FUNCTIONAL ANALYSIS OF THE RELATIONSHIP BETWEEN RAF AND OTHER GROWTH REGULATORS

RAF 与其他生长调节因子之间关系的功能分析

• 批准号: 3838391
• 负责人: U R RAPP
• 金额: --
• 依托单位: DIVISION OF CANCER EPIDEMIOLOGY AND GENETICS
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/3838391
• 关键词: 3T3 cells; antisense nucleic acid; biological signal transduction; cell growth regulation; genetic regulation; growth factor; immunoprecipitation; mitogens; molecular site; monoclonal antibody; neoplastic transformation; oncogenes; phorbols; phosphorylation; protein tyrosine kinase; pyruvate kinase; regulatory gene

Cytoplasmic serine/threonine-specific kinases such as raf, protein kinase C, and mitogen activated protein (MAP) kinases play an integrative role in the transduction of signals from the cell membrane to the nucleus. To dissect raf-specific signaling pathways, we designed Raf-1 inhibition experiments employing monoclonal antibodies as well as expression of antisense RNA. The isolation of cells resistant to transformation by v- raf constituted a second strategy to identify factors that regulate raf- dependent signaling pathways. We conclude that Raf kinases function downstream of Ras, and that Raf-1 signaling is essential for transformation of NIH/3T3 cells by peripheral oncogenes and for regulation of a subset of early response genes by 12-0-tetradecanoylphorbol-13- acetate and serum growth factors. We have now determined the relative position of Raf and another Ras-controlled protein kinase family, the MAP or extracellular signal regulated kinase (ERK) kinases. c-Raf-1 and MAP kinases are serine/threonine protein kinases activated by numerous mitogens and extracellular agonists. The MAP kinases are regulated by phosphorylation at tyrosine and threonine, and are catalyzed by a novel kinase provisionally named MAP kinase-kinase (MAP-KK) c-Raf-1 is also regulated by phosphorylation and upon activation often complexes with receptor tyrosine kinases. We found that NIH/3T3 cells, stably transformed with v-raf, display constitutively active MAP kinases (42 and 44 kilodaltons) and MAP-KK. Moreover, c-Raf-1, immunoprecipitated from mitogen-treated cells or purified after baculoviral expression, can directly reactivate phosphatase-2A- inactivated MAP-KK in vitro. These results indicate that c-Raf-1 is the immediate proximal activator of MAP- KK.

细胞质丝氨酸/苏氨酸特异性激酶，如raf、蛋白激酶 C和丝裂原活化蛋白（MAP）激酶在 信号从细胞膜到细胞核的传递。 到 分析raf特异性信号通路，我们设计了Raf-1抑制剂， 使用单克隆抗体的实验以及 反义RNA 抗V-转化细胞的分离 RAF构成了第二个战略，以确定调节RAF的因素， 依赖信号通路。 我们的结论是Raf激酶的功能 Ras下游，Raf-1信号传导对于 外周癌基因对NIH/3 T3细胞的转化及其调节 12-0-tetradecanoylphorbol-13- 醋酸盐和血清生长因子。 我们现在已经确定了 Raf和另一个Ras控制的蛋白激酶家族MAP的位置 或细胞外信号调节激酶（ERK）激酶。 c-Raf-1和MAP 激酶是丝氨酸/苏氨酸蛋白激酶，其被多种 有丝分裂原和细胞外激动剂。 MAP激酶受以下因素调节： 在酪氨酸和苏氨酸磷酸化，并由一种新的 暂时命名为MAP激酶-激酶（MAP-KK）c-Raf-1的激酶也 受磷酸化调节且激活后通常与 受体酪氨酸激酶。 我们发现NIH/3 T3细胞， 用v-raf转化，显示组成型活性MAP激酶（42和 44千道尔顿）和MAP-KK。 此外，c-Raf-1，免疫沉淀从 丝裂原处理的细胞或杆状病毒表达后纯化的细胞可 在体外直接再活化磷酸酶-2A-失活的MAP-KK。 这些 结果表明，c-Raf-1是MAP的近端激活剂。 KK.