Tài liệu Báo cáo khoa học: Endogenous expression and protein kinase A-dependent phosphorylation of

Endogenous expression and protein kinase A-dependent

phosphorylation of the guanine nucleotide exchange

factor Ras-GRF1 in human embryonic kidney 293 cells

Jens Henrik Norum1

, Trond Me´thi1

, Raymond R. Mattingly2 and Finn Olav Levy1

1 Department of Pharmacology, University of Oslo, Norway

2 Department of Pharmacology, Wayne State University, Detroit, MI, USA

Introduction

Signals mediated through receptor tyrosine kinases [1]

and G-protein-coupled receptors (GPCRs) can induce

the activation of intracellular cascades such as the

mitogen-activated protein (MAP) kinase – also called

extracellular signal-regulated kinase (ERK) – cascade.

The serine ⁄threonine kinases ERK1 and ERK2 are

activated by dual phosphorylation by the MAP kinase

kinase, MEK, which becomes phosphorylated and

activated by MEK kinases of the Raf family. All

three Raf isoforms [A-Raf, B-Raf and Raf-1 (C-Raf)]

Keywords

5-HT7, cAMP, ERK, GEF, serotonin

Correspondence

F. O. Levy, Department of Pharmacology,

University of Oslo, PO Box 1057 Blindern,

N-0316 Oslo, Norway

Fax: +47 22840202

Tel: +47 22840237 or +47 22840201

E-mail: [email protected]

(Received 2 December 2004, revised 1

February 2005, accepted 10 March 2005)

doi:10.1111/j.1742-4658.2005.04658.x

We have previously reported the Ras-dependent activation of the mitogen￾activated protein kinases p44 and p42, also termed extracellular signal￾regulated kinases (ERK)1 and 2 (ERK1 ⁄ 2), mediated through Gs-coupled

serotonin receptors transiently expressed in human embryonic kidney

(HEK) 293 cells. Whereas Gi- and Gq-coupled receptors have been shown

to activate Ras through the guanine nucleotide exchange factor (GEF)

called Ras-GRF1 (CDC25Mm) by binding of Ca2+ ⁄ calmodulin to its

N-terminal IQ domain, the mechanism of Ras activation through Gs-cou￾pled receptors is not fully understood. We report the endogenous expres￾sion of Ras-GRF1 in HEK293 cells. Serotonin stimulation of HEK293

cells transiently expressing Gs-coupled 5-HT7 receptors induced protein

kinase A-dependent phosphorylation of the endogenous human Ras-GRF1

on Ser927 and of transfected mouse Ras-GRF1 on Ser916. Ras-GRF1

overexpression increased basal and serotonin-stimulated ERK1 ⁄ 2 phos￾phorylation. Mutations of Ser916 inhibiting (Ser916Ala) or mimicking

(Ser916Asp ⁄ Glu) phosphorylation did not alter these effects. However, the

deletion of amino acids 1–225, including the Ca2+ ⁄ calmodulin-binding IQ

domain, from Ras-GRF1 reduced both basal and serotonin-stimulated

ERK1 ⁄ 2 phosphorylation. Furthermore, serotonin treatment of HEK293

cells stably expressing 5-HT7 receptors increased [Ca2+]i, and the sero￾tonin-induced ERK1 ⁄ 2 phosphorylation was Ca2+-dependent. Therefore,

both cAMP and Ca2+ may contribute to the Ras-dependent ERK1 ⁄ 2 acti￾vation after 5-HT7 receptor stimulation, through activation of a guanine

nucleotide exchange factor with activity towards Ras.

Abbreviations

5-HT, 5-hydroxytryptamine (serotonin); CaM, calmodulin; EGF, epidermal growth factor; Epac, exchange protein directly activated by cAMP;

ERK, extracellular signal-regulated kinase; GEF, guanine nucleotide exchange factor; GPCR, G protein-coupled receptor; H89,

N-[2-(p-bromocinnamylamino)ethyl]-5-isoquinolinesulfonamide dihydrochloride; HEK, human embryonic kidney; HRP, horseradish peroxidase;

MAP, mitogen-activated protein; MEK, mitogen-activated protein ⁄ extracellular signal-regulated kinase kinase; PKA, protein kinase A; Sos1,

son of sevenless 1.

2304 FEBS Journal 272 (2005) 2304–2316 ª 2005 FEBS