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Endogenous expression and protein kinase A‐dependent phosphorylation of the guanine nucleotide exchange factor Ras‐GRF1 in human embryonic kidney 293 cells
Author(s) -
Norum Jens Henrik,
Méthi Trond,
Mattingly Raymond R.,
Levy Finn Olav
Publication year - 2005
Publication title -
the febs journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.981
H-Index - 204
eISSN - 1742-4658
pISSN - 1742-464X
DOI - 10.1111/j.1742-4658.2005.04658.x
Subject(s) - guanine nucleotide exchange factor , embryonic stem cell , endogeny , phosphorylation , protein kinase a , microbiology and biotechnology , nucleotide , guanine , chemistry , kinase , biology , biochemistry , signal transduction , gene
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 G s ‐coupled serotonin receptors transiently expressed in human embryonic kidney (HEK) 293 cells. Whereas G i ‐ and G q ‐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, the mechanism of Ras activation through G s ‐coupled receptors is not fully understood. We report the endogenous expression of Ras‐GRF1 in HEK293 cells. Serotonin stimulation of HEK293 cells transiently expressing G s ‐coupled 5‐HT 7 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 Ca 2+ /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‐HT 7 receptors increased [Ca 2+ ] i , and the serotonin‐induced ERK1/2 phosphorylation was Ca 2+ ‐dependent. Therefore, both cAMP and Ca 2+ may contribute to the Ras‐dependent ERK1/2 activation after 5‐HT 7 receptor stimulation, through activation of a guanine nucleotide exchange factor with activity towards Ras.