FRINK Linked Data Fragments server

Matches in SemOpenAlex for { <https://semopenalex.org/work/W1981122961> ?p ?o ?g. }

• W1981122961 endingPage "39095" @default.
• W1981122961 startingPage "39090" @default.
• W1981122961 abstract "Gα-interacting protein (GAIP) is a regulator of G protein signaling (RGS) that accelerates the rate of GTP hydrolysis by the α-subunit of the trimeric Gi3 protein. Both proteins are part of a signaling pathway that controls lysosomal-autophagic catabolism in human colon cancer HT-29 cells. Here we show that GAIP is phosphorylated by an extracellular signal-regulated (Erk1/2) MAP kinase-dependent pathway sensitive to amino acids, MEK1/2 (PD098059), and protein kinase C (GF109203X) inhibitors. An in vitro phosphorylation assay demonstrates that Erk2-dependent phosphorylation of GAIP stimulates its GTPase-activating protein activity toward the Gαi3 protein (k = 0.187 ± 0.001 s−1, EC50 = 1.12 ± 0.10 μm) when compared with unphosphorylated recombinant GAIP (k = 0.145 ± 0.003 s−1, EC50 = 3.16 ± 0.12 μm) or to GAIP phosphorylated by other Ser/Thr protein kinases (protein kinase C, casein kinase II). This stimulation and the phosphorylation of GAIP by Erk2 were abrogated when serine at position 151 in the RGS domain was substituted by an alanine residue using site-directed mutagenesis. Furthermore, the lysosomal-autophagic pathway was not stimulated in S151A-GAIP mutant-expressing cells when compared with wild-type GAIP-expressing cells. These results demonstrate that the GTPase-activating protein activity of GAIP is stimulated by Erk2 phosphorylation. They also suggested that Erk1/2 and GAIP are engaged in the signaling control of a major catabolic pathway in intestinal derived cells. Gα-interacting protein (GAIP) is a regulator of G protein signaling (RGS) that accelerates the rate of GTP hydrolysis by the α-subunit of the trimeric Gi3 protein. Both proteins are part of a signaling pathway that controls lysosomal-autophagic catabolism in human colon cancer HT-29 cells. Here we show that GAIP is phosphorylated by an extracellular signal-regulated (Erk1/2) MAP kinase-dependent pathway sensitive to amino acids, MEK1/2 (PD098059), and protein kinase C (GF109203X) inhibitors. An in vitro phosphorylation assay demonstrates that Erk2-dependent phosphorylation of GAIP stimulates its GTPase-activating protein activity toward the Gαi3 protein (k = 0.187 ± 0.001 s−1, EC50 = 1.12 ± 0.10 μm) when compared with unphosphorylated recombinant GAIP (k = 0.145 ± 0.003 s−1, EC50 = 3.16 ± 0.12 μm) or to GAIP phosphorylated by other Ser/Thr protein kinases (protein kinase C, casein kinase II). This stimulation and the phosphorylation of GAIP by Erk2 were abrogated when serine at position 151 in the RGS domain was substituted by an alanine residue using site-directed mutagenesis. Furthermore, the lysosomal-autophagic pathway was not stimulated in S151A-GAIP mutant-expressing cells when compared with wild-type GAIP-expressing cells. These results demonstrate that the GTPase-activating protein activity of GAIP is stimulated by Erk2 phosphorylation. They also suggested that Erk1/2 and GAIP are engaged in the signaling control of a major catabolic pathway in intestinal derived cells. regulators of G protein signaling bovine serum albumin 5,6-dichloro-1-β-d-ribofuranosyl benzimidazole extracellular signal-regulated kinase Gα-interacting protein GTPase-activating protein Hanks' balanced salt solution lactate dehydrogenase mitogen-activated protein polymerase chain reaction wild-type polyacrylamide gel electrophoresis cAMP-dependent protein kinase protein kinase C Regulators of G protein signaling proteins (RGS)1 are a family of proteins that control the activity of trimeric G proteins (1Dohlman H.G. Thorner J. J. Biol. Chem. 1997; 272: 3871-3874Abstract Full Text Full Text PDF PubMed Scopus (450) Google Scholar, 2Berman D.M. Gilman A.G. J. Biol. Chem. 1998; 273: 1269-1272Abstract Full Text Full Text PDF PubMed Scopus (445) Google Scholar, 3Koelle M.R. Curr. Opin. Cell Biol. 1997; 9: 143-147Crossref PubMed Scopus (175) Google Scholar, 4De Vries L. Zheng B. Fischer T. Elenko E. Farquhar M.G. Annu. Rev. Pharmacol. Toxicol. 2000; 40: 235-271Crossref PubMed Scopus (508) Google Scholar). More than 20 mammalian RGS proteins have been identified generally by reference to a conserved domain of about 115 amino acid residues known as the RGS box (5Zheng B. De Vries L. Farquhar M.G. Trends Biochem. Sci. 1999; 24: 411-414Abstract Full Text Full Text PDF PubMed Scopus (118) Google Scholar). RGS proteins are involved in modulating a variety of cell functions such as proliferation, differentiation, response to neurotransmitters, membrane trafficking, and embryonic development (4De Vries L. Zheng B. Fischer T. Elenko E. Farquhar M.G. Annu. Rev. Pharmacol. Toxicol. 2000; 40: 235-271Crossref PubMed Scopus (508) Google Scholar, 6Arshavsky V.Y. Pugh Jr., E.N. Neuron. 1998; 20: 11-14Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar). RGS act as negative regulators of several G proteins by accelerating the rate of GTP hydrolysis by the Gα proteins, thereby promoting their association with the βγ dimer (7Watson N. Linder M.E. Druey K.M. Kehrl J.H. Blumer K.J. Nature. 1996; 383: 172-175Crossref PubMed Scopus (476) Google Scholar, 8Hunt T.W. Fields T.A. Casey P.J. Peralta E.G. Nature. 1996; 383: 175-177Crossref PubMed Scopus (310) Google Scholar, 9Berman D.M. Wilkie T.M. Gilman A.G. Cell. 1996; 86: 445-452Abstract Full Text Full Text PDF PubMed Scopus (653) Google Scholar). This GTPase-activating protein (GAP) activity is engaged in the desensitization of signaling by the trimeric G proteins, but it can also speed up the transmission of signals in some cases (10Doupnik C.A. Davidson N. Lester H.A. Kofuji P. