Kinase Suppressor of Ras Couples Ras to the ERK Cascade during T Cell Development Micheline N. Laurent, Danny Maria Ramirez, and Jose ´ Alberola-Ila 1 Ras signaling is critical for many developmental processes and requires the precise coordination of interactions among multiple downstream components. One mechanism by which this regulation is achieved is through the use of scaffolding molecules that coordinate the assembly of multimolecular complexes. Recently, the scaffolding molecule kinase suppressor of Ras (KSR) was isolated in genetic screens as a modifier of Ras signaling, although its contribution to regulating Ras-mediated activation of its different downstream effectors is not well understood. We have analyzed the role of KSR in linking Ras to the ERK cascade during positive selection. Our results demonstrate that KSR overexpression interferes with T cell development, an effect that requires the direct interaction between KSR and MEK. This functional effect correlates with the ability of KSR to uncouple Ras from the ERK cascade when overexpressed. The Journal of Immunology, 2004, 173: 986 –992. R as and its effectors are important during T cell develop- ment in the thymus. In particular, several lines of evi- dence indicate the requirement for the Ras/Raf/MEK/ ERK pathway in directing positive selection, the transition that a small percentage of immature double-positive (DP 2 ; CD4 + CD8 + ) cells undergo to become either mature CD4 single-positive (CD4 + SP), or mature CD8 SP cells (CD8 + SP) (1). The expression of dominant-negative forms of either Ras or MEK in transgenic an- imals inhibits the generation of mature single-positive thymocytes due to a specific blockade in positive selection (2, 3). Furthermore, ERK1 null mutant animals exhibit similar defects in thymocyte maturation, and the inhibition of ERK activity with chemical in- hibitors is sufficient to block positive selection in a fetal thymic organ culture assay (4, 5). The Ras signaling cascade is essential for many growth and developmental processes. One well-characterized pathway through which Ras mediates its effects is via the sequential activation of the cytoplasmic kinases Raf, MEK, and ERK (6). Upon Ras signaling, membrane-localized Raf is activated. Activated Raf then phos- phorylates and activates MEK, which in turn phosphorylates and activates the MAPK, ERK. However, the regulation of the inter- actions among these effectors is not completely understood. Fur- thermore, Ras also uses other downstream effector pathways to elicit its multiple effects (7, 8). A central and not well-character- ized aspect of Ras signaling concerns how the multiple down- stream effector pathways are coordinately activated to induce highly specific effects. One mechanism that is used to activate specific signaling path- ways among closely related molecules involves scaffolding mole- cules. Scaffolding molecules selectively bind multiple molecular components and specifically enhance signaling by these molecules by facilitating their association with appropriate components and/or by reducing nonproductive interactions (9, 10). Although scaffolding molecules have been well documented in yeast, only recently has it been shown in mammalian cells that molecules such as JNK-interacting protein 1 and MEK partner 1 are used as scaf- folds to enhance signaling through the MAPKs JNK and ERK, respectively (11–14). Kinase suppressor of Ras (KSR) was initially identified as a positive regulator of Ras signaling in genetic screens performed in Caenorhabditis elegans and Drosophila melanogaster (15–17), and homologous proteins were discovered in mammals (15). The protein sequence of KSR is most similar to Raf, with the highest homology corresponding to the kinase domains. However, the ne- cessity of the putative kinase domain for KSR function is unclear (18). Epistasis studies suggest that KSR functions either in parallel to, or downstream of Raf (15). KSR is constitutively associated with MEK1 and 2 and can directly interact with ERK1 and 2 (19 –25). KSR is also able to interact with Raf as well as other signaling molecules such as 14-3-3 (21, 22, 25–27). Association with 14-3-3 may be important for the negative regulation of KSR activity because this interaction prevents KSR membrane translo- cation (28, 29). Although KSR was originally found to augment Ras signaling, ensuing experiments demonstrated that KSR can also negatively regulate Ras signaling when expressed at high levels (19, 20, 22, 24, 25, 30, 31). These observations are consistent with a scaffold- ing function because scaffolds enhance positive signaling by in- creasing molecular interactions until the point at which the scaffold is in excess, thereby resulting in the formation of nonfunctional complexes (10, 32). Recently, it has been demonstrated in a Dro- sophila cell line that KSR initiates complex formation between Raf and MEK, thereby allowing for the activation of MEK by Raf (27). These results, in combination with the findings that KSR associates with a number of molecules, indicate that KSR can act as a scaffold to form a molecular bridge between Raf and MEK to facilitate activation of this cascade. To determine whether KSR expression might play a role in modulating Ras function during T cell development, we overex- pressed it in T cell precursors using retroviral infection, and its effect on T cell development was tested in reaggregate fetal thymic Division of Biology, California Institute of Technology, Pasadena, CA 91125 Received for publication November 13, 2003. Accepted for publication May 12, 2004. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1 Address correspondence and reprint requests to Dr. Jose ´ Alberola-Ila, MC147-75, California Institute of Technology, 1200 E. California Boulevard, Pasadena, CA 91125. E-mail address: alberola@caltech.edu 2 Abbreviations used in this paper: DP, double positive; CRM, cysteine-rich motif; dGuo, 2'deoxyguanosine; dMEK, dead MEK; KSR, kinase suppressor of Ras; Mig, murine stem cell virus-internal ribosomal entry site-GFP; rFTOC, reaggregate fetal thymic organ culture; SP, single positive. The Journal of Immunology Copyright © 2004 by The American Association of Immunologists, Inc. 0022-1767/04/$02.00