Proc. Nati. Acad. Sci. USA Vol. 89, pp. 5720-5724, July 1992 Biochemistry
Tyrosine phosphorylation of G protein a subunits by pp6Oc-src {guanosine 5'-[l-thio]triphosphate binding/GTPase activity/stimulatory guanine nucleotide-binding regulatory protein a subunit (H2la)}
WILLIAM P. HAUSDORFF*t, JULIE A. PITCHER*, DEIRDRE K. LUTTRELLt, MAURINE E. LINDER§, HITOSHI KUROSE*, SARAH J. PARSONS¶, MARC G. CARON*, AND ROBERT J. LEFKOWITZ*II *Howard Hughes Medical Institute, Departments of Medicine, Biochemistry and Cell Biology, Box 3821, Duke University Medical Center, Durham, NC 27710;
*Glaxo Research Laboratories, Research Triangle Park, NC 27709; §Department of Pharmacology, Southwestern Graduate School, University of Texas Health Science Center, Dallas, TX 75235; and IDepartment of Microbiology, Cancer Research Center and Molecular Biology Institute, University of Virginia Health Sciences Center, Charlottesville, VA 22908
Contributed by Robert J. Leflcowitz, March 19, 1992
the abilities of purified G proteins to serve as substrates for phosphorylation by pp60C-src.
A number of lines of evidence suggest that ABSTRACT cross-talk exists between the cellular signal transduction pathways involving tyrosine phosphorylation catalyzed by members of the pp6OC-srC kinase family and those mediated by guanine nucleotide regulatory proteins (G proteins). In this study, we explore the possibility that direct interactions between pp60' and G proteins may occur with functional consequences. Preparations of pp6cc isolated by immunoprecipitation phosphorylate on tyrosine residues the purified G-protein a subunits (Ga) of several heterotrimeric G proteins. Phosphorylation is highly dependent on G-protein conformation, and Ga(GDP) uncomplexed by Pr subunits appears to be the preferred substrate. In functional studies, phosphorylation of stimulatory Ga (Ga.) modestly increases the rate of binding of guanosine 5'-[y-[35S]thiojtriphosphate to G. as well as the receptor-stimulated steady-state rate of GTP hydrolysis by Gs. Heterotrimeric G proteins may represent a previously unappreciated class of potential substrates for ppW.SrI.
EXPERIMENTAL PROCEDURES Purification and Reconstitution of PrAdrenergic Receptors (fi2AR) and G Proteins. Purification of /32AR from hamster lung (18), rhodopsin, and transducin from bovine retina (19), pfy subunits from bovine brain (20, 21), and G-protein a subunits (Ga) expressed in Escherichia coli (22-24) were as described. Receptors were reconstituted in phosphatidyicholine vesicles by using octyl glucoside (25). pp6(' Immunoprecipitations and in Vitro Kinase Assays. C3HT1O2 (C5) or Syrian hamster (4A) cells overexpressing wild-type avian pp60c-src at levels >25-fold over nontransfected cells (15) were pretreated with 1.6 ,uM phorbol 12myristate 13-acetate for 15 min, the cells were lysed (1.5 ml per 75-cm2 flask) in RIPA [150 mM NaCI/SO mM Tris'HCI, pH 7.5/1% Nonidet P-40/0.25% deoxycholate/peptide inhibitors (18)/1 ,M microcystin (Calbiochem)] plus 0.1% SDS, and the insoluble fraction was removed by 10 min of centrifugation at 12,000 x g. Phorbol pretreatment (26), although not required, enhanced the phosphorylating activity of pp6OC-src (data not shown). Immunoprecipitations were performed by using the rodent/avian pp60c-src-specific monoclonal antibody GD11 (27). Specifically, 20 1.l of a 1:10 dilution of GD11 in Dulbecco's phosphate-buffered saline (PBS) was incubated per 1.5 ml of solubilized extract on ice for 1 hr. At this point, 25 ILI of Pansorbin (Calbiochem) prewashed in bovine serum albumin (1.5 mg/ml) was added, and the mixture was incubated for 30 min on ice and centrifuged at 12,000 x g for 3 min at 4TC. Immunoprecipitates were washed sequentially with RIPA plus 0.1% SDS, with RIPA without SDS, with 500 mM LiCl/0.1% Nonidet P-40/50 mM Tris HCI, pH 7.5 (twice), and with PBS. They were then resuspended in 100 A1 of 20 mM Hepes, pH 7.0/1-5 mM MnCI2/5 mM GDP/10% (vol/ vol) glycerol/3-8 pmol of purified Ga. Reactions were started by addition of 600 A.M MgCl2/60 p.M [y-32P]ATP (1000-2000 cpm/pmol) and were conducted at 30TC. For determinations of phosphorylation stoichiometry, reactions were terminated by addition of SDS/PAGE sample buffer, and stoichiometry was assessed by excision of gel bands after PAGE and was quantitated by Cerenkov counting. Before incubation in the kinase assay mixture, 30-70o of the Ga