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 10461-10466Crossref PubMed Scopus (297) Google Scholar, 11Saitoh O. Kubo Y. Miyatani Y. Asano T. Nakata H. Nature. 1997; 390: 525-529Crossref PubMed Scopus (191) Google Scholar). Recently, the key role of RGS in the regulation of G protein-coupled receptor signaling has been demonstrated in vivo (12Druey K.M. Blumer K.J. Kang V.H. Kehrl J.H. Nature. 1996; 379: 742-746Crossref PubMed Scopus (407) Google Scholar, 13Chen C.K. Burns M.E. He W. Wensel T.G. Baylor D.A. Simon M.I. Nature. 2000; 403: 557-560Crossref PubMed Scopus (333) Google Scholar). However, recent evidence supports the notion that RGS proteins may be engaged in functions distinct from the regulation of G protein-activity (14De Vries L. Gist Farquhar M. Trends Cell Biol. 1999; 9: 138-144Abstract Full Text Full Text PDF PubMed Scopus (180) Google Scholar). GAIP (Gα-interacting protein) is an RGS protein, which is known to interact with Gαi3 protein (15De Vries L. Mousli M. Wurmser A. Farquhar M.G. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 11916-11920Crossref PubMed Scopus (266) Google Scholar). GAIP has been located to the Golgi apparatus membrane and newly budding Golgi vesicles (16Wylie F. Heimann K. Le T.L. Brown D. Rabnott G. Stow J.L. Am. J. Physiol. 1999; 276: C497-C506Crossref PubMed Google Scholar, 17Petiot A. Ogier-Denis E. Bauvy C. Cluzeaud F. Vandewalle A. Codogno P. Biochem. J. 1999; 337: 289-295Crossref PubMed Google Scholar) and associated with clathrin-coated vesicles (18De Vries L. Elenko E. McCaffery J.M. Fischer T. Hubler L. McQuistan T. Watson N. Farquhar M.G. Mol. Biol. Cell. 1998; 9: 1123-1134Crossref PubMed Scopus (88) Google Scholar), suggesting its potential role in vesicular transport. Recently, it has been demonstrated that posttranslational modifications of RGS can modulate their properties. Palmitoylation of conserved cysteines in RGS boxes has been shown to modify the GAP activity of RGS4 and RGS10 (19Tu Y. Popov S. Slaughter C. Ross E.M. J. Biol. Chem. 1999; 274: 38260-38267Abstract Full Text Full Text PDF PubMed Scopus (88) Google Scholar). In addition, phosphorylation has been reported to influence the stability and the membrane association of the yeast RGS Sst2 and human GAIP, respectively (20Garrison T.R. Zhang Y. Pausch M. Apanovitch D. Aebersold R. Dohlman H.G. J. Biol. Chem. 1999; 274: 36387-36391Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar, 21Fischer T. Elenko E. Wan L. Thomas G. Farquhar M.G. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 4040-4045Crossref PubMed Scopus (41) Google Scholar). However, it is not known whether or not phosphorylation could modulate the GAP activity of RGS. In the present work, we show that the phosphorylation of GAIP is in part dependent upon the activation of the Erk1/2 MAP kinases in the human intestinal HT-29 cells, and both of these events were sensitive to PKC inhibitors and amino acids. Using a panel of Ser/Thr protein kinases in an in vitro assay, we demonstrate that the phosphorylation of GAIP by a recombinant Erk2 stimulated its GAP activity toward the Gαi3 protein when compared with the activity of unphosphorylated GAIP. This stimulation was abolished when serine at position 151 in the RGS domain was replaced by an alanine residue by site-directed mutagenesis. Previously, we have shown that GAIP and the Gαi3 protein are engaged in a signaling pathway that controls the lysosomal-autophagic route in HT-29 cells (22Ogier-Denis E. Couvineau A. Maoret J.J. Houri J.J. Bauvy C. De Stefanis D. Isidoro C. Laburthe M. Codogno P. J. Biol. Chem. 1995; 270: 13-16Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar, 23Ogier-Denis E. Houri J.J. Bauvy C. Codogno P. J. Biol. Chem. 1996; 271: 28593-28600Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar, 24Ogier-Denis E. Petiot A. Bauvy C. Codogno P. J. Biol. Chem. 1997; 272: 24599-24603Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar). A hallmark of autophagy in many mammalian cells is its sensitivity to extracellular amino acid levels, which reduce the formation of autophagic vacuoles containing cytoplasmic material destined to lysosomal degradation (25Blommaart E.F. Luiken J.J. Meijer A.J. Histochem. J. 1997; 29: 365-385Crossref PubMed Scopus (216) Google Scholar, 26Mortimore G.E. Miotto G. Venerando R. Kadowaki M. Lloyd J.B. Mason R.W. Subcellular Biochemistry. Plenium Publishing Corp., New York1996: 93-136Google Scholar). The inhibition of autophagy by the addition of amino acids was correlated with the inhibition of the Erk1/2 MAP kinases and a low level of GAIP phosphorylation in HT-29 cells. By contrast to cells expressing the wild-type GAIP, those expressing the S151A mutant were unable to increase their rate of autophagy in response to amino acid deprivation and Erk1/2 activation. In conclusion, these results demonstrate that an Erk1/2 MAP kinase-dependent phosphorylation stimulates the GAP activity of GAIP and they also identify GAIP as a target for amino acid-regulated catabolism in intestinal cells. cDNA were synthesized from mRNA isolated from HT-29 cells by reverse transcription and were used to amplify by PCR full-length cDNA encoding the wild-type GAIP (24Ogier-Denis E. Petiot A. Bauvy C. Codogno P. J. Biol. Chem. 1997; 272: 24599-24603Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar). Full-length GAIP mutant S151A was generated by oligonucleotide-directed