Farnesylation of Ras is important for the interaction with phosphoinositide 3‐kinase γ

Abstract

The correct functioning of Ras proteins requires post-translational modification of the GTP hydrolases (GTPases). These modifications provide hydrophobic moieties that lead to the attachment of Ras to the inner side of the plasma membrane. In this study we investigated the role of Ras processing in the interaction with various putative Ras-effector proteins. We describe a specific, GTP-independent interaction between post-translationally modified Ha-and Ki-Ras4B and the G-protein responsive phosphoinositide 3-kinase p110g. Our data demonstrate that post-translational processing increases markedly the binding of Ras to p110g in vitro and in Sf9 cells, whereas the interaction with p110a is unaffected under the same conditions. Using in vitro farnesylated Ras, we show that farnesylation of Ras is sufficient to produce this effect. The complex of p110g and farnesylated RasGTP exhibits a reduced dissociation rate leading to the efficient shielding of the GTPase from GTPase activating protein (GAP) action. Moreover, Ras processing affects the dissociation rate of the RasGTP complex with the Ras binding domain (RBD) of Raf-1, indicating that processing induces alterations in the conformation of RasGTP. The results suggest a direct interaction between a moiety present only on fully processed or farnesylated Ras and the putative target protein p110g.Keywords: Ras; post-translational modifications; phosphoinositide 3-kinase; farnesylation.The Ras GTP hydrolases (GTPases) are a family of related proteins involved in the regulation of numerous cellular processes such as mitogen-induced G0/G1 phase transition, apoptosis, cytoskeletal rearrangements and regular cell-cycle progression. Ras proteins cycle between a GDP-bound and a GTP-bound state, the latter being the active, signal transducing version of the GTPase.The correct subcellular localization of Ras is critical for the activation of its downstream targets. Immunocytochemical studies have shown, that the majority of Ras is localized to the inner side of the plasma membrane. This association with the plasma membrane has been causally connected to a series of post-translational modifications of Ras. Inhibition of the posttranslational modification reactions abolishes the plasma membrane localization and leads to the loss of the biological activity of Ras [1,2]. The transforming potential of oncogenic Ras variants is severely affected by interfering with their post-translational modifications [2±4]. The Raf kinase isoform Raf-1 binds to, and is activated by, Ras in insect Sf9 cells [5] and vertebrate cell lines [6,7] and this activation, although not the association, is lost if post-translational processing of Ras is prevented [7]. A farnesylation-deficient point mutant of oncogenic Ha-Ras has been identified that can no longer activate the phosphoinositide 3-kinase (PI3K) isoform p110a in COS7 cells [8]. These and other findings have been interpreted in such a way that the post-translational processing of Ras functions to localize both Ras and, eventually, its target pro...

High Affinity for Farnesyltransferase and Alternative Prenylation Contribute Individually to K-Ras4B Resistance to Farnesyltransferase Inhibitors

“…Although it is not clear why this might be the case, it is possible that geranylgeranylation targets Ras to different membrane subdomains than does farnesylation, bringing it into closer proximity with effector proteins. The demonstration that the Ras prenyl group is involved in effector association and activation (29) supports the possibility that geranylgeranylated Ras more effectively activates effectors through enhanced association. An FTI-induced increase in Ras signaling via Elk-1 cannot be the result of inhibition of other farnesylated proteins that might normally inhibit Ras function or else all Ras constructs would be similarly affected, but H-Ras, N-(K 6 )-CVLS, and K-(Q 5 )-CVLS are not.…”

“…This phenomenon, well documented here and elsewhere for N-Ras (CVVM) and K-Ras (CVIM) (8,9,21), may also apply to other farnesylated proteins such as RhoE (CTVM) to render these proteins FTI-resistant regardless of the presence or absence of a polybasic motif. There is also evidence implicating the prenyl group in the association of Ras family proteins with both downstream effectors (29) and upstream guanine nucleotide exchange factors (49), suggesting that geranylgeranylated proteins may not always be functionally equivalent to their farnesylated counterparts and, therefore, that alternatively prenylated proteins including N-Ras and K-Ras may be relevant FTI targets. However, our observation that both farnesylated and geranylgeranylated K-Ras12V produce nearly identical gene expression profiles when stably expressed in human embryonic kidney cells supports the widely held belief in the functional equivalence of the farnesylated and geranylgeranylated forms of this protein and clearly implicates other farnesylated proteins in the inhibition of K-Ras-transformed cells by FTIs.…”

“…Alternative Prenylation of N-or K-Ras Is Functionally Tolerated-Finally, in light of evidence that the identity of the prenyl group may influence signaling and/or subcellular localization of Ras (29,39,40), we wanted to confirm the functional equivalence of farnesylated and geranylgeranylated Ras. If Ras-F and Ras-GG do not have identical functions in cellular signaling, then the well established effect of FTIs on K-Rastransformed cells may result from this difference rather than (or in addition to) FTI inhibition of non-Ras farnesylated proteins.…”

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

“…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). Binding of Ras to the p110 subunit, Rho, Rac or Cdc42 to the p85 subunit or specific phosphotyrosine proteins to the SH2 or SH3 domains of p85 can result in PI3-K activation (Sjolander et al, 1991;Zheng et al, 1994;RodriquezViciana et al, 1997;Chan et al, 1999;Rubio et al, 1999;Rebello and Ayllon, 2000). The prostate and breast carcinoma cell lines examined in this study displayed binding of activated Ras, as well as Cdc42, Rho, Rac and EGFR to PI3-K; 3-Cl-AHPC did not modulate the binding of Ras, Rho, Cdc42 to PI3-K in any of the cell lines.…”

H-Ras and Phosphoinositide 3-Kinase Cooperate to Induce α(1,3)-Fucosyltransferase VII Expression in Jurkat T Cells

“…FucT-VII regulation could be mediated by H-Ras activation on the Golgi apparatus, with the involvement of phospholipase C␥ and RasGRP1. Differences in posttranslational modifications in Ras isoforms, such as palmitoylation (18), affect not only subcellular localization, as mentioned before, but also interactions with certain regulators and/or effectors (55,56). Thus, the specific ability of H-Ras to induce FucT-VII expression may be due to the distinct localization of H-Ras in microdomains in the plasma membrane or Golgi apparatus, where interactions take place with specific effectors that colocalize in those domains.…”