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A Direct Interaction between Oncogenic Ha-Ras and Phosphatidylinositol 3-Kinase Is Not Required for Ha-Ras-dependent Transformation of Epithelial Cells*

Open AccessPublished:October 26, 2001DOI:https://doi.org/10.1074/jbc.M102401200
      Cells expressing oncogenic Ras proteins transmit a complex set of signals that ultimately result in constitutive activation of signaling molecules, culminating in unregulated cellular function. Although the role of oncogenic Ras in a variety of cellular responses including transformation, cell survival, differentiation, and migration is well documented, the direct Ras/effector interactions that contribute to the different Ras biological end points have not been as clearly defined. Observations by other groups in which Ras-dependent transformation can be blocked by expression of either dominant negative forms of Phosphatidylinositol (PI) 3-kinase or PTEN, a 3-phosphoinositide-specific phosphatase, support an essential role for PI 3-kinase and its lipid products in the transformation process. These observations coupled with the in vitro observations that the catalytic subunits of PI 3-kinase, the p110 isoforms, bind directly to Ras-GTP foster the implication that a direct interaction between an oncogenic Ras protein and PI 3-kinase are causal in the oncogenicity of mutant Ras proteins. Using an activated Ha-Ras protein (Y64G/Y71G/F156L) that fails to interact with PI 3-kinase, we demonstrate that oncogenic Ha-Ras does not require a direct interaction with PI 3-kinase to support anchorage-independent growth of IEC-6 epithelial cells. We do find, however, that IEC-6 cells expressing an oncogenic Ha-Ras protein that no longer binds PI 3-kinase are greatly impaired in their ability to migrate toward fibronectin.
      PI
      phosphatidylinositol
      EGF
      epidermal growth factor
      EGFR
      EGF receptor
      DMEM
      Dulbecco's modified Eagle's medium
      FBS
      fetal bovine serum
      MOPS
      4-morpholinepropanesulfonic acid
      CHAPS
      3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid
      PAGE
      polyacrylamide gel electrophoresis
      TBS
      Tris-buffered saline
      MAPK
      mitogen-activated protein kinase
      PAS
      protein A-Sepharose
      HPLC
      high pressure liquid chromatography
      gPI
      glycerophosphoinositide
      GMPPNP
      guanylyl-5′-imidodiphosphate
      PI (3
      4)P2, phosphatidylinositol 3,4 bisphosphate
      PI (3
      4,5)P3, phosphatidylinositol 3,4,5 trisphosphate
      The Ras proteins exert their effects on cells by directly associating with downstream effector molecules, thereby initiating the activation of the signaling networks responsible for Ras-mediated biological events. A number of molecules have been identified as potential Ras effectors, including Raf-1, B-Raf, RalGDS, PI1 3-kinase, Rin 1, AF6, and Nore 1 (
      • Campbell S.L.
      • Khosravi-Far R.
      • Rossman K.L.
      • Clark G.J.
      • Der C.J.
      ,
      • Mangues R.
      • Corral T.
      • Kohl N.E.
      • Symmans W.F.
      • Lu S.Y.
      • Malumbres M.
      • Gibbs J.B.
      • Oliff A.
      • Pellicer A.
      ). Among these putative effector molecules, three major candidates, Raf-1, RalGDS, and PI 3-kinase, have been implicated as direct downstream Ras effectors that may contribute to Ras-dependent transformation (
      • Moodie S.A.
      • Willumsen B.M.
      • Weber M.J.
      • Wolfman A.
      ,
      • Warne P.H.
      • Viciana P.R.
      • Downward J.
      ,
      • Zhange Z.
      • Settleman J.
      • Kyriakis J.M.
      • Takeuchi-Suzuki E.
      • Elledge S.J.
      • Marshall M.S.
      • Bruder J.T.
      • Rapp U.R.
      • Avruch J.
      ,
      • Rodriguez-Viciana P.
      • Warne P.H.
      • Dhand R.
      • Vanhaesebroeck B.
      • Gout I.
      • Fry M.J.
      • Waterfield M.D.
      • Downward J.
      ,
      • Urano T.
      • Emkey R.
      • Feig L.A.
      ,
      • Rodriguez-Viciana P.
      • Warne P.H.
      • Vanhaesebroeck B.
      • Waterfield M.D.
