Abstract
Kinase suppressor of Ras (KSR) is a loss-of-function allele that suppresses the rough eye phenotype of activated Ras in Drosophila and the multivulval phenotype of activated Ras in Caenorhabditis elegans. Genetic and biochemical studies suggest that KSR is a positive regulator of Ras signaling that functions between Ras and Raf or in a pathway parallel to Raf. We examined the effect of mammalian KSR expression on the activation of extracellular ligand-regulated (ERK) mitogen-activated protein (MAP) kinase in fibroblasts. Ectopic expression of KSR inhibited the activation of ERK MAP kinase by insulin, phorbol ester, or activated alleles of Ras, Raf, and mitogen and extracellular-regulated kinase. Expression of deletion mutants of KSR demonstrated that the KSR kinase domain was necessary and sufficient for the inhibitory effect of KSR on ERK MAP kinase activity. KSR inhibited cell transformation by activated RasVal-12 but had no effect on the ability of RasVal-12 to induce membrane ruffling. These data indicate that KSR is a potent modulator of a signaling pathway essential to normal and oncogenic cell growth and development.
Highlights
Ras proteins are integrators of diverse extracellular signals that regulate cell growth and development [1]
To determine whether the inhibitory effect of Kinase suppressor of Ras (KSR) on ERK mitogen-activated protein (MAP) kinase activation is unique to the insulin signaling system, we tested the effect of KSR expression on ERK MAP kinase activation by the phorbol ester PMA
In this report we have examined the ability of ectopic KSR to affect ERK MAP kinase activation by extracellular stimuli and upstream regulators
Summary
Cell Culture—Cells were maintained in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal bovine serum and incubated at 37 °C. 293T cells were grown in an atmosphere of 5% CO2. 293T cells were grown in an atmosphere of 5% CO2. REF-52 cells were grown in 6% CO2. The PCR product was digested with NsiI, KpnI, and PvuII to yield a 383-base pair fragment containing the COOH terminus of KSR and the FLAG epitope. This fragment was ligated back into pMA57 after NsiI and KpnI digestion to give pBSKSRFLAG. The pBSKSRFLAG cDNA was cut with EcoRI and KpnI to remove KSRFLAG from pBS This 2724-base pair fragment was ligated into the same sites of pCMV5 [13] to produce pCMV5KSR.
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