Cell Death and Differentiation (1999) 6, 722 ± 728 ã 1999 Stockton Press All rights reserved 13509047/99 $15.00 http://www.stockton-press.co.uk/cdd Review Novel aspects of Ras proteins biology: regulation and implications Dolores PeÂrez-Sala1 and Angelita Rebollo*,2 kinase; MAPK, mitogen-activated protein kinase; NO, nitric oxide; PH, pleckstrin homology; PI3 K, phosphatidylinositol-3-kinase; 1 Departamento de Estructura y FuncioÂn de ProteõÂnas, Centro de Investigaciones PPMTase, prenylated protein methyltransferase; SAPK, stress- BioloÂgicas, CSIC, VelaÂzquez 144, E-28006 Madrid, Spain activated protein kinase 2 Department of Immunology and Oncology, Centro Nacional de BiotecnologõÂa, Universidad AutoÂnoma, Campus de Cantoblanco, E-28049 Madrid, Spain * corresponding author: Angelita Rebollo, Department of Immunology and Oncology, Centro Nacional de BiotecnologõÂa, Universidad AutoÂnoma, Campus Introduction de Cantoblanco, E-28049 Madrid, Spain. Tel: (+34-91) 585 4655; fax: (+34-91) Ras proteins are critical components of signaling pathways 585 4506; email: [email protected] that link the activation of cell surface receptors with Received 14.4.99; revised 16.6.99; accepted 21.6.99 transcriptional events leading to the control of proliferation, Edited by R. Knight differentiation and apoptosis. The ras genes codes for 21 kDa proteins (H-Ras, N-Ras, K-Ras4A and K-Ras4B) that function as GDP/GTP-regulated switches1 (Figure 1). The two K-Ras Abstract forms diverge in the C terminal as a consequence of alternate The importance of Ras proteins as crucial crossroads in exon utilization. Ras proteins are activated transiently in response to diverse extracellular signals such as growth cellular signaling pathways has been well established. In spite factors, cytokines, hormones and neurotransmitters, which of the elucidation of the mechanism of RAS activation by stimulate cell surface receptors. Ras uses several functionally growth factors and the delineation of MAP kinase cascades, diverse effectors that in turn activate multiple pathways. the overall framework of Ras interactions is far from being Several excellent reviews have recently dealt with the topics complete. Novel regulators of Ras GDP/GTP exchange have of Ras-mediated signalling pathways and cross-talk with other been identified that may mediate the activation of Ras in low molecular weight GTP-binding proteins.2,3 The aim of this response to changes in intracellular calcium and diacylgly- article is to survey emerging aspects of Ras protein biology cerol. The direct activation of Ras by free radicals such as focusing on novel mechanisms of Ras activation, as well as nitric oxide also suggests potential regulation of Ras function on the involvement of Ras in pro-apoptotic or anti-apoptotic by the cellular redox state. In addition, the array of Ras signaling pathways. effectors continues to expand, uncovering links between Ras and other cellular signaling pathways. Ras is emerging as a Regulation of GDP/GTP exchange dual regulator of cellular functions, playing either positive or The activity of Ras proteins is controlled by a cycle between a negative roles in the regulation of proliferation and apoptosis. GDP-bound inactive state and a GTP-bound active state. The signals transmitted by Ras may be modulated by other Although Ras proteins possess intrinsic GTPase exchange pathways triggered in parallel, resulting in the final order for activity, it is too low to account for the rapid, transient GDP/ proliferation or apoptosis. The diversity of ras-mediated GTP cycling. A complete model for Ras function includes effects may be related in part to differential involvement of Ras regulatory proteins that control the GTP/GDP cycle, compris- homologues in distinct cellular processes. The study of Ras ing the guanine nucleotide exchange factors (GEF) and the posttranslational modifications has yielded a broad battery of GTPase activating proteins (GAP) (Figure 2). Several GEFs inhibitors that have been envisaged as anti-cancer agents. act on Ras, including Sos1 and 2, which couple tyrosine Although an irreversible modification, Ras isoprenylation kinase receptors with Ras activation, and GRF1 and 2, which are activated by some G protein-coupled receptors and appears to be modulated by growth factors and by the activity calcium signals.4 of the isoprenoid biosynthetic pathway, which may lead to Several recent studies have addressed the mechanism changes in Ras activity. of action and regulation of Sos. The solution of the crystal structure of H-Ras coupled to the Ras guanine-nucleotide- Keywords: Ras proteins; guanine nucleotide exchange; Ras exchange factor region of Sos5 has shown that Sos inserts effectors; apoptosis; isoprenylation an a-helix into Ras that opens