title: Erk1/2-dependent phosphorylation of Galpha-interacting protein stimulates its GTPase accelerating activity and autophagy in human colon cancer cells.
abstract: Galpha-interacting protein (GAIP) is a regulator of G protein signaling (RGS) that accelerates the rate of GTP hydrolysis by the alpha-subunit of the trimeric G(i3) 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 Galpha(i3) protein (k = 0.187 +/- 0.001 s(-)(1), EC(50) = 1.12 +/- 0.10 microm) when compared with unphosphorylated recombinant GAIP (k = 0.145 +/- 0.003 s(-)(1), EC(50) = 3.16 +/- 0. 12 microm) 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.
authors.affiliation: INSERM U504, Glycobiologie et Signalisation Cellulaire, 16, avenue Paul-Vaillant Couturier, 94807 Villejuif cedex, France.
authors.forename: E
authors.initials: E
authors.lastname: Ogier-Denis
authors.forename: S
authors.initials: S
authors.lastname: Pattingre
authors.forename: J
authors.initials: J
authors.lastname: El Benna
authors.forename: P
authors.initials: P
authors.lastname: Codogno
mesh_terms: D000409:Alanine / Q000737:chemistry; D047390:Casein Kinase II; D003110:Colonic Neoplasms / Q000378:metabolism*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005419:Flavonoids / Q000494:pharmacology; D020558:GTP Phosphohydrolases / Q000378:metabolism*; D006801:Humans; D015151:Immunoblotting; D007211:Indoles / Q000494:pharmacology; D007700:Kinetics; D020935:MAP Kinase Signaling System; D008301:Maleimides / Q000494:pharmacology; D019950:Mitogen-Activated Protein Kinase 1 / Q000378:metabolism*; D048052:Mitogen-Activated Protein Kinase 3; D020928:Mitogen-Activated Protein Kinases / Q000378:metabolism*; D016297:Mutagenesis, Site-Directed; D010750:Phosphoproteins / Q000235:genetics / Q000378:metabolism*; D010766:Phosphorylation; D011233:Precipitin Tests; D011493:Protein Kinase C / Q000037:antagonists & inhibitors / Q000378:metabolism; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism; D017434:Protein Structure, Tertiary; D020710:RGS Proteins / Q000378:metabolism; D011994:Recombinant Proteins / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012694:Serine / Q000737:chemistry; D015398:Signal Transduction; D014162:Transfection; D014407:Tumor Cells, Cultured
chemical_list: D004791:Enzyme Inhibitors; D005419:Flavonoids; D007211:Indoles; D008301:Maleimides; D010750:Phosphoproteins; D020710:RGS Proteins; D011994:Recombinant Proteins; C097187:regulator of G-protein signalling 19; D012694:Serine; D047390:Casein Kinase II; D017346:Protein Serine-Threonine Kinases; D011493:Protein Kinase C; D019950:Mitogen-Activated Protein Kinase 1; D048052:Mitogen-Activated Protein Kinase 3; D020928:Mitogen-Activated Protein Kinases; D020558:GTP Phosphohydrolases; C070515:bisindolylmaleimide I; D000409:Alanine; C093973:2-(2-amino-3-methoxyphenyl)-4H-1-benzopyran-4-one