      • Downward J.
      ,
      • Rodriguez-Viciana P.
      • Warne P.H.
      • Khwaja A.
      • Marte B.M.
      • Pappin D.
      • Das P.
      • Waterfield M.D.
      • Ridley A.J.
      • Downward J.
      ). Raf-1 is the only known effector, however, that has been detected in stable complexes with Ras in vivo, at physiological levels of expression (
      • Hallberg B.
      • Rayter S.I.
      • Downward J.
      ,
      • Finney R.E.
      • Robbins S.M.
      • Bishop J.M.
      ,
      • Hamilton M.
      • Wolfman A.
      ). The consequences of such Ras/Raf interactions on cellular function have been extensively studied and found to result in the activation of the Raf/MEK/MAPK cascade. Ras-stimulated MAPK activity is not only required for cellular proliferation, but elevated MAPK activity is commonly associated with Ras transformation of both fibroblasts and epithelial cells. Although elevated MAPK is absolutely required to maintain the transformed phenotype, the current model for Ras function implicates the requirement of additional signaling networks to produce the full spectrum of Ras-dependent biological end points. Several lines of evidence, in support of this model, indicate that oncogenic Ras facilitates the activation of multiple Ras effector pathways that cooperate to establish cellular transformation. The most direct evidence comes from Ras effector domain mutants that have been characterized to bind only a single Ras target (
      • White M.A.
      • Nicolette C.
      • Minden A.
      • Polverino A.
      • Van Aelst L.
      • Karin M.
      • Wigler M.H.
      ). Rodriguez-Viciana et al. (
      • Rodriguez-Viciana P.
      • Warne P.H.
      • Khwaja A.
      • Marte B.M.
      • Pappin D.
      • Das P.
      • Waterfield M.D.
      • Ridley A.J.
      • Downward J.
      ) demonstrated that Ras effector domain mutants, which interact solely with Raf (G12V/T35S/E37G) or RalGDS (G12V/G37) or PI 3-kinase (G12V/Y40C) are not by themselves sufficient to transform fibroblasts. Co-expression, however, of any two of these Ras effector domain mutants induced the formation of foci, suggesting that Ras must activate more than one effector pathway to generate a transformed phenotype (
      • Rodriguez-Viciana P.
      • Warne P.H.
      • Khwaja A.
      • Marte B.M.
      • Pappin D.
      • Das P.
      • Waterfield M.D.
      • Ridley A.J.
      • Downward J.
      ).
      Transient expression of G12V Ha-Ras results in elevated levels of 3-phosphorylated phosphatidylinositides that are potentiated by the co-transfection of the p110 catalytic subunit of PI 3-kinase (
      • Rodriguez-Viciana P.
      • Warne P.H.
      • Vanhaesebroeck B.
      • Waterfield M.D.
      • Downward J.
      ,
      • Rubio I.
      • Rodriguez-Viciana P.
      • Downward J.
      • Wetzker R.
      ). The role of PI 3-kinase lipid products in the transformation process is further highlighted by observations that PTEN, a phosphatase that specifically dephosphorylates 3-phosphorylated phosphatidylinositides, has significantly reduced activity in a number of human tumors (
      • Steck P.A.
      • Pershouse M.A.
      • Jasser S.A.
      • Yung W.K.
      • Lin H.
      • Ligon A.H.
      • Langford L.A.
      • Baumgard M.L.
      • Hattier T.
      • Davis T.
      • Frye C.
      • Hu R.
      • Swedlund B.
      • Teng D.H.
      • Tavtigian S.V.
      ,
      • Li J.
      • Yen C.
      • Liaw D.
      • Podsypanina K.
      • Bose S.
      • Wang S.I.
      • Puc J.
      • Miliaresis C.
      • Rodgers L.
      • McCombie R.
      • Bigner S.H.
      • Giovanella B.C.
      • Ittmann M.
      • Tycko B.
      • Hibshoosh H.
      • Wigler M.H.
      • Parsons R.
      ,
      • Cantley L.C.
      • Neel B.G.
      ). Ectopic expression of PTEN in Ras transformed cells reduces the number of foci in a focus formation assay. It is not clear, however, whether the role of PI 3-kinase in Ras-dependent transformation arises from a direct association with Ras-GTP or through other unidentified indirect mechanisms. This uncertainty is amplified by the failure to detect a stable complex between Ras and PI 3-kinase in vivo, even when both are transiently overexpressed (
      • Rubio I.
