TI - Kinase suppressor of Ras signals through Thr269 of c-Raf-1 . AB - We recently established a two-stage in vitro assay for KSR kinase activity in which KSR never comes in contact with any recombinant kinase other than c-Raf-1 and defined the epidermal growth factor ( EGF ) as a potent activator of KSR kinase activity . That study , however , did not address the mechanism of c-Raf-1 stimulation by activated KSR . Here we show that phosphorylation of c-Raf-1 on Thr(269) by KSR is necessary for optimal activation in response to EGF stimulation . In vitro , KSR specifically phosphorylated c-Raf-1 on threonine residues during the first stage of the two-stage kinase assay . Using purified wild-type and mutant c-Raf-1 proteins , we demonstrate that Thr(269) is the major c-Raf-1 site phosphorylated by KSR in vitro and that phosphorylation of this site is essential for c-Raf-1 activation by KSR . KSR acts via transphosphorylation , not by increasing c-Raf-1 autophosphorylation , as kinase -inactive c-Raf-1 (K375M) served as an equally effective KSR substrate . In vivo , low physiologic doses of EGF ( 0.001-0.1 ng/ml ) stimulated KSR activation and induced Thr(269) phosphorylation and activation of c-Raf-1 . Low dose EGF did not induce serine or tyrosine phosphorylation of c-Raf-1 . High dose EGF ( 10-100 ng/ml ) induced no additional Thr(269) phosphorylation , but rather increased c-Raf-1 phosphorylation on serine residues and Tyr(340)/Tyr(341) . A Raf-1 mutant with valine substituted for Thr(269) was unresponsive to low dose EGF , but was serine - and Tyr(340)/Tyr(341) -phosphorylated and partially activated at high dose EGF . This study shows that Thr(269) is the major c-Raf-1 site phosphorylated by KSR . Furthermore , phosphorylation of this site is essential for c-Raf-1 activation by KSR in vitro and for optimal c-Raf-1 activation in response to physiologic EGF stimulation in vivo .