mutagenesis using the following mismatched oligonucleotide: 5′-GTATCCATCCTGGCCCCCAAGGAGGTG-3′. Identification of the mutation was obtained by direct DNA sequencing. Inserts encoding wild-type GAIP and S151A mutant were subcloned into the eucaryote expression vector pcDNA3 (Invitrogen) at the BamHI/XbaI sites. Wild-type and S151A mutant cDNAs were amplified by PCR using the following primers: forward primer, 5′-CGCAAGCTTATGCCCACCCCGCATGAG-3′; and reverse primer, 5′-CGCGGATCCCAAGGCCTCGGAGGAGGA-3′. PCR products were subcloned into pcDNA3.1/c-Myc/His-tagged vector (Invitrogen) at the HindIII/BamHI sites. Recombinant proteins were obtained as described previously (24Ogier-Denis E. Petiot A. Bauvy C. Codogno P. J. Biol. Chem. 1997; 272: 24599-24603Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar). HT-29 cells were cultured as described previously (22Ogier-Denis E. Couvineau A. Maoret J.J. Houri J.J. Bauvy C. De Stefanis D. Isidoro C. Laburthe M. Codogno P. J. Biol. Chem. 1995; 270: 13-16Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar, 23Ogier-Denis E. Houri J.J. Bauvy C. Codogno P. J. Biol. Chem. 1996; 271: 28593-28600Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar). His-tagged wild-type GAIP and S151A mutant were introduced into exponentially growing HT-29 cells by the Effectene™ kit according to the supplier's conditions (Qiagen). Cells were used 72 h after cell transfection. All experiments were carried out in nutrient-free HBSS medium supplemented with 0.1% BSA, and when appropriate amino acids or drugs were added. The final concentrations of amino acids in the mixture were multiples (4×) of the normal plasma concentrations and were as follows (in μm): asparagine, 60; isoleucine, 100; leucine, 250; lysine, 300; methionine, 40; phenylalanine, 50; proline, 100; threonine, 180; tryptophan, 70; valine, 180; alanine, 400; aspartate, 30; glutamate, 100; glutamine, 350; glycine, 300; cysteine, 60; histidine, 60; serine, 200; tyrosine, 75; ornithine, 100. Metabolic labeling of HT-29 cells with 0.5 mCi of [32P]orthophosphoric acid (Amersham Pharmacia Biotech) was carried out for 3 h in nutrient-free medium (HBSS), in complete medium, or in the absence or in the presence of amino acids. When used, protein kinase inhibitors (100 μm H-89 (Calbiochem), 5 μm GF103209X (Calbiochem), 10 μm SB203580 (Calbiochem), 50 μm PD098059 (Calbiochem), 100 μm DRB (Alexis Biochemicals)) were added in the same time. Cells were then rinsed three times with phosphate-buffered saline and then scraped into buffer A (20 mm Tris-HCl, pH 8.0, 150 mm NaCl, 5 mm EDTA, 1% Triton X-100, 0.2% BSA containing a mixture of protease and phosphatase inhibitors). 32P-Labeled cell extracts were lysed for 1 h at 4 °C. The anti-GAIP antibody (1/200) (17Petiot A. Ogier-Denis E. Bauvy C. Cluzeaud F. Vandewalle A. Codogno P. Biochem. J. 1999; 337: 289-295Crossref PubMed Google Scholar) or the anti-His antibody (1/500; Invitrogen) was bound to protein A-Sepharose under agitation at 4 °C. 32P-Labeled lysates were incubated with the anti-GAIP/protein A-Sepharose (Amersham Pharmacia Biotech) complex for 16 h at 4 °C. Sepharose beads were sequentially washed with buffer A, three times with buffer B (buffer A containing 0.1% SDS), three times with buffer C (20 mm Tris-HCl, pH 8.0, 500 mm NaCl, 0.5% Triton X-100, 0.2% BSA), and once with buffer D (50 mm Tris-HCl, pH 8.0). Immunoprecipitates were analyzed by SDS-PAGE, transferred to nitrocellulose membranes, which were exposed to Kodak X-Omat film for 16 h at −80 °C. The same blots were used to perform immunoblotting experiments with anti-GAIP and developed using the ECL kit. Phosphoamino acid analysis of GAIP was performed using one-dimensional analysis by the method of Boyle et al. (27Boyle D.M. van der Walt L.A. J. Steroid Biochem. 1988; 30: 239-244Crossref PubMed Scopus (24) Google Scholar). 32P-Labeled GAIP band was located by autoradiography, excised, and then hydrolyzed in 0.25 ml of 5.7 n HCl for 1 h at 110 °C. The sample was dried using a Speed-Vac concentrator and then resuspended in 10 μl of 10/1/189 (v/v/v) acetic acid/pyridine/water. The sample was spotted on a cellulose-coated thin layer electrophoresis plate and subjected to electrophoresis at 1100 V for 45 min with water cooling. Phosphoserine, phosphothreonine, and phosphotyrosine (1 mg/ml) were used as markers. The plate was dried, sprayed with ninhydrin to localize the phosphoamino acid standards, and then subjected to autoradiography. In all cases, kinetics were done with each kinase to determine the optimum reaction time. Recombinant PKC, PKA, casein kinase II, p38 MAP kinase, and Erk2 were used for in vitro phosphorylation assays according the supplier's instructions. Erk2, p38 MAP kinase, and PKA were used in reaction buffer (50 mm Tris-HCl, 10 mmMgCl2, 1 mm EGTA, 2 mmdithiothreitol, 0.01% Brij 35). PKC was used in reaction buffer (20 mm HEPES, pH 7.4, 10 mm MgCl2, 2 mm MnCl2, 0.1 mm dithiothreitol, 0.5 mm CaCl2, 1 μg/ml diacylglycerol, and 18.6 μg/ml phosphatidylserine), and casein kinase II was used in 20 mm Tris-HCl, pH 7.5, 50 mm KCl, 10 mm MgCl2. PKC (0.03 units; Promega), PKA (215 picomolar units; Sigma), casein kinase II (500 units; New England Biolabs), p38 MAP kinase (0.2 units; Upstate Biotechnology), or Erk2 (100 units; New England Biolabs) was incubated with 5 μg of recombinant GAIP in the appropriate reaction buffer and 0.5 mm ATP containing 1 μCi of [γ-32P]ATP (3,000 Ci/mmol, Amersham Pharmacia Biotech) at 30 °C for 1 h. Reactions were stopped by boiling in Laemmli buffer, and proteins were resolved by 