tailieunhanh - Báo cáo khoa học: Endogenous expression and protein kinase A-dependent phosphorylation of the guanine nucleotide exchange factor Ras-GRF1 in human embryonic kidney 293 cells

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 G i- and Gq-coupled receptors have been shown to activate Ras through the guanine nucleotide exchange factor (GEF) called Ras-GRF1 (CDC25 Mm ) by binding of Ca 2+ ⁄calmodulin to its N-terminal IQ domain, . | iFEBS Journal 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 Methi1 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 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 Received 2 December 2004 revised 1 February 2005 accepted 10 March 2005 doi 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 expression 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 phosphorylation. 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 .