sst2 Somatostatin Receptor Inhibits Cell Proliferation through Ras-, Rap1-, and B-Raf-dependent ERK2 Activation

doi:10.1074/jbc.M304524200

sst2 Somatostatin Receptor Inhibits Cell Proliferation through Ras-, Rap1-, and B-Raf-dependent ERK2 Activation*

^‡INSERM U531, IFR31, Centre Hospitalier Universitaire Rangueil, 1 avenue Jean Poulhès, 31403 Toulouse Cedex and ^¶CNRS Unité Mixte de Recherche 146, Institut Curie, Centre Universitaire, 91405 Orsay Cedex, France

∥ To whom correspondence should be addressed. Tel.: 33-5-61-32-24-07; Fax: 33-5-61-32-24-03; E-mail: susinich{at}toulouse.inserm.fr.

Abstract

The G protein-coupled sst2 somatostatin receptor is a critical negative regulator of cell proliferation. sstII prevents growth factor-induced cell proliferation through activation of the tyrosine phosphatase SHP-1 leading to induction of the cyclin-dependent kinase inhibitor p27^Kip1. Here, we investigate the signaling molecules linking sst2 to p27^Kip1. In Chinese hamster ovary-DG-44 cells stably expressing sst2 (CHO/sst2), the somatostatin analogue RC-160 transiently stimulates ERK2 activity and potentiates insulin-stimulated ERK2 activity. RC-160 also stimulates ERK2 activity in pancreatic acini isolated from normal mice, which endogenously express sst2, but has no effect in pancreatic acini derived from sst2 knock-out mice. RC-160-induced p27^Kip1 up-regulation and inhibition of insulin-dependent cell proliferation are both prevented by pretreatment of CHO/sst2 cells with the MEK1/2 inhibitor PD98059. In addition, using dominant negative mutants, we show that sst2-mediated ERK2 stimulation is dependent on the pertussis toxin-sensitive G_i/o protein, the tyrosine kinase Src, both small G proteins Ras and Rap1, and the MEK kinase B-Raf but is independent of Raf-1. Phosphatidylinositol 3-kinase (PI3K) and both tyrosine phosphatases, SHP-1 and SHP-2, are required upstream of Ras and Rap1. Taken together, our results identify a novel mechanism whereby a G_i/o protein-coupled receptor inhibits cell proliferation by stimulating ERK signaling via a SHP-1-SHP-2-PI3K/Ras-Rap1/B-Raf/MEK pathway.

Footnotes

↵1 The abbreviations used are: GPCR, G protein-coupled receptor; CHO, Chinese hamster ovary; CKI, cyclin-dependent kinase inhibitor; MAPK, mitogen-activated protein kinase; ERK, extracellular signal-regulated kinase; MAPKK, MAPK kinase; MEK, MAPK kinase or ERK kinase; HA, hemagglutinin epitope; PI3K, phosphatidylinositol 3-kinase; MBP, myelin basic protein; PTX, Bordetella pertusis toxin; GST, glutathione S-transferase; EGF, epidermal growth factor; NGF, nerve growth factor; MEM, minimum essential medium; DN, dominant negative; RBD, Ras or Rap1 binding domain; PMSF, phenylmethylsulfonyl fluoride; MBP, myelin basic protein; DTT, dithiothreitol; GEF, guanine nucleotide exchange factor.
↵2 C. Bousquet, personal communication.
↵* This work was supported in part by grants from Association pour la Recherche contre le Cancer (4505, ARECA) and Ligue Nationale contre le Cancer. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
↵§ Supported by a doctoral fellowship from Association pour la Recherche contre le Cancer.
- Received April 30, 2003.
- Revision received July 11, 2003.
The American Society for Biochemistry and Molecular Biology, Inc.