Phosphorylation-regulated Inhibition of the Gz GTPase-activating Protein Activity of RGS Proteins by Synapsin I
2003; Elsevier BV; Volume: 278; Issue: 52 Linguagem: Inglês
10.1074/jbc.m309626200
ISSN1083-351X
AutoresYaping Tu, Surendra Kumar Nayak, Jimmy Woodson, Elliott M. Ross,
Tópico(s)Lipid Membrane Structure and Behavior
ResumoSynapsins are neuronal proteins that bind and cluster synaptic vesicles in the presynaptic space, presumably by anchoring to actin filaments, but specific regulatory functions of the synapsins are unknown. We found that a sub-population of brain synapsin Ia, a splice variant of one of three synapsin isoforms, inhibits the GTPase-activating protein (GAP) activity of several RGS proteins. Inhibition is highly selective for Gαz, a member of the Gi family that is found in neurons, platelets, adrenal chromaffin cells, and a few other neurosecretory cells. Gz has been indirectly implicated in the regulation of secretion. Synapsin Ia constitutes a major fraction of the total GAP-inhibitory activity in brain, and its inhibitory activity is absent from the brains of synapsin I-/-/II-/- mice. Inhibition depends on the cationic D/E domain of synapsin. Phosphorylation of synapsin Ia at serine 9 by either cyclic AMP-dependent protein kinase or p21-activated protein kinase (PAK1) attenuates its potency as a GAP inhibitor more than 7-fold. Synapsin can thus act as a phosphorylation-modulated mediator of feedback regulation of Gz signaling by the synaptic machinery. Synapsins are neuronal proteins that bind and cluster synaptic vesicles in the presynaptic space, presumably by anchoring to actin filaments, but specific regulatory functions of the synapsins are unknown. We found that a sub-population of brain synapsin Ia, a splice variant of one of three synapsin isoforms, inhibits the GTPase-activating protein (GAP) activity of several RGS proteins. Inhibition is highly selective for Gαz, a member of the Gi family that is found in neurons, platelets, adrenal chromaffin cells, and a few other neurosecretory cells. Gz has been indirectly implicated in the regulation of secretion. Synapsin Ia constitutes a major fraction of the total GAP-inhibitory activity in brain, and its inhibitory activity is absent from the brains of synapsin I-/-/II-/- mice. Inhibition depends on the cationic D/E domain of synapsin. Phosphorylation of synapsin Ia at serine 9 by either cyclic AMP-dependent protein kinase or p21-activated protein kinase (PAK1) attenuates its potency as a GAP inhibitor more than 7-fold. Synapsin can thus act as a phosphorylation-modulated mediator of feedback regulation of Gz signaling by the synaptic machinery. Gz is a member of the Gi family of heterotrimeric G proteins that is expressed primarily in neurons, platelets, and adrenal chromaffin cells (1Fields T.A. Casey P.J. Biochem. J. 1997; 321: 561-571Crossref PubMed Scopus (247) Google Scholar, 2Ho M.K.C. Wong Y.H. Oncogene. 2001; 20: 1615-1625Crossref PubMed Scopus (63) Google Scholar). Consistent with its expression in committed secretory cells, Gz is apparently involved in signaling from cell surface receptors to the secretory machinery. It is required for normal platelet degranulation in response to α-adrenergic stimulation (3Yang J. Wu J. Kowalska M.A. Dalvi A. Prevost N. O'Brien P.J. Manning D. Poncz M. Lucki I. Blendy J.A. Brass L.F. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 9984-9989Crossref PubMed Scopus (164) Google Scholar), contributes significantly to adrenocorticotropic hormone secretion in response to serotonin (4Serres F. Li Q. Garcia F. Raap D.K. Battaglia G. Muma N.A. Van de Kar L.D. J. Neurosci. 