13% SDS-PAGE and submitted to nitrocellulose transfer and autoradiography. The same membranes were used for immunoblotting with the GAIP antibody. The activity of each kinase was monitored using 10 μg of histones or 10 μg of MBP as control substrates (Sigma). The calculation of the stoichiometry of GAIP phosphorylation by Erk2 was based on the specific activity of the [γ-32P]ATP, the radioactivity incorporated in GAIP determined by Cerenkov counting of the GAIP band excised from a Coomassie Blue SDS-PAGE, and the amount of GAIP estimated by comparison to bovine serum albumin used as a standard. HT-29 cells were cultured for 4 h at 37 °C in HBSS with or without amino acids, 5 μmGF109203X, or 50 μm PD098059. Cells were scrapped in lysis buffer (20 mm Tris, pH 7.5, 150 mm NaCl, 0.25 m sucrose, 5 mm EDTA, 5 mmEGTA, 0.5% Triton X-100, 25 mm NaF, 5 mmNa3VO4, 5 mm β-glycerophosphate, 1 mm levamisole, 1 mm para-nitrophenylphosphate, 1.5 mmphenylmethylsulfonyl fluoride, 10 mg/ml leupeptin, 10 mg/ml pepstatin, 10 mg/ml aprotinin, 1 mm diisopropylfluorophosphate, 1 mg/ml DNase I) and after sonication, the lysate was clarified by centrifugation at 50,000 × g for 15 min at 4 °C. Hundred-μg aliquots of proteins were submitted to 9% SDS-PAGE and transferred to nitrocellulose. The membrane was incubated for 1 h in blocking buffer (25 mm Tris, pH 7.5, 150 mmNaCl, 0.05% Tween 20) containing 5% milk. Antibodies phospho-Erk1/2 (1/4000; New England Biolabs) and phospho-p38 MAP kinase (1/2000; New England Biolabs) were incubated overnight at 4 °C in blocking buffer supplemented with 1% BSA. After washing in blocking buffer, membranes were incubated with the corresponding secondary antibodies for 1 h at room temperature. Bound antibodies were detected by enhanced chemoluminescence (ECL). The same membranes were then used with anti-Erk1 (1/1000; Santa Cruz) and anti-Erk2 (1/1000; Santa Cruz) or with anti-p38 MAP kinase (1/1000; Santa Cruz) to detect the complete pool of each MAP kinase. The secondary antibody (anti-rabbit) was linked to alkaline phosphatase. Single turnover GTPase activity measurements were carried out as follows; 250 nm recombinant Gαi3 were loaded with 1 μm[γ-32P]GTP (Amersham Pharmacia Biotech) for 30 min at 30 °C in 50 mm HEPES, pH 7.5, 5 mm EDTA, 1 mm dithiothreitol, 0.1% Lubrol PX, and reactions were next chilled at 4 °C. Free nucleotides were removed by size exclusion chromatography on microspin Sephadex G50. All hydrolysis experiments were done in solution at 4 °C under single turnover conditions. Reactions were started by addition of GAIP mix containing 15 mm MgSO4, 300 μm unlabeled GTP, and 30 nm to 30 μm recombinant proteins (WT GAIP or S151A mutant) when used. Aliquots (50 μl) were removed at different times, and reactions were stopped by addition of 750 μm 5% Norit activated charcoal in 50 mmNaH2PO4, pH 3.0. Charcoal was removed by centrifugation for 15 min at 12,000 × g, and 400 μl of free phosphate-containing supernatants were counted to determine the amount of Pi released per reaction. Zero time point was obtained by adding 30 μl of [γ -32P]GTP in Norit activated charcoal. No GAP activity could be detected by using boiled GAIP. The GTPase rate constants were calculated by fitting the experimental data to an exponential function: % GTP hydrolyzed = 100(1 − e −kt ), wherek is a rate constant for GTP hydrolysis. The results are expressed as the mean ± S.E. of triplicate measurements. Measurement of the degradation of [14C]valine-labeled long-lived proteins and LDH sequestration were monitored as reported previously (23Ogier-Denis E. Houri J.J. Bauvy C. Codogno P. J. Biol. Chem. 1996; 271: 28593-28600Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar). Briefly, degradation was measured by an extrapolation of the difference between soluble radioactivity and incorporated radioactivity in trichloroacetic acid-precipitable protein. To measure the sequestration of cytosolic LDH, we isolated autophagic vacuoles and determined the included LDH activity in sedimentable materials. Metabolic labeling of HT-29 cells with [32P]orthophosphoric acid followed by immunoprecipitation using an anti-GAIP antibody and SDS-PAGE showed that GAIP was a phosphoprotein (Fig. 1 a). Acid hydrolysis of32P-labeled GAIP and thin layer electrophoresis revealed only the presence of phosphoserine residues (Fig. 1 b). After a 3-h period of nutrient starvation in HBSS, the phosphorylation of GAIP was increased 1.5 times when compared with that observed in cells kept in complete medium. Addition of a mix amino acid to HBSS reduced by 60% the phosphorylation of GAIP, suggesting that an amino acid-dependent signaling pathway is involved in the control of the phosphorylation of GAIP.Figure 1GAIP is phosphorylated on serine residues. a, upper part, HT-29 cells were radiolabeled with 0.5 mCi of [32P]orthophosphoric acid for 3 h in complete medium, HBSS, or HBSS supplemented with amino acids (see “Experimental Procedures”). GAIP was immunoprecipitated using an anti-GAIP antibody and then submitted to SDS-PAGE. Middle part, Western blot (WB) of immunoprecipitates with an anti-GAIP antibody. -Lower part, the ratio [32P]GAIP/WB was determined after scanning.b, phosphoamino acid analysis after acid hydrolysis of immunoprecipitated [32P]GAIP by thin layer electrophoresis. Arrows indicate the mobility of the standards used: PS, phosphoserine; PT, phosphothreonine; PY, phosphotyrosine. Results are representative of four independent experiments.