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

Abstract

Ras activation of phosphoinositide 3-kinase (PI3K) is important for survival of transformed cells. We find that PI3Kgamma is strongly and directly activated by H-Ras G12V in vivo or by GTPgammaS-loaded H-Ras in vitro. We have determined a crystal structure of a PI3Kgamma/Ras.GMPPNP complex. A critical loop in the Ras binding domain positions Ras so that it uses its switch I and switch II regions to bind PI3Kgamma. Mutagenesis shows that interactions with both regions are essential for binding PI3Kgamma. Ras also forms a direct contact with the PI3Kgamma catalytic domain. These unique Ras/PI3Kgamma interactions are likely to be shared by PI3Kalpha. The complex with Ras shows a change in the PI3K conformation that may represent an allosteric component of Ras activation.

Structure, Dynamics, Lipid Binding, and Physiological Relevance of the Putative GTPase-binding Domain of Dictyostelium Formin C

“…The fraction of this region provided by ␤-strand 2 as well as part of ␤-strand 1 and potentially the C-terminal end of ␣-helix 1 may, as has been observed for the RalGDS (Fig. 3B) and the PI3K Ras-binding domain (31)(32)(33), mediate the interaction with complementary negatively charged patches in the switch I and II regions of a regulatory GTPase. The F0 domain of the protein talin, an actinbinding protein involved in integrin activation, also shows a ubiquitin-like fold, including a similar although smaller positive surface patch.…”

Involvement of PI3K/Akt pathway in cell cycle progression, apoptosis, and neoplastic transformation: a target for cancer chemotherapy

“…[19][20][21][22] Ras can then induce membrane localization and activation of the p110 subunit of PI3K, as determined by crystal structure studies. 23 Specifically, this interaction occurs between regions on Ras, known as switch I (residues 33-41) and switch II (residues 63, 64, and 73), which bind to a sequence on PI3K-g referred to as the Ras-binding domain (RBD). 24 It was recently shown that the PI3K regulatory subunit, p85, contains two additional regulatory domains: a GTPase-responsive domain (GRD) that can mediate PI3K activation through binding small GTPases (Ras, Rac1), and an inhibitory domain that can block these binding events.…”

Apoptosis signaling by the novel compound 3-Cl-AHPC involves increased EGFR proteolysis and accompanying decreased phosphatidylinositol 3-kinase and AKT kinase activities

“…The Class 1 PI3-Ks are responsible for the synthesis of the intracellular PtdIns (3,4)P 2 and Ptdlns (3,4,5)P 3 (Domain and Waterfield, 1997). The PI3-K-dependent synthesis of PtdIns (3,4)P 2 and PtdIns (3,4,5)P 3 results in their accumulation in the inner leaflet of the plasma membrane and the subsequent membrane localization and activation of proteins that specifically bind these phosphatidylinositols; this colocalization and activation of enzymes along with their substrates allows for enhanced signaling activity within the cell resulting in marked modulation of cell phenotype and proliferation (Pacold et al, 2000). We have shown that 3-Cl-AHPC inhibits both AKT and ILK activities in addition to PI3-K in agreement with the proposed cascade of PI3-K activation of ILK and AKT.…”

“…We have shown that 3-Cl-AHPC inhibits both AKT and ILK activities in addition to PI3-K in agreement with the proposed cascade of PI3-K activation of ILK and AKT. Class IA PI3-K is a heterodimer consisting of a 110 kDa (p110) catalytic subunit and a 85 kDa (p85) regulatory subunit (Pacold et al, 2000). Activation of PI3-K can occur through the binding of Ras, Rac, Rho, Cdc42 or specific phosphotyrosine proteins to specific domains (Carpentier et al, 1990;Yamamoto et al, 1992;Joly et al, 1994;Kapeller et al, 1994;Levy-Toledano et al, 1994).…”