2000; 20: 3095-3103Crossref PubMed Google Scholar), and the phenotype of Gaz knock-out mice suggests that it performs similar functions in multiple central nervous system neurons (5Hendry I.A. Kelleher K.L. Bartlett S.E. Leck K.J. Reynolds A.J. Heydon K. Mellick A. Megirian D. Matthaei K.I. Brain Res. 2000; 870: 10-19Crossref PubMed Scopus (62) Google Scholar). Although Gz can regulate the usual group of Gi effector proteins, it may also have unique targets of its own (2Ho M.K.C. Wong Y.H. Oncogene. 2001; 20: 1615-1625Crossref PubMed Scopus (63) Google Scholar, 6Meng J. Casey P.J. J. Biol. Chem. 2002; 277: 43417-43424Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar, 7Ammer H. Christ T.E. J. Neurochem. 2002; 83: 818-827Crossref PubMed Scopus (25) Google Scholar).Gz is unusual in that it deactivates (hydrolyzes bound GTP) very slowly (t½ ∼ 5 min at physiological temperature) (8Casey P.J. Fong H.K.W. Simon M.I. Gilman A.G. J. Biol. Chem. 1990; 265: 2383-2390Abstract Full Text PDF PubMed Google Scholar). To carry out cellular signaling with reasonable response kinetics, Gz relies on GTPase-activating proteins (GAPs) 1The abbreviations used are: GAPGTPase-activating proteinCaMcalmodulinPAKp21-activated protein kinasePKAprotein kinase AGSTglutathione S-transferasePMSFphenylmethylsulfonyl fluorideRecrecombinant synapsin IaNatsynapsin Ia purified from brainPP2Aprotein phosphatase 2aMOPS4-morpholinepropanesulfonic acidMES4-morpholineethanesulfonic acid.1The abbreviations used are: GAPGTPase-activating proteinCaMcalmodulinPAKp21-activated protein kinasePKAprotein kinase AGSTglutathione S-transferasePMSFphenylmethylsulfonyl fluorideRecrecombinant synapsin IaNatsynapsin Ia purified from brainPP2Aprotein phosphatase 2aMOPS4-morpholinepropanesulfonic acidMES4-morpholineethanesulfonic acid. of the RGS protein family. Members of the RGSZ subfamily can accelerate hydrolysis of Gαz-bound GTP over 600-fold (9Wang J. Ducret A. Tu Y. Kozasa T. Aebersold R. Ross E.M. J. Biol. Chem. 1998; 273: 26014-26025Abstract Full Text Full Text PDF PubMed Scopus (134) Google Scholar), and other RGS proteins probably stimulate to similar extents (10Tu Y. Woodson J. Ross E.M. J. Biol. Chem. 2001; 276: 20160-20166Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar). Such acceleration both allows Gz to respond to removal of receptor agonists on a reasonable physiological time scale and provides a way of inhibiting Gz-mediated signaling by decreasing its activation lifetime (11Ross E.M. Wilkie T.M. Annu. Rev. Biochem. 2000; 69: 795-827Crossref PubMed Scopus (918) Google Scholar). Relatively little is known about how GAP activity is regulated in cells. RGS proteins can be inhibited by phosphorylation or palmitoylation, by binding to phosphatidylinositol 4,5-bisphosphate and by phosphorylation or palmitoylation of their Gα substrates. However, the quantitative importance and physiological control of these processes are not well understood (see Refs. 11Ross E.M. Wilkie T.M. Annu. Rev. Biochem. 2000; 69: 795-827Crossref PubMed Scopus (918) Google Scholar, 12Hollinger S. Hepler J.R. Pharmacol. Rev. 2002; 54: 527-559Crossref PubMed Scopus (594) Google Scholar, 13De Vries L. Zheng B. Fischer T. Elenko E. Farquhar M.G. Annu. Rev. Pharmacol. Toxicol. 2000; 40: 235-271Crossref PubMed Scopus (504) Google Scholar for reviews).During the purification of the Gz-selective GAP RGSZ1 from brain, we noted that membrane-bound Gz GAP activity increased when a crude cerebral cortical membrane fraction was washed with high ionic strength buffer (14Wang J. Tu Y. Woodson J. Song X. Ross E.M. J. Biol. Chem. 1997; 272: 5732-5740Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar). We show here that the high salt extract contains protein inhibitors of Gz GAP activity as well as inhibitors of other G protein GAPs. By purifying the inhibitory factors, we found that synapsin Ia is a major and selective inhibitor of Gz GAP activity in brain.Synapsins are abundant neuronal phosphoproteins that bind both actin filaments and synaptic vesicles, and they are responsible for ordered vesicle packing in the reserve pool of the synaptic terminal (see Refs. 15Pieribone V.A. Shupliakov O. Brodin L. Hilfiker-Rothenfluh S. Czernik A.J. Greengard P. Nature. 