Abstract
Genetic screens for modifiers of activated Ras phenotypes have identified a novel protein, kinase suppressor of Ras (KSR), which shares significant sequence homology with Raf family protein kinases. Studies using Drosophila melanogaster and Caenorhabditis elegans predict that KSR positively regulates Ras signaling; however, the function of mammalian KSR is not well understood. We show here that two predicted kinase-dead mutants of KSR retain the ability to complement ksr-1 loss-of-function alleles in C. elegans, suggesting that KSR may have physiological, kinase-independent functions. Furthermore, we observe that murine KSR forms a multimolecular signaling complex in human embryonic kidney 293T cells composed of HSP90, HSP70, HSP68, p50(CDC37), MEK1, MEK2, 14-3-3, and several other, unidentified proteins. Treatment of cells with geldanamycin, an inhibitor of HSP90, decreases the half-life of KSR, suggesting that HSPs may serve to stabilize KSR. Both nematode and mammalian KSRs are capable of binding to MEKs, and three-point mutants of KSR, corresponding to C. elegans loss-of-function alleles, are specifically compromised in MEK binding. KSR did not alter MEK activity or activation. However, KSR-MEK binding shifts the apparent molecular mass of MEK from 44 to >700 kDa, and this results in the appearance of MEK in membrane-associated fractions. Together, these results suggest that KSR may act as a scaffolding protein for the Ras-mitogen-activated protein kinase pathway.
Highlights
Ras activation is an essential step in signaling in response to a variety of extracellular signals, including receptor tyrosine kinase ligands which bind and activate their corresponding tyrosine kinase receptors
Integrity of the kinase suppressor of Ras (KSR) kinase domain is not required for its biological function in C. elegans vulval induction
Mutations in the C. elegans ksr-1 gene were isolated as suppressors of the Multivulva (Muv) phenotype caused by activated Ras [17, 30], suggesting that in C. elegans, the KSR-1 protein normally plays a positive role in Ras-mediated signaling
Summary
Ras activation is an essential step in signaling in response to a variety of extracellular signals, including receptor tyrosine kinase ligands which bind and activate their corresponding tyrosine kinase receptors. One of the best-characterized Ras signaling pathways is the Raf-MEK-ERK pathway, known as the mitogen-activated protein (MAP) kinase cascade [20, 25]. Activated ERK directly phosphorylates and thereby activates the transcription activation potential of TCFs [8, 12, 18] It appears that TCFs, in association with the serum response factor, play an essential role in the activation of many mitogen-inducible genes [11]. Genetic screens for mutations that suppress constitutively active Ras mutants have identified numerous components of the MAP kinase pathway. Such screens identified KSR (for kinase suppressor of Ras), a putative protein kinase with significant sequence identity to Raf [17, 30, 31].
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