The oncogenes of many retroviruses encode tyrosine-specific protein kinases that are presumed to phosphorylate cellular proteins involved in the control of cell growth and differentiation. Many of these kinases belong to the src family of kinases by virtue of their structural homology with the oncogene product (pp60v-src) encoded by Rous sarcoma virus. Members of this family are cytoplasmic proteins that are myristoylated, associated with cellular membranes, and may mediate the actions of diverse cell-surface receptors (1-3). A number of lines of experimental evidence point to the existence of functional interactions between activated members of the pp6OC-src family and elements in the signal transduction pathways mediated by guanine nucleotide-binding regulatory proteins (G proteins). For example,. several growth factors and lymphocyte cell-surface antigens whose actions have been proposed to be mediated by members of the src subfamily (1-9) also modulate G-protein functioning (10, 11) and adenylyl cyclase activity (12, 13). In addition, two enzymes involved in inositol phospholipid metabolism, which is highly regulated by G proteins, may be targets of phosphorylation by activated src gene products (1, 14). Furthermore, overexpression .of avian pp6OC-src in fibroblasts results in enhanced intracellular cAMP levels and adenylyl cyclase activity in response to B-adrenergic hormones, which act via the stimulatory G protein (GJ) (15, 16). The molecular nature of these putative interactions has remained obscure. However, the observation that pp60c-src is a major contaminant in some highly purified G-protein preparations (17) suggests the possibility of a direct association between pp6Oc-src and G proteins. In this study, we examined
Abbreviations: G protein, guanine nucleotide-binding regulatory protein; G., stimulatory G protein; Gi, inhibitory G protein; Gt, retinal G-protein transducin; P2AR, 82-adrenergic receptor(s); Iso, (-)-isoproterenol; Alp, (+)-alprenolol; Ga, G-protein a subunit(s); Ga,,, a subunit (short form) of G,; GaL, a subunit (long form) of G.;
GTP[yS], guanosine 5'-[y-thio]triphosphate. tPresent address: Office of Health, U.S. Agency for International
The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.
Development, Washington, DC 20523.
ITo whom reprint requests should be addressed. 5720
Biochemistry: Hausdorff et al.
Proc. Natl. Acad. Sci. USA 89 (1992)
protein (by mass) was active-i.e., bound guanosine 5'-[ythio]triphosphate (GTP[yS]). Since inactive Ga is not a substrate for phosphorylation (see Fig. 2), all stoichiometries were calculated relative to the active protein initially present in the kinase mixture. For functional studies, nonradiolabeled phosphorylated Gas was prepared and reactions were terminated by addition of a 2x vol of ice-cold 10 mM Tris, pH 8/5 mM EDTA. To produce unphosphorylated Gas, Gas was incubated in one of three mock kinase mixtures; either AppNHp was substituted for ATP, or the GD11 antibody had not been preincubated with cell lysate, or the GD11 immune complex was replaced by the immune complex of the unrelated antibody SP20 (unable to precipitate pp60C-src; ref. 27). Pansorbin was removed by centrifugation at 12,000 x g for 3 min; under these conditions =70% of Gas remained in the supernatant. Equivalent amounts of phosphorylated or unphosphorylated Ga. (determined by the ultimate extent of GTP[y-35S] binding) were subsequently examined in functional assays. GTPase Assay. For assays of f32AR-catalyzed activity, vesicles containing 200 fmol of reconstituted P2AR were incubated with 200 fmol of recombinant Gas and 1 pmol of By subunits. For assays of rhodopsin-catalyzed activity, 200 fmol of bovine rhodopsin was preincubated with 0.25-1 pmol of retinal G-protein transducin (Gj) in the same buffer in the dark. GTPase assays were conducted at 30°C as described (18, 28). Stimulation by the 3-adrenergic agonist (-)isoproterenol (Iso) was generally 2- to 3-fold, and stimulation by light was 10- to 12-fold over basal in their respective systems. To ensure the absence ofactive rhodopsin, the basal GTPase activity of Gt was determined in the light with denatured rhodopsin (microwave on high for 3 min). GTP[yS] Binding. Fifty microliters of vesicles containing 600 fmol of reconstituted P2AR was incubated with 300 fmol of the short form of bovine Gas (Gass) and 300 or 1500 fmol of 8y subunits in 20 mM Hepes, pH 8.0/100 mM NaCl/0.1 mM ascorbic acid/1-10 mM MgCl2/0.1 ,uM GTP[
Tyrosine phosphorylation of G protein a subunits by pp6Oc-src {guanosine 5'-[l-thio]triphosphate binding/GTPase activity/stimulatory guanine nucleotide-binding regulatory protein a subunit (H2la)}
WILLIAM P. HAUSDORFF*t, JULIE A. PITCHER*, DEIRDRE K. LUTTRELLt, MAURINE E. LINDER§, HITOSHI KUROSE*, SARAH J. PARSONS¶, MARC G. CARON*, AND ROBERT J. LEFKOWITZ*II *Howard Hughes Medical Institute, Departments of Medicine, Biochemistry and Cell Biology, Box 3821, Duke University Medical Center, Durham, NC 27710;
*Glaxo Research Laboratories, Research Triangle Park, NC 27709; §Department of Pharmacology, Southwestern Graduate School, University of Texas Health Science Center, Dallas, TX 75235; and IDepartment of Microbiology, Cancer Research Center and Molecular Biology Institute, University of Virginia Health Sciences Center, Charlottesville, VA 22908
Contributed by Robert J. Leflcowitz, March 19, 1992
the abilities of purified G proteins to serve as substrates for phosphorylation by pp60C-src.
