Reimann T, Büscher D, Hipskind R A, Krautwald S, Lohmann-Matthes M L, Baccarini M
Department of Immunobiology, Fraunhofer Institute for Toxicology and Molecular Biology, Hannover, Germany.
J Immunol. 1994 Dec 15;153(12):5740-9.
Bacterial LPS is a potent macrophage activator. The early steps in LPS signal transduction involve the tyrosine phosphorylation and activation of a number of kinases of the src family, and inhibition of this pathway causes a severe impairment in the production of the cytokines TNF-alpha and IL-1 beta. We find that LPS-induced macrophages activation also involves the Raf-1 kinase, a key component in mitogenic signal transduction. Treatment of BAC-1.2F5 macrophages with LPS causes phosphorylation and activation of Raf-1. This is paralleled by the stimulation of MEK-1 and MAP-kinase activity and by the phosphorylation of the transcription factor Elk-1, a nuclear target of MAP-kinase. Activation of the Raf/MAP-kinase pathway was inhibited upon pretreatment of the cells with genistein, a tyrosine kinase inhibitor. Raf-1 must thus lie downstream of tyrosine kinase in LPS signal transduction. However, Raf-1 is not a direct substrate of a LPS-induced tyrosine kinase, because Raf-1 immunoisolated from LPS-induced cells contains only phosphoserine. This resembles the situation after CSF-1-stimulation of macrophages, in which Raf-1 clearly transduces a signal generated by the CSF-1 receptor kinase, but is phosphorylated exclusively in serine. Phosphopeptide maps of Raf-1 immunoprecipitated from LPS- or CSF-1-treated cells are indistinguishable, suggesting that these agents activate Raf-1 by similar mechanisms. Finally, v-raf-infected BAC-1.2F5 macrophages were found to constitutively express low levels of IL-1 beta and TNF-alpha. These data argue that Raf-1 functions downstream of tyrosine kinases in LPS-mediated macrophage activation and cytokine production.
细菌脂多糖（LPS）是一种强大的巨噬细胞激活剂。LPS信号转导的早期步骤涉及src家族多种激酶的酪氨酸磷酸化和激活，抑制该途径会导致细胞因子TNF-α和IL-1β的产生严重受损。我们发现LPS诱导的巨噬细胞激活还涉及Raf-1激酶，它是促有丝分裂信号转导中的关键成分。用LPS处理BAC-1.2F5巨噬细胞会导致Raf-1的磷酸化和激活。这与MEK-1和MAP激酶活性的刺激以及转录因子Elk-1（MAP激酶的核靶点）的磷酸化同时发生。在用酪氨酸激酶抑制剂染料木黄酮预处理细胞后，Raf/MAP激酶途径的激活受到抑制。因此，Raf-1必然位于LPS信号转导中酪氨酸激酶的下游。然而，Raf-1不是LPS诱导的酪氨酸激酶的直接底物，因为从LPS诱导的细胞中免疫分离的Raf-1仅含有磷酸丝氨酸。这类似于巨噬细胞受到CSF-1刺激后的情况，其中Raf-1明显转导由CSF-1受体激酶产生的信号，但仅在丝氨酸处磷酸化。从LPS或CSF-1处理的细胞中免疫沉淀的Raf-1的磷酸肽图谱无法区分，表明这些试剂通过类似机制激活Raf-1。最后，发现v-raf感染的BAC-1.2F5巨噬细胞组成性表达低水平的IL-1β和TNF-α。这些数据表明，在LPS介导的巨噬细胞激活和细胞因子产生过程中，Raf-1在酪氨酸激酶下游发挥作用。