      • Rodriguez-Viciana P.
      • Downward J.
      • Wetzker R.
      ).
      Our laboratory has previously published data showing that specific Ras isoforms have different affinities for Raf-1 in vivo. Using 10T1/2 fibroblasts transformed by minimal levels of G12V Ha-Ras, we observe that Raf-1 preferentially associates with c-N-Ras and not the ectopically expressed oncogenic Ha-Ras (
      • Hamilton M.
      • Wolfman A.
      ). We have extended these observations by demonstrating that G12V Ha-Ras induces the secretion of EGF receptor (EGFR) ligands that promote the activation of c-N-Ras·Raf-1 complexes and, hence, up-regulate MAPK activity (
      • Hamilton M.
      • Wolfman A.
      ). Together these observations suggest that G12V Ha-Ras does not interact with Raf-1 and requires the co-operative function of other signaling modules to generate transformation and elevate basal MAPK. Observations that Raf-CAAX does not substitute for oncogenic G12V Ha-Ras in the transformation of RIE-1 cells provide further evidence that Ha-Ras transformation is mediated by additional Raf-independent mechanisms (
      • Oldham S.M.
      • Clark G.J.
      • Gangarosa L.M.
      • Coffey Jr., R.J.
      • Der C.J.
      ). These observations prompted us to investigate whether oncogenic Ha-Ras requires a direct interaction with PI 3-kinase to mediate the transformation of epithelial cells. To address this issue we constructed a constitutively active Ha-Ras protein (Y64G/Y71G/F156L) that binds all known downstream Ras effectors except PI 3-kinase. Our results indicate that oncogenic Ha-Ras does not require a direct association with PI 3-kinase to transform rat intestinal epithelial (IEC-6) cells. Although a direct Ha-Ras/PI 3-kinase interaction is dispensable for the transforming activities of oncogenic Ha-Ras, we do find that loss of this direct interaction between Ha-Ras and PI 3-kinase greatly impairs migration of epithelial cells toward fibronectin.

      DISCUSSION

      The significance of constitutive Ras signaling through the Raf kinases, resulting in the up-regulation of the Raf/MEK/MAPK pathway, and its role in cellular transformation is well established (
      • Khosravi-Far R.
      • Campbell S.
      • Rossman K.L.
      • Der C.J.
      ). Although overexpression of activated Raf is sufficient to transform fibroblasts, several lines of evidence suggest that Ras-dependent transformation results from the constitutive signaling through at least two separate effector pathways. The most compelling data supporting this hypothesis were generated using partial loss of function Ras mutants that bind only Raf (G12V/T35S), only RalGDS (G12V,E37G), or PI 3-kinase (G12V/Y40C) (
      • Rodriguez-Viciana P.
      • Warne P.H.
      • Khwaja A.
      • Marte B.M.
      • Pappin D.
      • Das P.
      • Waterfield M.D.
      • Ridley A.J.
      • Downward J.
      ,
      • White M.A.
      • Nicolette C.
      • Minden A.
      • Polverino A.
      • Van Aelst L.
      • Karin M.
      • Wigler M.H.
      ). Studies using these mutants lead to the conclusion that expression of an oncogenic Ras protein that interacts with a single effector is not sufficient to transform cells. Further support for additional Ras/effector mechanisms that contribute to Ras-dependent transformation arise from reports demonstrating that constitutive Raf activation, by the overexpression of either ΔRaf-22W or Raf-CAAX, do not substitute for oncogenic Ras to establish the transformed phenotype of RIE-1 cells (
      • Oldham S.M.
      • Clark G.J.
      • Gangarosa L.M.
      • Coffey Jr., R.J.
      • Der C.J.
      ). These observations are quite different from those in fibroblasts where overexpression of activated Raf is sufficient for transformation (
      • Samuels M.L.
      • Weber M.J.
      • Bishop J.M.
      • McMahon M.
      ), suggesting that Ras signaling involves different mechanisms in epithelial cells.
      Although activated versions of all four Ras isoforms transform cells, we have previously published data supporting the hypothesis that they do so by utilizing distinct downstream effector molecules. Raf-1 preferentially associates with c-N-Ras in mouse fibroblasts minimally expressing an oncogenic Ha-Ras protein (
      • Hamilton M.
