Volmat V

Volmat V., Camps M., Arkinstall S., Pouyssgur J., Lenormand P. at Ser-112 but not at Ser-155. We find that this -arrestin 1-dependent ERK1/2 activation engaged by GLP-1 mediates the Ser-112 phosphorylation of Bad, through p90RSK activation, allowing the association of Bad with the scaffold protein 14-3-3, leading to its inactivation. -Arrestin 1 is usually further found to mediate the antiapoptotic effect of GLP-1 in -cells through the ERK1/2-p90RSK-phosphorylation of Bad. This new regulatory mechanism engaged by 25-hydroxy Cholesterol activated GLP-1R involving a -arrestin 1-dependent spatiotemporal regulation of the ERK1/2-p90RSK activity is now suspected to participate in the protection of -cells against apoptosis. Such signaling mechanism may serve as a prototype to generate new therapeutic GLP-1R ligands. nuclear location, and the ultimate functions of GLP-1-stimulated ERK1/2 activation remains totally unknown. Although many nuclear substrates have been shown to be phosphorylated by ERK1/2 in -cells, little is known about the identity of ERK1/2 cytosolic substrates. To address these issues, we examined the spatiotemporal regulation of ERK1/2 activation induced by GLP-1 and the potential requirement of ERK1/2 activity in phosphorylation of cytoplasmic targets. In this view, we studied Bad (Bcl-xL/Bcl-2-associated death promoter homolog), a well known ERK1/2 substrate in many cellular systems, localized in the -cell cytoplasm (24,C27). Bad (Bcl-xL/Bcl-2-associated death promoter homolog, or Bcl antagonist of cell death) was the first proapoptotic member of the Bcl-2 family to be described, and its proapoptotic activity is usually regulated by phosphorylation at several sites (24,C28). Phosphorylation of Bad at Ser-112 favors binding of Bad to the scaffold protein 14-3-3 in the cytoplasm, preventing the translocation of Bad to mitochondria (28,C30). This association further induces the release of Bcl-xL and Bcl-2 proteins from Bad, preventing accumulation of apoptotic proteins, such as Bax and Bak, around the mitochondrial membrane, blocking the release of cytochrome an antiapoptotic role (27). To date, the upstream signaling events that target Bad phosphorylation and thus control its functional behavior within the -cells are unknown. Here, we report that GLP-1 stimulates ERK1/2 by two temporally distinct pathways in -cells. The rapid and transient first phase is mainly mediated by the Gs/cAMP/PKA pathway and favors the nuclear translocation of the ERK1/2. The sustained and long lasting second phase of ERK1/2 activation is usually exclusively -arrestin 1 dependent, is restricted to the -cell cytoplasm, and enhances the p90 ribosomal S6 kinase (p90RSK) activity. We further report that GLP-1 significantly phosphorylates Bad at Ser-112 but not at Ser-155. The -arrestin 1-dependent ERK1/2-p90RSK signaling network engaged by GLP-1 phosphorylates Bad at Ser-112 and regulates its binding to the scaffold protein 25-hydroxy Cholesterol 14-3-3, revealing a new pathway in GLP-1-mediated antiapoptotic effects in -cells. EXPERIMENTAL PROCEDURES Antibodies and Reagents Glucagon-like peptide-1-(7C36) amide (GLP-1-(7C36) amide) was obtained from Bachem (Bubendorf, Switzerland). Anti-CREB, anti-phospho-Bad (Ser-112), anti-phospho-Bad (Ser-155), anti-Bad, anti-tubulin, anti-p44/42 mitogen-activated ENOX1 protein kinase (ERK1/2), anti-phospho-p90RSK (Thr-573), anti-cleaved caspase-3, and anti-rabbit IgG horseradish peroxidase-linked antibodies were from Cell Signaling Technology (New England Biolabs, Beverly, MA). Anti–arrestin 1 and anti-ERK1 antibodies were from Transduction Laboratories (Lexington, KY). Horseradish peroxidase-linked anti-mouse, goat polyclonal anti–arrestin 1, anti–arrestin 2, anti-14-3-3 (C-20) protein, anti-Bad (C7) antibodies, and protein A/G-Plus-agarose were obtained from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). LipofectamineTM2000 and StealthTM prevalidated small interfering RNA (siRNA) double-stranded duplexes were from Invitrogen. Plasmids expressing hemagglutinin-tagged mouse 25-hydroxy Cholesterol wild type (WT) and mutant Bad S112A were a generous gift from Dr. Michael Greenberg (Harvard Medical School, Boston, MA) and Dr. Robert J. Lefkowitz (Duke University, Durham, NC). Dulbecco’s altered Eagle’s medium (DMEM) and fetal calf serum were from Invitrogen. H89 and wortmannin were obtained from Calbiochem, and U0126 was from Promega (Madison, WI). 45CaCl2 was obtained from Amersham Biosciences. All other reagents were purchased from Sigma. Animals -Arrestin 1 knock-out (KO) mice, a nice gift from R. J. Lefkowitz, were generated on C57BL/6 background as described previously (32). WT littermate controls were C57BL/6 mice (5 weeks aged) purchased from Charles River (France). All mice used in this study were treated in accordance with European Community guidelines, and the local institution approved the experimentation. Pancreatic Islet Isolation, Western Blotting, and Insulin Secretion Pancreatic islets were isolated from WT and -arrestin 1 KO mice weighing 30C35 g on the day of killing using collagenase digestion followed by hand picking as described (33, 34). For Western blotting experiments, islets were stabilized for 2 h at 37 C in HEPES-balanced Krebs-Ringer bicarbonate buffer made up of 0.1% bovine serum albumin (KRB buffer) 25-hydroxy Cholesterol as described (33, 34) and supplemented with 1.4 mm glucose. Islets were washed and further incubated in groups of 100 islets for various occasions at 37 C in KRB buffer supplemented with effectors. Islets were then rapidly centrifuged, washed with cold phosphate-buffered saline, frozen in.