Crystal Structure and Functional Analysis of Ras Binding to Its Effector Phosphoinositide 3-Kinase γ

suppression of apoptosis (Franke et al., 1997), reorganization of the actin cytoskeleton (Rodriguez-Viciana et al., 1997), cardiac myocyte growth (Shioi et al., 2000), glycogen synthase stimulation by insulin (reviewed in Shepherd et al., 1998), TNFa-mediated neutrophil primMichael E. Pacold,* Sabine Suire,† Olga Perisic,* Samuel Lara-Gonzalez,* Colin T. Davis,* Edward H. Walker,* Phillip T. Hawkins,† Len Stephens,† John F. Eccleston,‡ and Roger L. Williams*§ *MRC Laboratory of Molecular Biology ing and superoxide generation (Condliffe et al., 1998), Hills Road and leukocyte migration and adhesion to endothelial Cambridge CB2 2QH cells (reviewed in Wymann et al., 2000). United Kingdom There are two types of class I PI3Ks. Class IA consists †The Babraham Institute of p110a, b, and d catalytic subunits that associate with Babraham an SH2 domain–containing subunit (p85a and splice Cambridge CB2 4AT variants of it, p85b or p55g) that is indispensable for United Kingdom activation by phosphotyrosine-containing proteins (Car‡National Institute for Medical Research penter et al., 1993). Consequently, the class IA PI3Ks are The Ridgeway typically activated by receptors that regulate tyrosine Mill Hill kinases. Class IB contains only one member, PI3Kg. This London NW7 1AA enzyme is regulated by G protein–coupled receptors via United Kingdom association with bg subunits of heterotrimeric G proteins (Stoyanov et al., 1995). A p101 regulatory subunit that tightly binds p110g is critical for Gbg-activated Summary PtdIns(3,4,5)P3 production (Stephens et al., 1997; Krugmann et al., 1999; Maier et al., 1999). All class I PI3Ks Ras activation of phosphoinositide 3-kinase (PI3K) is have a domain that is similar to a Ras binding domain important for survival of transformed cells. We find (RBD) present in other proteins that are regulated by that PI3Kg is strongly and directly activated by H-Ras the GTPase Ras. Furthermore, all class I PI3Ks bind Ras G12V in vivo or by GTPgS-loaded H-Ras in vitro. We in a GTP-dependent manner (Rodriguez-Viciana et al., have determined a crystal structure of a PI3Kg/ 1994; Rubio et al., 1997; Vanhaesebroeck et al., 1997; Ras·GMPPNP complex. A critical loop in the Ras bindDeora et al., 1998). ing domain positions Ras so that it uses its switch I The Ras family of membrane-localized GTPases and switch II regions to bind PI3Kg. Mutagenesis transduces a variety of signals in eukaryotic cells (Lowy shows that interactions with both regions are essential and Willumsen, 1993; Shields et al., 2000). The exchange for binding PI3Kg. Ras also forms a direct contact with of GDP for GTP causes two regions of Ras, known as the PI3Kg catalytic domain. These unique Ras/PI3Kg switch I (residues 32–40) and switch II (residues 60–76), interactions are likely to be shared by PI3Ka. The comto change conformation (Pai et al., 1989; Milburn et al., plex with Ras shows a change in the PI3K conforma1990). This GTP-induced conformational change allows tion that may represent an allosteric component of Ras to bind and activate multiple downstream effectors, Ras activation. which can cooperate to produce the diverse phenotypes characteristic of Ras transformation (White et al., 1995; Introduction Marshall, 1996) or function independently in activating survival pathways (Xue et al., 2000). Most Ras-depenThe phosphoinositide 3-kinases are a family of ubiquident signaling is mediated by three downstream eftous multidomain signaling proteins that phosphorylate fectors: the protein kinase Raf, the exchange factor the 3-hydroxyl of phosphoinositides. Mammalian PI3Ks RalGDS, and PI3K (reviewed in Wittinghofer and Herrare divided into three classes based on their structure mann, 1995; Feig et al., 1996; Rodriguez-Viciana et al., and substrate specificity (Domin and Waterfield, 1997). 1996a). Class I enzymes are acutely activated by a variety of Although all class I PI3Ks bind Ras, only PI3Ka has cell-surface receptors, and are responsible for synthesis been shown to be directly stimulated by Ras·GTP (Rodriof intracellular phosphatidylinositol (3,4,5)-trisphosphate guez-Viciana et al., 1994, 1996b). Ras-induced activation (PtdIns(3,4,5)P3). Thus, stimulation of many cells results in is synergistic with p85-mediated stimulation of PI3Ka by the PI3K-dependent accumulation of PtdIns(3,4,5)P3 in the phosphotyrosine peptides. Direct Ras activation of PI3Ka inner leaflet of the plasma membrane. This usually transtimulates actin rearrangement and inhibits programmed sient production of PtdIns(3,4,5)P3 initiates a multitude cell death upon detachment from the extracellular matrix of further downstream signals by causing membrane (anoikis) (Khwaja et al., 1997; Rodriguez-Viciana et al., recruitment of proteins that specifically bind PtdIns 1997). Other consequences of Ras activation of PI3Ks (3,4,5)P3. Typically, this results in a colocalization of eninclude cell transformation (Gire et al., 2000), T cell adhezymes and substrates that fires further signaling activity. sion and migration (Tanaka et al., 1999), and blocking Cellular processes in which PI3Ks are essential include of apoptosis induced by c-Myc (Kauffmann-Zeh et al., 1997) or by NGF deprivation (Xue et al., 2000). A recent crystal structure of PI3Kg (Walker et al., 1999) § To whom correspondence should be addressed (e-mail: rlw@mrc-