Identification of a novel human kinase supporter of Ras (hKSR-2) that functions as a negative regulator of Cot (Tpl2) signaling

Kinase suppressor of Ras ( KSR) is an integral and conserved component of the Ras signaling pathway. Although KSR is a positive regulator of the Ras/ mitogen-activated protein ( MAP) kinase pathway, the role of KSR in Cot- mediated MAPK activation has not been identified. The serine/ threonine kinase Cot ( also known as Tp12) is a member of the MAP kinase kinase kinase ( MAP3K) family that is known to regulate oncogenic and inflammatory pathways; however, the mechanism( s) of its regulation are not precisely known. In this report, we identify an 830- amino acid novel human KSR, designated hKSR- 2, using predictions from genomic data base mining based on the structural profile of the KSR kinase domain. We show that, similar to the known human KSR, hKSR- 2 co- immunoprecipitates with many signaling components of the Ras/ MAPK pathway, including Ras, Raf, MEK- 1, and ERK- 1/ 2. In addition, we demonstrate that hKSR- 2 co- immunoprecipitates with Cot and that co- expression of hKSR- 2 with Cot significantly reduces Cot- mediated MAPK and NF-kappaB activation. This inhibition is specific to Cot, because Ras- induced ERK and IkappaB kinase- induced NF-kappaB activation are not significantly affected by hKSR- 2 co- expression. Moreover, Cot- induced interleukin- 8 production in HeLa cells is almost completely inhibited by the concurrent expression of hKSR- 2, whereas transforming growth factor beta- activated kinase 1 ( TAK1)/ TAK1- binding protein 1 ( TAB1)- induced interleukin- 8 production is not affected by hKSR- 2 co- expression. Taken together, these results indicate that hKSR- 2, a new member of the KSR family, negatively regulates Cot- mediated MAP kinase and NF-kappaB pathway signaling.