1995; 375: 493-497Crossref PubMed Scopus (418) Google Scholar, 16Hilfiker S. Pieribone V.A. Czernik A.J. Kao H.-T. Augustine G.J. Greengard P. Phil. Trans. R. Soc. Lond. B. 1999; 354: 269-279Crossref PubMed Scopus (434) Google Scholar, 17Südhof T.C. Czernik A.J. Kao H.-T. Takei K. Johnston P.A. Horiuchi A. Kanazir S.D. Wagner M.A. Perin M.S. DeCamilli P. Greengard P. Science. 1989; 245: 1474-1480Crossref PubMed Scopus (415) Google Scholar for reviews). Three synapsin genes exist in mammals, and each is expressed as at least two splice products, but unique roles for the individual synapsin proteins have been hard to ascertain. Disruption of the genes for either or both major synapsin proteins I and II causes disorganization of synaptic vesicles and leads to a decrease in vesicle number. Consequent physiological effects of synapsin gene disruption include deficient vesicle recycling, altered adaptive response to paired or repetitive stimulation and a tendency to seizures (18Ryan T.A. Li L. Chin L.S. Greengard P. Smith S.J. J. Cell Biol. 1996; 134: 1219-1227Crossref PubMed Scopus (144) Google Scholar, 19Rosahl T.W. Geppert M. Spillane D. Herz J. Hammer R.E. Malenka R.C. Südhof T.C. Cell. 1993; 75: 661-670Abstract Full Text PDF PubMed Scopus (280) Google Scholar, 20Rosahl T.W. Spillane D. Missler M. Herz J. Selig D.K. Wolff J.R. Hammer R.E. Malenka R.C. Südhof T.C. Nature. 1995; 375: 488-493Crossref PubMed Scopus (615) Google Scholar, 21Li L. Chin L.-S. Shupliakov O. Brodin L. Sihra T.S. Hvalby Ø. Jensen V. Zheng D. McNamara J.O. Greengard P. Andersen P. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 9235-9239Crossref PubMed Scopus (294) Google Scholar). Long term and developmental anomalies caused by synapsin gene disruption may result from loss of synaptic vesicle organization but may also reflect novel regulatory roles of the synapsins (22Ferreira A. Rapoport M. Cell. Mol. Life Sci. 2002; 59: 589-595Crossref PubMed Scopus (133) Google Scholar).Diverse protein kinases phosphorylate synapsins at multiple sites in response to a wide variety of extracellular inputs (16Hilfiker S. Pieribone V.A. Czernik A.J. Kao H.-T. Augustine G.J. Greengard P. Phil. Trans. R. Soc. Lond. B. 1999; 354: 269-279Crossref PubMed Scopus (434) Google Scholar). Both CaM kinase II and p21-regulated kinase (PAK) catalyze phosphorylation of two serine residues, and phosphorylation at these sites results in the inability of synapsin to bind to actin (23Czernik A.J. Pang D.T. Greengard P. Proc. Natl. Acad. Sci. U. S. A. 1987; 84: 7518-7522Crossref PubMed Scopus (136) Google Scholar, 24Sakurada K. Kato H. Nagumo H. Hiraoka H. Furuya K. Ikuhara T. Yamakita Y. Fukunaga K. Miyamoto E. Matsumura F. Matsuo Y.-I. Naito Y. Sasaki Y. J. Biol. Chem. 2002; 277: 45473-45479Abstract Full Text Full Text PDF PubMed Scopus (27) Google Scholar, 25Bähler M. Greengard P. Nature. 