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Phosphorylation of GAIP was investigated after metabolic labeling with [32P]orthophosphoric acid in the presence of a panel of inhibitors of Ser/Thr protein kinases (Fig. 2). The phosphorylation of GAIP was inhibited by 60% and 70% when bisindolylmaleimide (GF109203X), a broad spectrum PKC inhibitor, and PD098059, an inhibitor of the Erk1/2 pathway, were used, respectively. Inhibitors of casein kinase II (DRB) and p38 MAP kinase (SB203580) have only moderate inhibitory effects on the phosphorylation of GAIP (10% and 25%, respectively), whereas H-89, an inhibitor of PKA, has no effect. From the above results, we reasoned that the signaling pathway responsible for the phosphorylation of GAIP is controlled by Erk1/2 and/or PKC. Using antibodies directed against the activated forms of Erk1/2, we showed that amino acids and PD098059 were able to reduce the activation of Erk1/2 (Fig. 3 a). By contrast, these products have no effect on the activity of the closely related p38 MAP kinase (Fig. 3 b). These results are in agreement with the inhibition of GAIP phosphorylation stimulated by either amino acids or PD098059. It is well known that PKCs can stimulate the Erk1/2 pathway in different cell types (28Hawes B.E. van Biesen T. Koch W.J. Luttrell L.M. Lefkowitz R.J. J. Biol. Chem. 1995; 270: 17148-17153Abstract Full Text Full Text PDF PubMed Scopus (413) Google Scholar) including HT-29 cells (29Andre F. Rigot V. Remacle-Bonnet M. Luis J. Pommier G. Marvaldi J. Gastroenterology. 1999; 116: 64-77Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar). According to these data, GF109203X greatly impaired the activation of Erk1/2 (Fig. 3 a) but has only a moderate effect on p38 MAP kinase (Fig. 3 b). This result would explain the inhibitory effect of GF109203X on the phosphorylation of GAIP without totally excluding the possibility that GAIP could be a PKC substrate (see below). In order to examine whether the GAP activity of GAIP is influenced by its phosphorylation, we performed an in vitro phosphorylation assay using recombinant GAIP and a panel of Ser/Thr protein kinases (corresponding to the inhibitors used in Fig. 2). We then tested the GAP activity of each recombinant phosphorylated GAIP toward the recombinant Gαi3 protein. Incubation of GAIP with purified kinases in the presence of [γ -32P]ATP revealed labeling by PKC, casein kinase II, and Erk2 but not by PKA and the p38 MAP kinase (Fig. 4 a). In parallel control experiments were conducted on histones and myelin basic protein whose substrate characteristics with respect to the different kinases used are well known (see Fig. 4 b and “Experimetnal Procedures”). The ability of recombinant GAIP incubated with PKC, casein kinase II, or Erk2 to accelerate the GTPase activity of Gαi3 protein was determined during a single round of [γ -32P]GTP hydrolysis experiment. At each concentration of GAIP used (from 30 nm to 30 μm), the rate of GTP hydrolysis (k), corresponding to the Pi liberation as a function of time, was calculated. These values were plotted as a function of GAIP concentration (Fig. 4 c). In all assays the Gαi3 protein was present at a concentration of 250 nm. The intrinsic rate of GTP hydrolysis by Gαi3 was 0.022 ± 0.001 s−1. Addition of 30 μm GAIP resulted in acceleration of the GTPase activity by more than 6.5 times (k = 0.145 ± 0.003 s−1) with an EC50 value of 3.16 ± 0.12 μm. This stimulating effect was abolished when boiled GAIP was used. The casein kinase II- and PKC-mediated phosphorylation of GAIP did not change the rate of GTP hydrolysis when compared with that of the recombinant GAIP (k = 0.146 ± 0.002 s−1, EC50 = 3.15 ± 0.11 μm andk = 0.158 ± 0.002 s−1, EC50 = 3.09 ± 0.12 μm, respectively). However, an increase in the rate of GTP hydrolysis and a 3-fold reduction of EC50 for GAIP were observed when was phosphorylation was effectuated by Erk2 (k = 0.187 ± 0.01 s−1, EC50 = 1.12 ± 0.1 μm) as compared with the unphosphorylated recombinant GAIP. The calculation of the estimated stoichiometry of in vitro Erk2 phosphorylation of GAIP indicates an average incorporation of 0.4 mol of phosphate/mol of protein, suggesting the presence of a single Erk1/2 phosphorylation site. The primary sequence of GAIP contains two consensus sites for PKC and seven consensus sites for casein kinase II (15De Vries L. Mousli M. Wurmser A. Farquhar M.G. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 11916-11920Crossref PubMed Scopus (266) Google Scholar). However, several SP motifs that are potential phosphorylation sites for MAP kinases are also present. Among them, serine 151 in the RGS domain appears to be the most appropriate consensus site for Erk1/2 (ILSP) (30Kreegipuu A. Blom N. Brunak S. Jarv J. FEBS Lett. 1998; 430: 45-50Crossref PubMed Scopus (112) Google Scholar). This sequence is highly conserved among the GAIP subfamily (GAIP, Ret-RGS1, and RGSZ1) and absent among the five other RGS subfamilies (5Zheng B. De Vries L. Farquhar M.G. Trends Biochem. Sci. 1999; 24: 411-414Abstract Full Text Full Text PDF PubMed Scopus (118) Google Scholar) (Fig. 5 a). For this reason we performed site-directed mutagenesis on Ser151 in order to generate a recombinant S151A GAIP mutant. Both GAIP and the S151A GAIP mutant were then examined in an in vitrophosphorylation assay. The absence of phosphorylation of S151A GAIP mutant by Erk2, shown in Fig. 5 b, is not a consequence of gross modifications of the protein structure because: (i) the GAIP activity of the S151A GAIP protein is close to that of the wild-type GAIP (k = 0.119 ± 0.002 s−1, EC50 = 4.17 ± 0.09 μm versus k= 0.145 ± 0.003 s−1, EC50 = 3.16 ± 0.12 μm), (ii) PKC phosphorylates