the nucleotide binding site. In turn, Sos activity has been proposed to be regulated by Abbreviations: EGF, epidermal growth factor; ERK, extracellular intramolecular interactions. However, the data concerning signal-regulatedkinase;FTase,farnesyltransferase;GAP,GTPase the role of Sos N- and C-terminal domains are conflicting; activating protein; GEF, guanine nucleotide exchange factor; GRF, while some studies suggest that both regions exert an guanine nucleotide releasing factor; IL-2, interleukin 2; JNK, Jun inhibitory effect on the activity of the catalytic domain,6 Novel aspects of Ras proteins biology DPeÂrez-Sala and A Rebollo 723 others propose that the Dbl and pleckstrin homology (PH) does not occur in vitro.10,11 It has therefore been proposed domains of the N-terminal region of Sos are critical for its that the effect of Ca2+-CaM in vivo may be indirect, function.7 Sos activity is also regulated by phosphorylation modulating the interaction of RasGRF with its targets.10 after growth factor stimulation of cells. In addition to MAP Calcium signals could also contribute to GRF activation by kinases ERK1 and 2, it has been recently shown that promoting the proteolytic removal of the N-terminal domain, p90Rsk-2 can phosphorylate Sos.8 This phosphorylation is as it has been shown in vitro.11 A novel member of the believed to exert a negative feedback effect on Ras family of Ras-GEFs has recently been cloned from brain12 stimulation. The observation that Sos activity can be and lymphoid tissues.4 This factor, named RasGRP, inhibited in vitro by binding of phosphatidylinositol 4,5-P2 possesses calcium and diacyglycerol-binding domains and 9 (PI4,5P2) to the Sos PH domain has led to propose that may directly couple changes in diacylglycerol and possibly PI4,5P2 may serve as a negative regulator of Sos in the calcium concentrations to Ras activation. The activation of 4 cell, and that removal of PI4,5P2 may cause Sos and Ras RasGRP is associated with membrane partitioning of the activation. factor, which suggests that recruitment of RasGRP to The mechanism of GRF activation has not been fully membranes may be a primary mechanism for its activation. elucidated. GRFs contain cdc25 and Dbl homology regions GEFs act on G proteins other than Ras, thus functioning plus an IQ region, considered the calmodulin (CaM) as bifunctional signaling proteins that may coordinate the interaction site. The interaction of GRP with Ca2+-CaM activation of various cellular pathways. Ras GRF1 also leads to GRF activation in intact cells, however, this effect serves as a GEF for the Ras-related protein R-Ras,13 whereas Ras GRF2 has been reported to bind and activate Rac, thereby coordinating the activation of the ERK and SAPK pathways.10 Ras and Rac-activated pathways are also potentially coupled through Sos.14 Reactive free radicals and cellular redox stress have been proposed to directly activate Ras. Nitric oxide (NO) promotes the direct posttranslational modification of Ras by Figure 1 Scheme of Ras showing the GTP-binding sites, which mediate binding and hydrolysis of the guanine nucleotide, the effector loop, which single S-nitrosylation at Cys118. This results in stimulation interacts with effector molecules, and the C-terminal CAAX motif of guanine nucleotide exchange, possibly by destabilization Figure 2 Schematic view of Ras regulatory factors. Ras proteins cycle between the active GTP-bound and the inactive GDP-bound states. Exchange factors (Ras GRF, Sos, GRP) catalyze Ras activation by facilitating the dissociation of bound GDP. Free radicals such as nitric oxide (NO) may also promote Ras-GTP formation. Ras remains active until it hydrolyzes bound GTP to GDP. This process is accelerated by GTPase activating proteins (p120GAP, NF1), which complement the Ras active site Novel aspects of Ras proteins biology DPeÂrez-Sala and A Rebollo 724 of the interaction between residues in the GDP-binding ates with other effectors, leading to transduction of Ras- pocket and the nucleotide, and leads to activation of mediated signals through multiple pathways. downstream signaling.15 This suggests that Ras function Among other candidate Ras effectors is phosphatidyli- may be regulated directly by changes in the redox state of nositol-3-kinase (PI3 K). PI3 K is a lipid kinase that the cell and provide a mechanism for the GEF-independent phosphorylates phosphoinositides at the 3' position of the activation of Ras. inositol ring. PI3 K is composed of a p110 catalytic and a The mechanism of action of Ras-GAP has recently been p85 regulatory subunit. Multiple PI3 K isoforms have been elucidated in a series of biochemical and structural isolated and some can associate with Ras.23 The PI3 K studies,16 and will thus be addressed here only briefly.