      • Wolfman A.
      ). Using IEC-6 cells transformed by G12V Ha-Ras, Raf-1 co-immunoprecipitated with c-N-Ras and not with the oncogenic G12V Ha-Ras (data not shown). These data suggest that Raf-1 may not act as a direct downstream effector for oncogenic Ha-Ras. The functional consequences of these observations are supported by reports that the transformed phenotype of RIE-1 epithelial cells expressing G12V Ha-Ras can be reverted by blocking EGFR function, indicating that oncogenic Ha-Ras mediates transformation in part by stimulating the production of EGFR ligands (
      • Gangarosa L.M.
      • Sizemore N.
      • Graves-Deal R.
      • Oldham S.M.
      • Der C.J.
      • Coffey R.J.
      ). We have extended those observations by demonstrating that blocking the EGFR in Ha-Ras transformed cells down-regulates MAPK activity, failing to activate the endogenous c-N-Ras·Raf-1 complex (
      • Hamilton M.
      • Wolfman A.
      ).
      Taken together these findings point out the relevance of additional Ras/effector interactions that may contribute to Ras transformation. PI 3-kinase is a likely effector molecule that has the potential to function directly downstream of the Ras proteins and participate in the transformation process. Cells transformed by oncogenic Ras proteins can be partially reverted by blocking PI 3-kinase activity using dominant negative p85 constructs or by the overexpression of PTEN, a phosphatase that specifically dephosphorylates the 3 position on the inositol ring (
      • Tolkacheva T.
      • Chan A.M.
      ,
      • Zhang Q.X.
      • Davis I.D.
      • Baldwin G.S.
      ). There is some ambiguity, however, as to whether this increase in PI 3-kinase-dependent lipid products results from a direct interaction with oncogenic Ras-GTP. In this report, we focus on the specific question concerning the requirement of a direct interaction between oncogenic Ha-Ras and PI 3-kinase for cellular transformation.
      We use an activated Ha-Ras mutant (Y64G/Y71G/F156L Ha-Ras) that interacts with known Ras effectors (B-Raf, AF6, Rin 1, RalGDS, and Raf-1) similarly to wild-type Ha-Ras but does not interact with PI 3-kinase. We demonstrate that minimal protein expression of Y64G/Y71G/F156L Ha-Ras supports both a transformed phenotype- and anchorage-independent growth of IEC-6 epithelial cells. Interestingly, all of the Ras expressing IEC cell lines exhibited identical changes in steady-state PI 3-kinase-dependent lipid products. All of the Ras-transformed cell lines possessed about a 2-fold increase in steady-state PI (3,4,5)P3 and a rather striking absence of PI (3,4)P2 compared with the easily detectable levels in the control cultures. These observations suggest that the two predominant lipid products of PI 3-kinase might have opposing effects on the transformation process. Because we did not detect increased basal phosphorylation of AKT, this might also suggest that it is the PI (3,4)P2 PI 3-kinase-dependent lipid product that is responsible for the activation of AKT through its phosphorylation by PDK1 (
      • Alessi D.R.
      • Deak M.
      • Casamayor A.
      • Caudwell F.B.
      • Morrice N.
      • Norman D.G.
      • Gaffney P.
      • Reese C.B.
      • MacDougall C.N.
      • Harbison D.
      • Ashworth A.
      • Bownes M.
      ). Our data also imply, because the PI 3-kinase-defective mutant (Y64G/Y71G/F156L Ha-Ras) showed changes in PI 3-kinase-dependent lipids identical to those of the control transforming Ras proteins, that the observed changes in steady-state lipids do not arise from the direct interaction between oncogenic Ha-Ras and PI 3-kinase.
      We did not detect significant changes in cellular (or plasma membrane-associated) PI 3-kinase activity in Ras transformed IEC-6 cells, even though we documented a 2-fold increase in the steady-state level of the PI 3-kinase product, PI (3,4,5)P3. We did find, however, that inhibition of the basal PI 3-kinase activity was sufficient to revert the transformed morphology. As with the basal MAPK activity in this cell type, the basal PI 3-kinase activity is both sufficient and required to generate the transformed phenotype. These observations are in agreement with work by another group suggesting that the basal PI 3-kinase activity is required for low, mitogenic doses of EGF to activate both Ras and the MAPK cascade (
      • Wennstrom S.
