LIPOPOLYSACCHARIDE INDUCES ACTIVATION OF THE RAF-1 MAP KINASE PATHWAY- A PUTATIVE ROLE FOR RAF-1 IN THE INDUCTION OF THE IL-1-BETA AND THETNF-ALPHA GENES/

Bacterial LPS is a potent macrophage activator. The early steps in LPS signal transduction involve the tyrosine phosphorylation and activati on of a number of kinases of the src family, and inhibition of this pa thway causes a severe impairment in the production of the cytokines TN F-alpha and IL-1 beta. We find that LPS-induced macrophage activation also involves the Raf-l kinase, a key component in mitogenic signal tr ansduction. Treatment of BAC-1.2F5 macrophages with LPS causes phospho rylation and activation of Raf-1. This is paralleled by the stimulatio n of MEK-1 and MAP-kinase activity and by the phosphorylation of the t ranscription factor Elk-l, a nuclear target of MAP-kinase. Activation of the Raf/MAP-kinase pathway was inhibited upon pretreatment of the c ells with genistein, a tyrosine kinase inhibitor. Raf-l must thus lie downstream of tyrosine kinases in LPS signal transduction. However, Ra f-l is not a direct substrate of a LPS-induced tyrosine kinase, becaus e Raf-l immunoisolated from LPS-induced cells contains only phosphoser ine. This resembles the situation after CSF-l-stimulation of macrophag es, in which Raf-l clearly transduces a signal generated by the CSF-1 receptor kinase, but is phosphorylated exclusively in serine. Phosphop eptide maps of Raf-l immunoprecipitated from LPS- or CSF-l-treated cel ls are indistinguishable, suggesting that these agents activate Raf-l by similar mechanisms. Finally, v-raf-infected BAC-1.2F5 macrophages w ere found to constitutively express low levels of IL-1 beta and TNF-al pha. These data argue that Raf-l functions downstream of tyrosine kina ses in LPS-mediated macrophage activation and cytokine production.