1987; 326: 704-707Crossref PubMed Scopus (342) Google Scholar). One of these sites, serine 9, is also phosphorylated by cyclic AMP-dependent protein kinase (PKA) (23Czernik A.J. Pang D.T. Greengard P. Proc. Natl. Acad. Sci. U. S. A. 1987; 84: 7518-7522Crossref PubMed Scopus (136) Google Scholar). The complexity of synapsin phosphorylation suggests that it may mediate signaling events in addition to actin binding, and some of these events may account for yet unexplained effects of synapsin gene disruption. As an initial step toward understanding the functional interaction between synapsin I, Gz, and RGS proteins in regulating synaptic activity, we characterize here the ability of synapsin Ia to inhibit Gz GAPs and the control of inhibition by synapsin phosphorylation.EXPERIMENTAL PROCEDURESMaterials—RGS proteins, Gαi1 and Gαo were expressed in Escherichia coli and purified as described previously (9Wang J. Ducret A. Tu Y. Kozasa T. Aebersold R. Ross E.M. J. Biol. Chem. 1998; 273: 26014-26025Abstract Full Text Full Text PDF PubMed Scopus (134) Google Scholar, 10Tu Y. Woodson J. Ross E.M. J. Biol. Chem. 2001; 276: 20160-20166Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar, 14Wang J. Tu Y. Woodson J. Song X. Ross E.M. J. Biol. Chem. 1997; 272: 5732-5740Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar). Mixed synapsin isoforms were purified from bovine brain according to the method of Bähler and Greengard (25Bähler M. Greengard P. Nature. 1987; 326: 704-707Crossref PubMed Scopus (342) Google Scholar). Protein phosphatase 2A was a gift from Marc Mumby (University of Texas Southwestern). GST-PAK1(both full-length and residues 232-544) (26Wang J. Frost J.A. Cobb M.H. Ross E.M. J. Biol. Chem. 1999; 274: 31641-31647Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar) was a gift from Melanie Cobb (University of Texas Southwestern). The catalytic subunit of PKA was purchased from Sigma. Gαz was purified from Sf9 cells as described previously (14Wang J. Tu Y. Woodson J. Song X. Ross E.M. J. Biol. Chem. 1997; 272: 5732-5740Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar)C-terminally His6-tagged rat synapsin Ia and a truncated form that contained only the A, B, and C domains (synABC; residues 2-419) were expressed in Sf9 cells using baculovirus vectors that were a gift from Johann Deisenhofer (University of Texas Southwestern) (27Esser L. Wang C.-R. Hosaka M. Smagula S.C. Südhof T.C. Deisenhofer J. EMBO J. 1998; 17: 977-984Crossref PubMed Scopus (108) Google Scholar). cDNAs that encode His6-tagged synABCD (residues 1-657) and synABCE (residues 419-659 deleted) were prepared by polymerase chain reaction and transferred to baculovirus vectors as described (9Wang J. Ducret A. Tu Y. Kozasa T. Aebersold R. Ross E.M. J. Biol. Chem. 1998; 273: 26014-26025Abstract Full Text Full Text PDF PubMed Scopus (134) Google Scholar). Sf9 cell pellets were homogenized at 4 °C in lysis buffer (25 mm Tris-Cl (pH 8), 1 mm 2-mercaptoethanol, 1 mm MgCl2, 0.02 mg/ml DNase I, and protease inhibitors (0.4 mm phenylmethylsulfonyl fluoride, 0.01 mg/ml leupeptin, 0.01 mg/ml aprotinin, 0.002 mg/ml pepstatin)). After 10 min, 250 mm NaCl and 1% Triton X-100 were added and the cell lysates were stirred for 30 min. After centrifugation (100,000 × g, 30 min), the supernatant was diluted 4-fold with buffer A (20 mm NaHepes (pH 8), 10 mm 2-mercaptoethanol, 10% glycerol, and protease inhibitors) and applied to nickel-nitrilotriacetic acid-agarose that was equilibrated with buffer A plus 0.2% Triton X-100. The resin was washed with 200 ml of buffer A plus 1 m NaCl, 0.1% Triton X-100, and 7.5 mm imidazole and then with 50 ml of buffer A plus 100 mm NaCl, and 0.05% Triton X-100. Synapsin was eluted with buffer A plus 100 mm NaCl, 0.05% Triton X-100, and 150 mm imidazole. Purified synapsin was exchanged into storage buffer (50 mm NaHepes (pH 8), 0.05% Triton X-100, 5 mm dithiothreitol, 10% glycerol), flash-frozen in liquid nitrogen, and stored at -80 °C. Protein was estimated according to the method of Schaffner and Weismann (28Schaffner W. Weissmann C. Anal. Biochem. 