Kinase suppressor of ras is necessary for tumor necrosis factor alpha activation of extracellular signal-regulated kinase/mitogen-activated protein kinase in intestinal epithelial cells

Mitogen-activated protein (MAP) kinase activity is essential for tumor necr osis factor (TNF) alpha receptor 1 regulation of intestinal epithelial cell proliferation, However, the mechanism of TNF-alpha mediated activation of extracellular signal-regulated kinase (ERK)/MAP kinase has not been establi shed dearly. Both TNF-alpha and cell-permeable ceramide have been reported to increase the kinase activity of kinase suppressor of Pas (KSR1, To deter mine the role of KSR in TNF-alpha -induced ERK1/ERK2 activation, we studied young adult mouse colon cells expressing a dominant-negative. kinase-inact ive (ki) KSR, We report that TNF-alpha, a cell-permeable ceramide, and sphi ngomyelinase stimulate ERK1/ERK2 activation and increase the phosphoserine content of KSR, which are inhibited by kiKSR expression in intact cells. Fu rthermore, TNF-alpha -induced Raf-1 threonine phosphorylation, kinase activ ity toward MEK1, and association with KSR are also inhibited by RiKSR expre ssion. Our data also show by sequential in vitro kinase assays that TNF-alp ha enhances KSR phosphorylation of Raf-1 on threonine, enhancing Raf-1 kina se activity toward MAP kinase kinase, We therefore conclude that KSR is an essential upstream regulator of TNF alpha -stimulated ERK1/ERK2 activation, most likely mediated via direct phosphorylation of Raf-1.