both the S151A GAIP mutant and wild-type GAIP in a similar manner (Fig. 5 b), and (iii) the heat-denatured S151A GAIP mutant is not a substrate for Erk2 (data not shown). After incubation with PKC or Erk2, the rate of GTP hydrolysis observed in the presence of S151A GAIP mutant was reduced compared with that observed with phosphorylated wild-type GAIP (Fig. 5 c). These data strongly suggest that phosphorylation of serine 151 residue by Erk2 is required for the increase of the GAP activity of GAIP. We have reported that the autophagic pathway is dependent upon the activity of the Gαi3protein and GAIP in HT-29 cells. To study the relationship between GAIP phosphorylation and macroautophagy, we have measured the autophagic sequestration of the cytosolic enzyme LDH in sedimentable material (Table I) and the degradation of long-lived [14C]valine-labeled proteins (Fig. 6) in HT-29 cells transfected with His-tagged expression vectors containing either the wild-type GAIP cDNA or the S151A GAIP mutant cDNA. According to our previous studies (24Ogier-Denis E. Petiot A. Bauvy C. Codogno P. J. Biol. Chem. 1997; 272: 24599-24603Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar), the overexpression of wild-type GAIP stimulates both autophagic parameters in HT-29 cells (Fig. 6 and Table I). These parameters were greatly reduced when amino acids or PD098059 were added (Fig. 6 and Table I). These results are in line with those shown in Figs. 1 and 2 on the effect of amino acids and PD098059 on both the phosphorylation of GAIP and Erk1/2 activation.Table IEffect of the S151A mutation of GAIP and Erk1/2 activity on autophagic sequestration of LDH in HT-29 cellsHis-tagged vectorGAIP expression 1-aThe ratio of overexpressed GAIP (WT or S151A mutant)/endogenous GAIP was calculated after scanning of a Western blot using an antibody directed against GAIP. The overexpression of His-tagged WT GAIP or His-tagged S151A GAIP was then detected by Western blot using an antibody directed against His tag.Autophagic sequestration of LDH 1-bThe values reported are the mean ± S.D. of four determinations.HBSSHBSS + amino acidsHBSS + PD098059%/hEmpty14.45 ± 0.672.15 ± 0.372.81 ± 0.42WT GAIP2.59.52 ± 1.352.75 ± 0.512.94 ± 0.58S151A GAIP2.44.25 ± 0.512.07 ± 0.352.31 ± 0.401-a The ratio of overexpressed GAIP (WT or S151A mutant)/endogenous GAIP was calculated after scanning of a Western blot using an antibody directed against GAIP. The overexpression of His-tagged WT GAIP or His-tagged S151A GAIP was then detected by Western blot using an antibody directed against His tag.1-b The values reported are the mean ± S.D. of four determinations. Open table in a new tab By contrast to the stimulatory effect of wild-type GAIP on autophagy, S151A GAIP failed to increase the rate of autophagy in the absence of amino acids under conditions where Erk1/2 MAP kinases were activated in S151 GAIP-expressing cells (data not shown) and the same level of transfected proteins were expressed (Table I). Several studies have shown that the activity of RGS is controlled at the transcriptional level (4De Vries L. Zheng B. Fischer T. Elenko E. Farquhar M.G. Annu. Rev. Pharmacol. Toxicol. 2000; 40: 235-271Crossref PubMed Scopus (508) Google Scholar). Posttranslational modifications of RGS (including palmitoylation and phosphorylation) have been reported to be involved in their cellular localization and stability (19Tu Y. Popov S. Slaughter C. Ross E.M. J. Biol. Chem. 1999; 274: 38260-38267Abstract Full Text Full Text PDF PubMed Scopus (88) Google Scholar, 20Garrison T.R. Zhang Y. Pausch M. Apanovitch D. Aebersold R. Dohlman H.G. J. Biol. Chem. 1999; 274: 36387-36391Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar, 21Fischer T. Elenko E. Wan L. Thomas G. Farquhar M.G. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 4040-4045Crossref PubMed Scopus (41) Google Scholar, 31De Vries L. Elenko E. Hubler L. Jones T.L. Farquhar M.G. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 15203-15208Crossref PubMed Scopus (156) Google Scholar). To our knowledge, our results provide the first evidence thatin vitro phosphorylation of GAIP by the Erk2 MAP kinase increases its GAP activity toward the Gαi3 protein. Substitution of Ser151 by Ala abrogates this stimulation. The GAP activity of S151A GAIP is comparable to that observed with unphosphorylated wild-type GAIP. This result strongly suggests that this GAIP mutant is still functional and able to interact with the Gαi3 protein. Ser151 is located in a loop connecting helices V and VI of GAIP in its RGS domain (32de Alba E. De Vries L. Farquhar M.G. Tjandra N. J. Mol. Biol. 1999; 291: 927-939Crossref PubMed Scopus (64) Google Scholar). Crystallographic data have shown that this loop is involved in the interaction of RGS4 and the Gαi1 protein (33Tesmer J.J. Berman D.M. Gilman A.G. Sprang S.R. Cell. 1997; 89: 251-261Abstract Full Text Full Text PDF PubMed Scopus (687) Google Scholar). A critical residue in this loop is RGS4-Asn128. A serine residue (Ser156) in GAIP occupies this position. This characteristic defines a subfamily of RGS proteins, which includes GAIP, RET1-RGS, and RGSZ1 (5Zheng B. De Vries L. Farquhar M.G. Trends Biochem. Sci. 1999; 24: 411-414Abstract Full Text Full Text PDF PubMed Scopus (118) Google Scholar). The sequence upstream of Ser156 is also conserved in this subfamily149Ileu-Leu-Ser-Pro152, whereas in other RGS subfamilies this tetrapeptide is not conserved. The determination of the soluble structure of GAIP by NMR techniques has suggested that, upon binding to Gαi, conformational rearrangements of the