1973; 56: 502-514Crossref PubMed Scopus (1946) Google Scholar) throughout.Purification of Bovine Synapsin Ia according to Inhibition of GzGAP Activity—All procedures were performed at 0-4 °C. Cerebral cortices from two to three steers were washed in 0.9% NaCl and homogenized in a Waring blender in 1 liter of TEP buffer (25 mm Tris-HCl (pH7.5), 1 mm MgCl2, 1 mm EDTA, 0.3 mm PMSF, 1 μm pepstatin, 1 μm leupeptin, 2 μg/ml aprotinin). The homogenate was passed through cheesecloth and centrifuged at 30,000 × g for 30 min. The pellet was homogenized in TEP buffer and centrifuged again. The pellet was suspended in 300 ml of TEP buffer plus 1 mm dithiothreitol and 300 mm NaCl, homogenized by five strokes with a Dounce homogenizer and stirred at 4 °C for 30 min. The homogenate was centrifuged at 100,000 × g for 45 min. The clear supernatant was diluted 6-fold with Buffer I (20 mm Hepes, pH 7.0, 1 mm EDTA, 1 mm dithiothreitol, 0.3 mm PMSF, 0.05% Lubrol PX) and passed through a 200-ml column of DEAE-Sephacel that was equilibrated with Buffer I. The flowthrough was incubated with 30 ml of CM-Sepharose 6B for at least 1 h. The resin was washed in a column with 200 ml of Buffer I and 50 ml of Buffer I plus 30 mm NaCl. Protein was eluted with a 150-ml gradient of 30-300 mm NaCl in Buffer I. A broad peak of GAP-inhibitory activity was eluted between 150 and 250 mm NaCl. The pooled peak fractions (∼40 ml) were mixed with 4 m NaCl and 2 m (NH4)2SO4 to yield final concentrations of 1 m NaCl and 0.4 m (NH4)2SO4. The mixture was centrifuged, and the supernatant was mixed with 5 ml of phenyl-Sepharose for 45 min. The resin was washed in a column with Buffer I plus 1 m NaCl and 0.4 m (NH4)2SO4 and GAP-inhibitory activity was eluted with buffer I. Pooled active fractions were diluted 5-fold with Buffer II (20 mm Hepes, pH 8.0, 1 mm EDTA, 1 mm dithiothreitol, 0.05% Lubrol, 0.3 mm PMSF) and applied to a column of Mono S (1.5 ml) that had been equilibrated with Buffer II. The column was washed sequentially with Buffer II, Buffer II plus 0.05 m NaCl, and a gradient of 0.05-0.40 m NaCl in Buffer II. Active fractions were concentrated on an Amicon PM10 membrane. Table II summarizes one such preparation.Table IIPurification of GAP inhibitor from bovine brain membranes The inhibitor was purified and assayed as described under "Experimental Procedures." The last two rows refer to activity of the two major protein bands after elution from SDS polyacrylamide gels and renaturation.TotalSpecific activityTotalProteinActivityPurificationYieldmgUnits/mgUnits-fold%Extract4100.26107(1)(100)DEAE-Sephacel unadsorbed2400.40961.588CM-Sepharose481.1534.248Phenyl-Sepharose5.55.9322229Mono S0.4239.2898.6SDS (70–80 kDa)0.024451.11732.1SDS (∼20 kDa)0.014911.33502.4 Open table in a new tab GAP inhibitors were further purified by sequential SDS-polyacrylamide gel electrophoresis. Pooled inhibitor was denatured at room temperature in Tris-phosphate sample buffer (29Laemmli U.K. Nature. 1970; 227: 680-685Crossref PubMed Scopus (206048) Google Scholar) that contained 0.25% SDS and 10 mm dithiothreitol and fractionated by electrophoresis at 4 °C on 10% acrylamide gels (∼0.2 mg on a 180-× 150-× 0.75-mm gel). Proteins were extracted from gel slices by homogenization in 5-10 volumes of renaturation buffer (20 mm NaHepes (pH 7.5), 1 mm dithiothreitol, 0.1 mm EDTA, 0.2% Triton X-100) and shaking overnight at 4 °C. About 50-60% of GAP-inhibitory activity was recovered from the gel. Activity was broadly distributed between 15 and 100 kDa (data not shown; recoveries in Table II), with two peaks of activity reproducibly appearing at about 20 and 80 kDa. The 80-kDa region was cut and extracted again, and eluted protein was electrophoresed similarly on a 8% acrylamide gel. The highest inhibitory activity could be assigned to a single protein band with an apparent molecular mass of 75 kDa.To compare GAP-inhibitory activity in brains of wild type and synapsin I-/-/II-/- mice (20Rosahl T.W. Spillane D. Missler M. Herz J. Selig D.K. Wolff J.R. Hammer R.E. Malenka R.C. Südhof T.C. Nature. 