-G. J. Biol. Chem. 2002; 277: 19697-19702Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar), we used the GST-PH1 and GST-nPH2 fusion proteins in this experiment. As shown in Fig. 1A, the GST-nPH2 fusion protein specifically pulled down a prominent 48-kDa protein band that had been identified previously as EF-1α (26Chang J.-S. Seok H. Kwon T.K. Min D.S. Ahn B.H. Lee Y.H. Suh J.W. Kim J.W. Iwashita S. Omori A. Ichinose S. Numata O. Seo J.K. Oh Y.S. Suh P.-G. J. Biol. Chem. 2002; 277: 19697-19702Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar). In addition, both the GST-PH1 and GST-nPH2 fusion proteins specifically pulled down a 55-kDa protein that was not pulled down by the control GST fusion protein. This band was excised from the gel and analyzed by MALDI-TOF-mass spectrometry.Table I shows the peptide sequences and mass data derived from a trypsin digest of the 55-kDa protein. When the peptide mass maps were searched against the NCBI protein data base, the 55-kDa protein was identified as mouse β-tubulin. This identification was confirmed by Western blotting with monoclonal anti-β-tubulin (Fig. 1B). We further confirmed that the interaction between PLC-γ1 and β-tubulin occurs in vivo by reciprocal immunoprecipitations: anti-β-tubulin immunoprecipitated PLC-γ1 and vice versa (Fig. 1C and see Fig. 4B). To examine whether the binding is direct or not, we performed far Western blot analysis using purified tubulin. The result shows that GST-PH1 and GST-nPH2 directly bind to β-tubulin, whereas GST alone or GST-SH3 does not (Fig. 1D). Finally, we performed a sedimentation experiment to verify the interaction between PLC-γ1 and β-tubulin. Because self-assembled tubulin microtubules can be pelleted by centrifugation, we incubated purified tubulin (α- and β-tubulin heterodimers) and allowed them to polymerize into microtubules in the presence of PLC-γ1 or BSA (control). As shown in Fig. 1E, PLC-γ1 coprecipitated with polymerized tubulin, whereas BSA did not, indicating that PLC-γ1 binds to microtubules and tubulin heterodimers in vivo. Moreover, when further polymerization and depolymerization cycles were performed with the polymerized tubulin precipitates, PLC-γ1 was found to consistently coexist with tubulin in the supernatant and pellet fractions (Fig. 1E).Table ITryptic peptides of β-tubulin identified by MALDI-TOFPeptide sequenceaAmino acid residues derived from the 55-kDa proteinStart-endbPosition of the amino acid residue in the deduced peptide sequence of mouse β-tubulinMonoisotopic mass ([M + H+])TheoreticalExperimentalFWEVISDEHGIDPTGTYHGDSDLQLER11-373115.423115.37SGPFGQIFRPDNFVFGQSGAGNNWAK69-942797.342797.30GHYTEGAELVDSVLDVVR95-1121957.971957.97LTTPTYGDLNHLVSATMSGVTTCLR208-2322707.332707.29FPGQLNADLR233-2421129.591129.62LAVNMVPFPR244-2531142.631142.65LHFFMPGFAPLTSR254-2671619.831619.84YLTVAAVFR301-3091038.591038.61NSSYFVEWIPNNVK328-3411695.831695.83MSATFIGNSTAIQELFK354-3701856.931856.94ISEQFTAMFR372-3811228.591228.61a Amino acid residues derived from the 55-kDa proteinb Position of the amino acid residue in the deduced peptide sequence of mouse β-tubulin Open table in a new tab Fig. 4Immunofluorescence of transfected FLAG-Mβ-tubulin isotypes in COS-7 cells.A, cells transfected with N-terminal FLAG-tagged mouse α- and β-tubulin isotypes were visualized by immunofluorescence microscopy. cDNAs encoding FLAG-Mα1(a), -Mβ1(b), -Mβ2(c), -Mβ3(d), and -Mβ6 (e) were transiently transfected into COS-7 cells. After 48 h, the cells were fixed and stained with anti-FLAG antibodies. B, FLAG-Mβ2-tubulin coimmunoprecipitates with FLAG-PLC-γ1 in COS-7 cell lysates. COS-7 cells transfected with FLAG-tagged mouse β2-tubulin and rat PLC-γ1 were used for immunoprecipitations (IP) of FLAG-Mβ2-tubulin and FLAG-PLC-γ1 using their antibodies. The membrane was probed with a monoclonal anti-FLAG antibody followed by HRP-conjugated anti-mouse IgG. C, to examine the domain specificity of β-tubulin association, various GST fusion proteins were incubated with COS-7 cell lysates containing each FLAG-Mβ tubulin protein. The bound proteins were resolved by 10% SDS-PAGE followed by immunoblotting with an anti-FLAG antibody. WCL, whole cell lysates.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Colocalization of PLC-γ1 and β-Tubulin—To examine the subcellular localizations of PLC-γ1 and β-tubulin in COS-7 cells, we performed double immunostaining by using anti-PLC-γ1 and anti-β-tubulin antibodies. As shown in Fig. 2A, PLC-γ1 and β-tubulin were localized in the cytoplasm of resting cells. Overlays of the fluorescent microscopy images showed that both proteins dominantly colocalized in the perinuclear region of the COS-7 cells. Generally, agonist treatment results in translocation of PLC-γ1 from the cytosol to the plasma membrane, where it binds to receptor tyrosine kinase (30Rhee S.G. Annu. Rev. Biochem. 2001; 70: 281-312Crossref PubMed Scopus (1209) Google Scholar, 31Schlessinger J. Cell. 2000; 103: 193-200Abstract Full Text Full Text PDF PubMed Scopus (3484) Google Scholar). To examine the localizations of PLC-γ1 and β-tubulin following agonist stimulation, we performed double immunostaining with anti-PLC-γ1 and anti-β-tubulin antibodies. In cells treated with 20% FBS or EGF for 10 min, both PLC-γ1 and β-tubulin were localized in the plasma membrane (Fig. 2, B and C).Fig. 2Colocalization of PLC-γ1 and β-tubulin in COS-7 cells.A, double immunostaining of β-tubulin and PLC-γ1. Serum-deprived quiescent COS-7 cells were fixed and stained with a monoclonal anti-β-tubulin and a polyclonal anti-PLC-γ1 antibody followed by rhodamine- and fluorescein isothiocyanate-labeled secondary antibody staining, respectively. B and C, COS-7 cells treated with either 20% FBS (B) or 0.2 μg/ml EGF (C) for 10 min were stained with anti-β-tubulin and anti-PLC-γ1 antibodies as described above. D, double immunofluorescence of mitotic COS-7 cells, prepared as in A.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Furthermore, in mitotic cells, PLC-γ1 was hi" @default.
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• W1848900308 date "2005-02-01" @default.
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• W1848900308 title "Pleckstrin Homology Domains of Phospholipase C-γ1 Directly Interact with β-Tubulin for Activation of Phospholipase C-γ1 and Reciprocal Modulation of β-Tubulin Function in Microtubule Assembly" @default.
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• W1848900308 doi "https://doi.org/10.1074/jbc.m406350200" @default.
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