loop V-VI may facilitate the formation of electrostatic interactions that stabilize the RGS protein structure (32de Alba E. De Vries L. Farquhar M.G. Tjandra N. J. Mol. Biol. 1999; 291: 927-939Crossref PubMed Scopus (64) Google Scholar). Whether phosphorylation of Ser151 could directly interfere with the residues of Gαi actively involved in GTP hydrolysis or stabilize GAIP structure to optimize its GAP activity remains to be elucidated by structural studies. According to the presence of potential phosphorylation sites in its sequence, GAIP was in vitro phosphorylated by casein kinase II and PKC but not by PKA and p38 MAP kinase. These results are in good agreement with the inhibition profile of GAIP phosphorylation observedin vivo. Recently it has been reported that the casein kinase II-dependent phosphorylation of GAIP on Ser24 (outside of RGS domain) could regulate its membrane association (21Fischer T. Elenko E. Wan L. Thomas G. Farquhar M.G. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 4040-4045Crossref PubMed Scopus (41) Google Scholar). Although Ser151 is a potential phosphorylation site for PKC, it is not likely to be a substrate for this kinase in vitro because the PKC-dependent phosphorylation of the S151A GAIP mutant was similar to that of the wild-type GAIP. However, it is interesting to note that a PKC site is located in the C-terminal part of GAIP (Thr201) in the vicinity of a GIPC, a PDZ domain-containing protein, interacting site (34De Vries L. Lou X. Zhao G. Zheng B. Farquhar M.G. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 12340-12345Crossref PubMed Scopus (189) Google Scholar). These data suggest that depending on the acting Ser/Thr protein kinase, the occupancy of different phosphorylation sites regulates different functional properties of GAIP. Following up the above reported results, we have shown that GAIP is phosphorylated in a Erk1/2-dependent manner in a cellular environment. This adds a novel mammalian non-nuclear substrate for Erk1/2 MAP kinases (35Seger R. Krebs E.G. FASEB J. 1995; 9: 726-735Crossref PubMed Scopus (3207) Google Scholar). The list of non-nuclear MAP kinase substrates includes several proteins involved in the interruption of G protein signaling pathways (members of G protein-coupled receptor kinases and arrestins (20Garrison T.R. Zhang Y. Pausch M. Apanovitch D. Aebersold R. Dohlman H.G. J. Biol. Chem. 1999; 274: 36387-36391Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar, 36Lin F.T. Miller W.E. Luttrell L.M. Lefkowitz R.J. J. Biol. Chem. 1999; 274: 15971-15974Abstract Full Text Full Text PDF PubMed Scopus (121) Google Scholar). This suggests that MAP kinases can act as feedback regulators of trimeric G protein signaling. The recent demonstration of this feedback control of MAP kinases on G protein signaling via RGS in yeast emphasizes the importance of this regulation loop, which has been conserved during evolution (20Garrison T.R. Zhang Y. Pausch M. Apanovitch D. Aebersold R. Dohlman H.G. J. Biol. Chem. 1999; 274: 36387-36391Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar). Finally, our work concerns the signal control of the macroautophagic pathway. Previously, we have demonstrated that GAIP is a regulator of the Gi3 protein-dependent macroautophagic pathway in intestinal derived HT-29 cells (24Ogier-Denis E. Petiot A. Bauvy C. Codogno P. J. Biol. Chem. 1997; 272: 24599-24603Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar). A hallmark of macroautophagy in many mammalian cells including HT-29 cells is to be inhibited by amino acids (26Mortimore G.E. Miotto G. Venerando R. Kadowaki M. Lloyd J.B. Mason R.W. Subcellular Biochemistry. Plenium Publishing Corp., New York1996: 93-136Google Scholar). This inhibition has been demonstrated to be dependent upon the phosphorylation of the ribosomal S6 protein by the activation p70S6 kinase in rat hepatocytes (37Blommaart E.F. Luiken J.J. Blommaart P.J. van Woerkom G.M. Meijer A.J. J. Biol. Chem. 1995; 270: 2320-2326Abstract Full Text Full Text PDF PubMed Scopus (571) Google Scholar). This signaling pathway is also operative in HT-29 cells. 2E. Ogier-Denis, S. Pattingre, J. El Benna, and P. Codogno, unpublished results. Here we report that amino acids can control autophagy by inhibiting Erk1/2-dependent GAIP phosphorylation. This control is dependent upon the presence of Ser151 in GAIP suggesting that the Erk1/2-dependent phosphorylation of Ser151 accelerates the GTP hydrolysis by the Gαi3 protein. This would be in line with our previous data showing that the GDP-bound form of the Gαi3 protein increases the rate of autophagy (23Ogier-Denis E. Houri J.J. Bauvy C. Codogno P. J. Biol. Chem. 1996; 271: 28593-28600Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar). In HT-29 cells amino acids have a coordinated inhibitory effect on autophagy by activating the p70S6 kinase and inhibiting the Erk1/2 pathway. The mechanism by which amino acids control the Erk1/2 pathway in this cell line remains to be investigated. The control of macroautophagy by the p38 MAP kinase has been reported in rat hepatocytes in response to change in cell volume (38Haussinger D. Schliess F. Dombrowski F. Vom Dahl S. Gastroenterology. 1999; 116: 921-935Abstract Full Text Full Text PDF PubMed Scopus (93) Google Scholar). Although care must be taken in extrapolating data from different experimental models, a role for the MAP kinase family in the control of the signaling of a major catabolic pathway could be a new function for these kinases. We thank Dr. S. E. H. Moore for critical reading of the manuscript." @default.