A number of lines of evidence suggest that ABSTRACT cross-talk exists between the cellular signal transduction pathways involving tyrosine phosphorylation catalyzed by members of the pp6OC-srC kinase family and those mediated by guanine nucleotide regulatory proteins (G proteins). In this study, we explore the possibility that direct interactions between pp60' and G proteins may occur with functional consequences. Preparations of pp6cc isolated by immunoprecipitation phosphorylate on tyrosine residues the purified G-protein a subunits (Ga) of several heterotrimeric G proteins. Phosphorylation is highly dependent on G-protein conformation, and Ga(GDP) uncomplexed by Pr subunits appears to be the preferred substrate. In functional studies, phosphorylation of stimulatory Ga (Ga.) modestly increases the rate of binding of guanosine 5'-[y-[35S]thiojtriphosphate to G. as well as the receptor-stimulated steady-state rate of GTP hydrolysis by Gs. Heterotrimeric G proteins may represent a previously unappreciated class of potential substrates for ppW.SrI.
EXPERIMENTAL PROCEDURES Purification and Reconstitution of PrAdrenergic Receptors (fi2AR) and G Proteins. Purification of /32AR from hamster lung (18), rhodopsin, and transducin from bovine retina (19), pfy subunits from bovine brain (20, 21), and G-protein a subunits (Ga) expressed in Escherichia coli (22-24) were as described. Receptors were reconstituted in phosphatidyicholine vesicles by using octyl glucoside (25). pp6(' Immunoprecipitations and in Vitro Kinase Assays. C3HT1O2 (C5) or Syrian hamster (4A) cells overexpressing wild-type avian pp60c-src at levels >25-fold over nontransfected cells (15) were pretreated with 1.6 ,uM phorbol 12myristate 13-acetate for 15 min, the cells were lysed (1.5 ml per 75-cm2 flask) in RIPA [150 mM NaCI/SO mM Tris'HCI, pH 7.5/1% Nonidet P-40/0.25% deoxycholate/peptide inhibitors (18)/1 ,M microcystin (Calbiochem)] plus 0.1% SDS, and the insoluble fraction was removed by 10 min of centrifugation at 12,000 x g. Phorbol pretreatment (26), although not required, enhanced the phosphorylating activity of pp6OC-src (data not shown). Immunoprecipitations were performed by using the rodent/avian pp60c-src-specific monoclonal antibody GD11 (27). Specifically, 20 1.l of a 1:10 dilution of GD11 in Dulbecco's phosphate-buffered saline (PBS) was incubated per 1.5 ml of solubilized extract on ice for 1 hr. At this point, 25 ILI of Pansorbin (Calbiochem) prewashed in bovine serum albumin (1.5 mg/ml) was added, and the mixture was incubated for 30 min on ice and centrifuged at 12,000 x g for 3 min at 4TC. Immunoprecipitates were washed sequentially with RIPA plus 0.1% SDS, with RIPA without SDS, with 500 mM LiCl/0.1% Nonidet P-40/50 mM Tris HCI, pH 7.5 (twice), and with PBS. They were then resuspended in 100 A1 of 20 mM Hepes, pH 7.0/1-5 mM MnCI2/5 mM GDP/10% (vol/ vol) glycerol/3-8 pmol of purified Ga. Reactions were started by addition of 600 A.M MgCl2/60 p.M [y-32P]ATP (1000-2000 cpm/pmol) and were conducted at 30TC. For determinations of phosphorylation stoichiometry, reactions were terminated by addition of SDS/PAGE sample buffer, and stoichiometry was assessed by excision of gel bands after PAGE and was quantitated by Cerenkov counting. Before incubation in the kinase assay mixture, 30-70o of the Ga