      • Downward J.
      ).
      The use of selective Ras mutants that fail to interact with a single putative downstream effector molecule have distinct advantages over the previously well used selective effectors that were characterized to interact with only a single target. The original “single” interacting Ras mutants (the T35S/E37G, 37G, and Y40C) were only characterized relative to “known” Ras binding partners. Identification of additional Ras binding partners and the possibility that each or all of these single interacting Ras mutants might interact with an unidentified target does leave data obtained with these mutants somewhat in question. We have found that the G12V/Y40C Ha-Ras protein interacts with Nore 1 in a manner identical to that of wild-type Ha-Ras (data not shown). This might suggest that previously identified PI 3-kinase-specific biological outcomes might also be attributed to the interactions between oncogenic Ha-Ras and Nore 1 or a co-operative effect arising from the interaction between both PI 3-kinase and Nore 1. Data obtained using the Y64G/Y71G/F156L Ha-Ras mutant are not subject to this limited interpretation. If we were to find that the Y64G/Y71G/F156L Ha-Ras protein fails to interact with a novel Ras binding partner, this would only add to our general conclusions that the putative novel binding partner and PI 3-kinase need not directly interact with oncogenic Ha-Ras to generate a transformed phenotype. Although it is attractive to speculate that a direct interaction between Ha-Ras and PI 3-kinase is important in cell migration, it is also possible that another Ras effector that fails to interact with the Y64G/Y71G/F156L Ha-Ras protein might also play a role in cell migration. The recently identified phospholipase Cε (
      • Lopez I.
      • Mak E.C.
      • Ding J.
      • Hamm H.E.
      • Lomasney J.W.
      ,
      • Song C.
      • Hu C.-D.
      • Masago M.
      • Kariya K.-I.
      • Yamawaki-Kataoka Y.
      • Shibatohge M.
      • Wu D.
      • Satoh T.
      • Kataoka T.
      ,
      • Kelley G.G.
      • Reks S.E.
      • Ondrako J.M.
      • Smrcka A.V.
      ), whose binding properties to the Y64G/Y71G/F156L Ha-Ras protein have not been characterized, is certainly a candidate for this Ras-dependent biological end point.
      The results presented in this report raise two important issues concerning the direct interactions of oncogenic Ha-Ras and putative effector proteins. First, if Ha-Ras does not require a physical interaction with either Raf-1 or PI 3-kinase to transform cells, which direct downstream effector(s) of oncogenic Ha-Ras is responsible for the initial events in the transformation process? The most obvious candidate is RalGDS. Although the expression of activated RalGDS in NIH3T3 cells can induce anchorage-independent growth and proliferation in low serum, its immediate target, Ral, does not behave as an oncogene when expressed in NIH3T3 cells (
      • Urano T.
      • Emkey R.
      • Feig L.A.
      ,
      • White M.A.
      • Vale T.
      • Camonis J.H.
      • Schaefer E.
      • Wigler M.H.
      ,
      • Bos J.L.
      ). Several studies, however, suggest that the role of RalGDS in Ras-dependent transformation is cooperative with other Ras effectors (
      • Rodriguez-Viciana P.
      • Warne P.H.
      • Khwaja A.
      • Marte B.M.
      • Pappin D.
      • Das P.
      • Waterfield M.D.
      • Ridley A.J.
      • Downward J.
      ,
      • White M.A.
      • Nicolette C.
      • Minden A.
      • Polverino A.
      • Van Aelst L.
      • Karin M.
      • Wigler M.H.
      ). Even if RalGDS is a direct and specific effector of Ha-Ras, our data implicate at least one other Ras target that might cooperate with RalGDS or function independently of RalGDS as the critical element mediating Ha-Ras-dependent transformation.

      Acknowledgments

      We thank Mark Hamilton for helpful discussion and review of the manuscript, Janice Wolfman for review of the manuscript, and Donna Driscoll for technical instruction. We thank Walter Kolch, Steve Martin, Franz Hofer, and K. Kaibuchi for generous contribution of antibodies. We also thank Stan Hazen and Dave Schmitt for help with the HPLC lipid analysis.

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