1995; 375: 488-493Crossref PubMed Scopus (615) Google Scholar), inhibitor was partially purified by DEAESephacel and CM-Sepharose as described above. The active fractions were concentrated by Centricon PM10 and electrophoresed on 10% gels at 4 °C. Activity was extracted from the 70- to 100-kDa and 15- to 25-kDa regions of the gels.Protein Analysis—The ∼75-kDa protein band from the 8% acrylamide gel described above was transferred electrophoretically to a polyvinylidene difluoride membrane and visualized by Amido Black staining. Protein bands were cut in 1-mm squares, and incubated for 18 h at 37 °C in 15-20 μl of 15 mmN-ethylmorpholine, 5 mm acetic acid, 1% Zwittergent 3-16 (Calbiochem) that contained 1 μg of sequencing grade-modified trypsin (Promega). The supernatant was collected, the membrane pieces were washed with another 15 μl of digestion buffer, and both fractions were pooled for peptide analysis.Electrophoresis and Immunoblotting—Samples were denatured, reduced with dithiothreitol, and alkylated by N-ethylmaleimide. Amounts of protein applied to gels for immunoblots were normalized according to Coomassie Blue staining of the specific band. Immunoblots from 8% acrylamide gels were probed with antiserum in 10% blocking solution and developed according to instructions in the ECL kit (Amersham Biosciences). Polyclonal synapsin antibody (E028), a gift from Thomas Südhof, was raised against a peptide common to all synapsin isoforms (30Hosaka M. Südhof T.C. J. Biol. Chem. 1999; 274: 16747-16753Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar). Anit-sera specific for individual phosphorylation sites in synapsin I (31Jovanovic J.N. Sihra T.S. Nairn A.C. Hemmings Jr., H.C. Greengard P. Czernik A.J. J. Neurosci. 2001; 21: 7944-7953Crossref PubMed Google Scholar) were a gift from Angus Nairn (Rockefeller University).Immunodepletion of Synapsin—Synapsin (350 ng of purified brain fraction or 1 μg of recombinant) was incubated with antibody (5 μg of E028 or control) overnight at 4 °Cin50 μl of 20 mm Hepes (pH 7.5)/0.15 m NaCl/0.2% Lubrol. Protein A/G-agarose (Santa Cruz Biotechnology, ∼20 μg), equilibrated in the same buffer, was added, and the mixture was rotated at 4 °C for 1 h. After centrifugation, 6 μl of each supernatant was assayed for GAP inhibition. The amount of synapsin remaining in the supernatant was estimated by immunoblot.GAP Assay—GAP activity was measured according to the acceleration of hydrolysis of [γ-32P]GTP that was pre-bound to a Gα subunit as described previously (14Wang J. Tu Y. Woodson J. Song X. Ross E.M. J. Biol. Chem. 1997; 272: 5732-5740Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar, 32Wang J. Tu Y. Mukhopadhyay S. Chidiac P. Biddlecome G.H. Ross E.M. Manning D.R. G Proteins: Techniques of Analysis. CRC Press, Boca Raton, FL1999: 123-151Google Scholar). GAP activities are described either as the increment in the first-order rate constant for GTP hydrolysis (1 GAP unit = 1 min-1 increment) or in terms of Vmax and Km for the overall reaction in which the GAP is formally assumed to catalyze the conversion of Gα-GTP to Gα-GDP. To quantitate inhibitory activity, we define a unit as the amount that decreases the Gz GAP activity of RGS4 by 50%. For these assays, the concentration of RGS4 was 0.4 nm and that of Gz-[γ-32P]GTP was usually ∼2.5 nm (Km ∼ 8 nm). To measure inhibition of RGS4 with other Gα substrates (see Table III), GAP assays were performed at 4 °C with 3 nm RGS4, 8.5 nm [γ-32P]GTP-Gαz, 10 nm [γ-32P]GTP-Gαi1, and 