Regulation of Ras·GTP Loading and Ras-Raf Association in Neonatal Rat Ventricular Myocytes by G Protein-coupled Receptor Agonists and Phorbol Ester

Abstract

The small G protein Ras has been implicated in hypertrophy of cardiac myocytes. We therefore examined the activation (GTP loading) of Ras by the following hypertrophic agonists: phorbol 12-myristate 13-acetate (PMA), endothelin-1 (ET-1), and phenylephrine (PE). All three increased Ras⅐GTP loading by 10 -15-fold (maximal in 1-2 min), as did bradykinin. Other G protein-coupled receptor agonists (e.g. angiotensin II, carbachol, isoproterenol) were less effective. Activation of Ras by PMA, ET-1, or PE was reduced by inhibition of protein kinase C (PKC), and that induced by ET-1 or PE was partly sensitive to pertussis toxin. 8-(4-Chlorophenylthio)-cAMP (CPT-cAMP) did not inhibit Ras⅐GTP loading by PMA, ET-1, or PE. The association of Ras with c-Raf protein was increased by PMA, ET-1, or PE, and this was inhibited by CPT-cAMP. However, only PMA and ET-1 increased Ras-associated mitogen-activated protein kinase kinase 1-activating activity, and this was decreased by PKC inhibition, pertussis toxin, and CPT-cAMP. PMA caused the rapid appearance of phosphorylated (activated) extracellular signal-regulated kinase in the nucleus, which was inhibited by a microinjected neutralizing anti-Ras antibody. We conclude that PKC-and G idependent mechanisms mediate the activation of Ras in myocytes and that Ras activation is required for stimulation of extracellular signal-regulated kinase by PMA.Members of the membrane-associated small (21-kDa) G protein Ras family (Ha-Ras, K-Ras, N-Ras) are important in eukaryotic signal transduction (reviewed in Ref. 1). In its GDPligated state, Ras is inactive. By increasing its GTP loading in a regulated manner, Ras acts as a "molecular switch" and transmits signals from a variety of cell surface receptors to downstream effector proteins. The best characterized effector is c-Raf, one of the mitogen-activated protein kinase (MAPK) 1 kinase kinases of the extracellular signal-regulated kinase (ERK) cascade. Ras⅐GTP (but not Ras⅐GDP) has a high affinity for c-Raf, causing it to translocate to the membrane (reviewed in Ref. 2), where, in a process that possibly involves its phosphorylation (3-5), c-Raf becomes fully activated. Several other potential Ras effectors have been identified. These include phosphatidylinositol 3Ј-kinase, other small G proteins, the Ral-GDS family, and other protein kinases (reviewed in Ref. 1). Ras is converted back to its inactive state by its innate GTPase activity, which may be enhanced by GTPase-activating proteins.In the terminally differentiated, cell cycle-arrested ventricular myocyte (in contrast to transformed cell lines and other dividing cells), G protein-coupled receptor (GPCR) agonists such as endothelin-1 (ET-1), bradykinin (BK), and ␣ 1 -adrenergic agonists promote a stronger activation of the ERK cascade members (c-Raf/A-Raf, MAPK kinase 1/2 (MKK1/2), and ERK1/ ERK2) than peptide growth factors (6 -9). This activation is mediated at least in part through the G q /G 11 group of GPCRs (recently also demonstrated in vivo (10)), stimulation of membrane phospho...

Ras Regulates NFAT3 Activity in Cardiac Myocytes

“…In particular, microinjection of constitutively active forms of Ras recapitulated the hypertrophic program with an increase in cell size and the enhanced expression of hypertrophic markers such as ANF (4). In addition, stimulation with hypertrophic agents such as phenylephrine leads to an increase in Ras-GTP levels (5). These in vitro studies have been complemented with in vivo experiments that demonstrated increased left ventricular mass and increased ANF expression in cardiac-targeted transgenics expressing a constitutively active ras gene product (6).…”

Regulation of phorbol ester-mediated TRAF1 induction in human colon cancer cells through a PKC/RAF/ERK/NF-κB-dependent pathway

“…In addition, PKC, independent of Ras, plays a critical role in angiotensininduced Raf1 and MEK activation in cardiac myocytes (Zou et al, 1996), and PKCd activates the MEK/ERK pathway, independent of Ras but dependent on Raf activation, in COS and NIH-3T3 cells (Ueda et al, 1996;Seternes et al, 1999). In contrast, other studies have shown that PMA/PKC regulates downstream gene induction and functions through sequential induction of Ras, Raf and MEK (Chiloeches et al, 1999;Verin et al, 2000;Vuong et al, 2000); these effects appear to be dependent on cellular context. In our present study, cotransfection with a dominant-negative Raf effectively inhibited TRAF1 promoter induction by PMA.…”

Involvement of the ERK Signaling Cascade in Protein Kinase C-mediated Cell Cycle Arrest in Intestinal Epithelial Cells

“…There has been some controversy in the literature (for example, see Refs. [42][43][44][45][46][47][48][49] regarding the ability of PKC signaling to activate Ras. Our study confirms the ability of PMA to promote PKC-dependent GTP loading of Ras and, to our knowledge, provides the first evidence that Bryo can also produce this effect.…”