Kolch W, Philipp A, Mischak H, Dutil E M, Mullen T M, Feramisco J R, Meinkoth J L, Rose D W
Department of Medicine, University of California at San Diego, La Jolla 92093, USA.
Oncogene. 1996 Sep 19;13(6):1305-14.
Raf-1 is a serine/threonine specific kinase that integrates signaling by a large number of mitogens to elicit a transcriptional response in the nucleus. Activated Raf-1 phosphorylates and activates MAPK/ERK kinase Mek), thus initiating the Mek--> MAP kinase cascade, which ultimately results in the phosphorylation and activation of transcription factors by MAP kinase. Here we have characterized the mechanism by which monoclonal antibody URP26K, which binds to an epitope in the Raf-1 kinase domain, inhibits intracellular signal transduction. This antibody preferentially immunoprecipitated the underphosphorylated, non-activated form of Raf-1 from quiescent cells. Baculovirus-expressed Raf-1 immunoprecipitated with URP26K was largely refractory to phosphorylation and activation mediated by protein kinase C (PKC)alpha or the tyrosine kinase Lck. In addition, URP26K reduced the binding of Raf-1 to its substrate Mek in vitro, but did not disturb the association of Raf-1 with Ras. Microinjection of URP26K into Rat-1 cells blocked DNA synthesis initiated by serum, insulin and various purified growth factors, but it did not block DNA synthesis initiated by v-ras. Microinjected URP26K also impaired the expression of stably transfected beta-galactosidase reporter genes regulated by minimal promoter elements. These results demonstrate, (i) that the URP26K monoclonal antibody inhibits Raf-1 by preventing activating Raf-1 phosphorylation and/or association with its substrate Mek, (ii) that inhibition of Raf-1 by URP26K does not interfere with Ras-induced DNA synthesis. In contrast to dominant negative Raf-1 mutants, which also block Ras signaling by binding to the Ras effector domain, antibody mediated Raf-1 inhibition thus reveals a branchpoint of mitogenic signaling at the level of Ras.
Raf-1是一种丝氨酸/苏氨酸特异性激酶，它整合多种有丝分裂原的信号，以引发细胞核中的转录反应。活化的Raf-1磷酸化并激活丝裂原活化蛋白激酶/细胞外信号调节激酶（MAPK/ERK激酶，即Mek），从而启动Mek→MAP激酶级联反应，最终导致MAP激酶对转录因子进行磷酸化和激活。在此，我们阐述了与Raf-1激酶结构域中的一个表位结合的单克隆抗体URP26K抑制细胞内信号转导的机制。该抗体优先从静止细胞中免疫沉淀未充分磷酸化的、未活化形式的Raf-1。用URP26K免疫沉淀的杆状病毒表达的Raf-1在很大程度上对蛋白激酶C（PKC）α或酪氨酸激酶Lck介导的磷酸化和激活具有抗性。此外，URP26K在体外降低了Raf-1与其底物Mek的结合，但并未干扰Raf-1与Ras的结合。将URP26K显微注射到大鼠-1细胞中可阻断由血清、胰岛素和各种纯化生长因子引发的DNA合成，但不阻断由v-ras引发的DNA合成。显微注射的URP26K还损害了由最小启动子元件调控的稳定转染的β-半乳糖苷酶报告基因的表达。这些结果表明，（i）URP26K单克隆抗体通过阻止Raf-1的激活磷酸化和/或与其底物Mek的结合来抑制Raf-1，（ii）URP26K对Raf-1的抑制不干扰Ras诱导的DNA合成。与通过结合Ras效应结构域也阻断Ras信号传导的显性负性Raf-1突变体不同，抗体介导的Raf-1抑制因此揭示了有丝分裂信号在Ras水平的一个分支点。