11 nm [γ-32P]GTP-Gαo. Crude extracts of cerebral cortex were prepared by homogenizing cortex in 20 mm Hepes, 0.2% Triton X-100, 3 mm dithiothreitol, 1 mm EDTA, 0.2 mm PMSF, 5 μg/ml pepstatin, 20 μg/ml leupeptin, 20 μg/ml aprotinin, and 300 mm NaCl, sonicating four times for 30 s and centrifuging at 30,000 × g for 30 min to remove particulate material.Table IIISynapsin Ia specifically inhibits Gz GAP activity GAP assays were performed at 4 °C as described previously (10Tu Y. Woodson J. Ross E.M. J. Biol. Chem. 2001; 276: 20160-20166Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar). Activities are shown as the increment in the hydrolytic rate constant of each Gα-[γ-32P]GTP (min-1) caused by 3 nm RGS4 in the absence of inhibitor and in the presence of either 300 nm recombinant synapsin Ia or 50 μg/ml of a crude extract of bovine cerebral cortex. Concentrations of substrates and their intrinsic GTPase rate constants were, for Gαz: 8.5 nm, <0.006 min-1; Gαo: 11 nm, 0.07 min-1; Gαi1: 10 nm, 0.24 min-1. Data shown are averages from two separate experiments with each determination in duplicate.SubstrateGAP activityControlSynapsinBrain extractmin-1Gαz0.140.0120.10Gαo0.370.360.22Gαi10.560.570.37 Open table in a new tab Synapsin Phosphorylation—To phosphorylate synapsin fractions, synapsin was incubated with or without 100 nm GST-PAK1 at 30 °C for 2 h in 50 mm NaHepes (pH 8.0), 10 mm MgCl2, 1 mm dithiothreitol, and either unlabeled ATP or [γ-32P]ATP (0.5 mm). For phosphorylation with the catalytic subunit of PKA, the concentration of MgCl2 was 5 mm and 0.1 mm EGTA was included. Reactions were terminated by addition of EDTA to 10 mm. Fractional phosphorylation was determined by scintillation counting of radioactive bands cut from polyacrylamide gels. For dephosphorylation with intestinal alkaline phosphatase or PP2A, either recombinant synapsin Ia (Rec) or synapsin Ia purified from brain (Nat) was treated as follows: 3.2 μg of Rec or 1.6 μg of Nat, 10 units of calf intestinal alkaline phosphatase (or phosphatase inactivated at 100 °C for 10 min), 25 mm Tris-Cl (pH 8.4), 1 mm EDTA, 1 mm dithiothreitol, 10 μg/ml leupeptin, 30 min, 30 °C; 8 μg of Rec or 2 μg of Nat, 1.4 μg of PP2A, 20 mm MOPS (pH 7.4), 20 μg/ml bovine serum albumin, 1 mm dithiothreitol, 25 min, 30 °C. For dephosphorylation with acid phosphatase, synapsin Ia (1 μm recombinant or 0.5 μm from bovine brain) was incubated at 30 °C for 1.5-2 h with potato acid phosphatase (Sigma type VII; 0.3 unit/μg of synapsin) in 25 mm MES-OH, pH 5.5, 2 mm MgCl2, 0.1 mm dithiothreitol, 10 μg/ml leupeptin, 10 μg/ml aprotinin, 0.3 mm PMSF, 2.5 mm benzamidine. In experiments where dephosphorylation followed in vitro phosphorylation reactions, hexokinase (1 unit) and 1 mm glucose were added to destroy ATP, and, after 20 min at 30 °C, pH was adjusted to 5.0-5.5 with MES before addition of acid phosphatase.Synapsin Ia was hyperphosphorylated by exposing baculovirus-infected Sf9 cells to 1 μm okadaic acid for 2 h before harvest. The lysis buffer included 25 mm Tris-Cl (pH 8.0), 1 mm 2-mercaptoethanol, 1 mm MgCl2, 20 μg/ml DNase I, 50 mm NaF, 3 μmp-nitrophenyl phosphate, 200 μm sodium orthovanadate, 10 mm sodium pyrophosphate, 0.4 mm phenylmethylsulfonyl fluoride, 10 μg/ml aprotinin, 2 μg/ml pepstatin, 250 mm NaCl, and 1% Triton X-100. Purification following lysis was as described above.RESULTSIdentification of Synapsin Ia as a GzGAP Inhibitor in Brain—In the course of purifying the Gz-selective GAP RGSZ1 from bovine brain, we observed that total Gz GAP activity in the membrane fraction increased about 2-fold after washing with high ionic strength buffer, which suggested that washing removed some inhibitory activity (14Wang J. Tu Y. Woodson J. Song X. Ross