The oncogenes of many retroviruses encode tyrosine-specific protein kinases that are presumed to phosphorylate cellular proteins involved in the control of cell growth and differentiation. Many of these kinases belong to the src family of kinases by virtue of their structural homology with the oncogene product (pp60v-src) encoded by Rous sarcoma virus. Members of this family are cytoplasmic proteins that are myristoylated, associated with cellular membranes, and may mediate the actions of diverse cell-surface receptors (1-3). A number of lines of experimental evidence point to the existence of functional interactions between activated members of the pp6OC-src family and elements in the signal transduction pathways mediated by guanine nucleotide-binding regulatory proteins (G proteins). For example,. several growth factors and lymphocyte cell-surface antigens whose actions have been proposed to be mediated by members of the src subfamily (1-9) also modulate G-protein functioning (10, 11) and adenylyl cyclase activity (12, 13). In addition, two enzymes involved in inositol phospholipid metabolism, which is highly regulated by G proteins, may be targets of phosphorylation by activated src gene products (1, 14). Furthermore, overexpression .of avian pp6OC-src in fibroblasts results in enhanced intracellular cAMP levels and adenylyl cyclase activity in response to B-adrenergic hormones, which act via the stimulatory G protein (GJ) (15, 16). The molecular nature of these putative interactions has remained obscure. However, the observation that pp60c-src is a major contaminant in some highly purified G-protein preparations (17) suggests the possibility of a direct association between pp6Oc-src and G proteins. In this study, we examined
Abbreviations: G protein, guanine nucleotide-binding regulatory protein; G., stimulatory G protein; Gi, inhibitory G protein; Gt, retinal G-protein transducin; P2AR, 82-adrenergic receptor(s); Iso, (-)-isoproterenol; Alp, (+)-alprenolol; Ga, G-protein a subunit(s); Ga,,, a subunit (short form) of G,; GaL, a subunit (long form) of G.;
GTP[yS], guanosine 5'-[y-thio]triphosphate. tPresent address: Office of Health, U.S. Agency for International
The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.
Development, Washington, DC 20523.
ITo whom reprint requests should be addressed. 5720
Biochemistry: Hausdorff et al.
Proc. Natl. Acad. Sci. USA 89 (1992)
protein (by mass) was active-i.e., bound guanosine 5'-[ythio]triphosphate (GTP[yS]). Since inactive Ga is not a substrate for phosphorylation (see Fig. 2), all stoichiometries were calculated relative to the active protein initially present in the kinase mixture. For functional studies, nonradiolabeled phosphorylated Gas was prepared and reactions were terminated by addition of a 2x vol of ice-cold 10 mM Tris, pH 8/5 mM EDTA. To produce unphosphorylated Gas, Gas was incubated in one of three mock kinase mixtures; either AppNHp was substituted for ATP, or the GD11 antibody had not been preincubated with cell lysate, or the GD11 immune complex was replaced by the immune complex of the unrelated antibody SP20 (unable to precipitate pp60C-src; ref. 27). Pansorbin was removed by centrifugation at 12,000 x g for 3 min; under these conditions =70% of Gas remained in the supernatant. Equivalent amounts of phosphorylated or unphosphorylated Ga. (determined by the ultimate extent of GTP[y-35S] binding) were subsequently examined in functional assays. GTPase Assay. For assays of f32AR-catalyzed activity, vesicles containing 200 fmol of reconstituted P2AR were incubated with 200 fmol of recombinant Gas and 1 pmol of By subunits. For assays of rhodopsin-catalyzed activity, 200 fmol of bovine rhodopsin was preincubated with 0.25-1 pmol of retinal G-protein transducin (Gj) in the same buffer in the dark. GTPase assays were conducted at 30°C as described (18, 28). Stimulation by the 3-adrenergic agonist (-)isoproterenol (Iso) was generally 2- to 3-fold, and stimulation by light was 10- to 12-fold over basal in their respective systems. To ensure the absence ofactive rhodopsin, the basal GTPase activity of Gt was determined in the light with denatured rhodopsin (microwave on high for 3 min). GTP[yS] Binding. Fifty microliters of vesicles containing 600 fmol of reconstituted P2AR was incubated with 300 fmol of the short form of bovine Gas (Gass) and 300 or 1500 fmol of 8y subunits in 20 mM Hepes, pH 8.0/100 mM NaCl/0.1 mM ascorbic acid/1-10 mM MgCl2/0.1 ,uM GTP[