• W1981122961 created "2016-06-24" @default.
• W1981122961 creator A5014280478 @default.
• W1981122961 creator A5051700932 @default.
• W1981122961 creator A5063537638 @default.
• W1981122961 creator A5075080951 @default.
• W1981122961 date "2000-12-01" @default.
• W1981122961 modified "2023-10-13" @default.
• W1981122961 title "Erk1/2-dependent Phosphorylation of Gα-interacting Protein Stimulates Its GTPase Accelerating Activity and Autophagy in Human Colon Cancer Cells" @default.
• W1981122961 cites W1531145879 @default.
• W1981122961 cites W1565088438 @default.
• W1981122961 cites W1968137432 @default.
• W1981122961 cites W1971584376 @default.
• W1981122961 cites W1974229569 @default.
• W1981122961 cites W1975915718 @default.
• W1981122961 cites W1989817592 @default.
• W1981122961 cites W1993240415 @default.
• W1981122961 cites W1993327137 @default.
• W1981122961 cites W1993485378 @default.
• W1981122961 cites W2009431295 @default.
• W1981122961 cites W2010861085 @default.
• W1981122961 cites W2011308475 @default.
• W1981122961 cites W2013381376 @default.
• W1981122961 cites W2020197246 @default.
• W1981122961 cites W2023009549 @default.
• W1981122961 cites W2029445497 @default.
• W1981122961 cites W2043764225 @default.
• W1981122961 cites W2050307742 @default.
• W1981122961 cites W2053217010 @default.
• W1981122961 cites W2056038118 @default.
• W1981122961 cites W2063419955 @default.
• W1981122961 cites W2071242185 @default.
• W1981122961 cites W2073223681 @default.
• W1981122961 cites W2091240754 @default.
• W1981122961 cites W2095013577 @default.
• W1981122961 cites W2114748778 @default.
• W1981122961 cites W2117918340 @default.
• W1981122961 cites W2119889843 @default.
• W1981122961 cites W2124829726 @default.
• W1981122961 cites W2140909611 @default.
• W1981122961 cites W2146386420 @default.
• W1981122961 cites W2148882351 @default.
• W1981122961 cites W2170755436 @default.
• W1981122961 cites W2174534121 @default.
• W1981122961 cites W4211012044 @default.
• W1981122961 cites W4292820908 @default.
• W1981122961 cites W4320800997 @default.
• W1981122961 doi "https://doi.org/10.1074/jbc.m006198200" @default.
• W1981122961 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/10993892" @default.
• W1981122961 hasPublicationYear "2000" @default.
• W1981122961 type Work @default.
• W1981122961 sameAs 1981122961 @default.
• W1981122961 citedByCount "277" @default.
• W1981122961 countsByYear W19811229612012 @default.
• W1981122961 countsByYear W19811229612013 @default.
• W1981122961 countsByYear W19811229612014 @default.
• W1981122961 countsByYear W19811229612015 @default.
• W1981122961 countsByYear W19811229612016 @default.
• W1981122961 countsByYear W19811229612017 @default.
• W1981122961 countsByYear W19811229612018 @default.
• W1981122961 countsByYear W19811229612019 @default.
• W1981122961 countsByYear W19811229612020 @default.
• W1981122961 countsByYear W19811229612021 @default.
• W1981122961 countsByYear W19811229612022 @default.
• W1981122961 countsByYear W19811229612023 @default.
• W1981122961 crossrefType "journal-article" @default.
• W1981122961 hasAuthorship W1981122961A5014280478 @default.
• W1981122961 hasAuthorship W1981122961A5051700932 @default.
• W1981122961 hasAuthorship W1981122961A5063537638 @default.
• W1981122961 hasAuthorship W1981122961A5075080951 @default.
• W1981122961 hasBestOaLocation W19811229611 @default.
• W1981122961 hasConcept C11960822 @default.
• W1981122961 hasConcept C121608353 @default.
• W1981122961 hasConcept C185592680 @default.
• W1981122961 hasConcept C190283241 @default.
• W1981122961 hasConcept C203522944 @default.
• W1981122961 hasConcept C207332259 @default.
• W1981122961 hasConcept C526805850 @default.
• W1981122961 hasConcept C54355233 @default.
• W1981122961 hasConcept C55493867 @default.
• W1981122961 hasConcept C86803240 @default.
• W1981122961 hasConcept C95444343 @default.
• W1981122961 hasConcept C96232424 @default.
• W1981122961 hasConceptScore W1981122961C11960822 @default.
• W1981122961 hasConceptScore W1981122961C121608353 @default.
• W1981122961 hasConceptScore W1981122961C185592680 @default.
• W1981122961 hasConceptScore W1981122961C190283241 @default.
• W1981122961 hasConceptScore W1981122961C203522944 @default.
• W1981122961 hasConceptScore W1981122961C207332259 @default.
• W1981122961 hasConceptScore W1981122961C526805850 @default.
• W1981122961 hasConceptScore W1981122961C54355233 @default.
• W1981122961 hasConceptScore W1981122961C55493867 @default.
• W1981122961 hasConceptScore W1981122961C86803240 @default.
• W1981122961 hasConceptScore W1981122961C95444343 @default.
• W1981122961 hasConceptScore W1981122961C96232424 @default.
• W1981122961 hasIssue "50" @default.
• W1981122961 hasLocation W19811229611 @default.
• W1981122961 hasOpenAccess W1981122961 @default.

• next