E.M. J. Biol. Chem. 1997; 272: 5732-5740Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar). As shown in Table I, the high salt extract inhibited both the endogenous Gz GAP activity of the washed brain membranes and the Gz GAP activity of purified RGS4. The extent of inhibition increased with the amount of extract added to the GAP assay, and maximal inhibition exceeded 90% (Fig. 1A). The extract could also inhibit the GAP activities of purified recombinant RGSZ1 or RGS10, although the extent and potency of inhibition varied depending on which GAP was used (see below). The inhibitory activity was sensitive to trypsin, was not found in cytosol, and was eluted from membranes maximally by 300 mm NaCl (data not shown). The inhibitor thus behaved like a cytoskeletal or peripheral membrane protein. GAP-inhibiting activity was found in several mammalian cell types, but was not found in Escherichia coli or in Sf9 insect cells (Fig. 1B).Table IGAP-inhibiting activity in bovine brain membranes Gz GAP activity was measured in crude bovine brain membranes before and after washing with 1 m NaCl. The mixture of washed membranes plus high salt extract displayed a total activity of 31 milliunits, and the activity of the membranes after subtraction of the GAP activity in the extract is shown. The ability of the extract to inhibit the GAP activity of RGS4 is also shown, again after subtraction of the activity in the extract alone. Data are averages of duplicate determinations in two separate experiments and are expressed as milliunits of GAP activity (14Wang J. Tu Y. Woodson J. Song X. Ross E.M. J. Biol. Chem. 1997; 272: 5732-5740Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar, 32Wang J. Tu Y. Mukhopadhyay S. Chidiac P. Biddlecome G.H. Ross E.M. Manning D.R. G Proteins: Techniques of Analysis. CRC Press, Boca Raton, FL1999: 123-151Google Scholar).AdditionGAP activityMilliunitsBrain membranes (1 μg)22Salt-washed membrane (1 μg)42Extract (5 μg)6Washed membrane plus extract25RGS4 (0.4 nm)33RGS4 plus extract11 Open table in a new tab Because the GAP-inhibiting activity was most abundant in brain, we purified it from a high salt extract of bovine brain membranes. We used the inhibition of RGS4, a soluble and convenient nonspecific GAP (10Tu Y. Woodson J. Ross E.M. J. Biol. Chem. 2001; 276: 20160-20166Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar), to follow purification of inhibitory activity. As shown in Table II, inhibitory activity did not bind to anion exchangers but did bind to cation exchangers and to phenyl-Sepharose, and initial chromatographic purification was about 100-fold. However, the chromatographic behavior of the inhibitory activity suggested that it was not a single species. Partially purified inhibitor was permanently deactivated by heating in SDS plus dithiothreitol, but could be reactivated after exposure to SDS if it was not heated. This behavior allowed further separation of inhibitory factors by SDS-polyacrylamide gel electrophoresis followed by renaturation in buffer that contained Triton X-100. GAP-inhibiting activity was separated into two fractions of apparent molecular masses of 70-80 and 15-20 kDa, neither of which was homogeneous according to protein staining. The two fractions contained about equal activity, and recovery of total activity was about 50% (Table II). The low molecular weight fraction was diffuse according to both protein staining and activity (not shown) and was not studied further. The high molecular weight fraction contained discrete protein bands according to protein staining, and inhibitory activity could be assigned to a single protein band after electrophoresis on large gels under optimized conditions (Fig. 2).Fig. 2Purificat
Referência(s)