CELL GROWTH REGULATION BY THE MEKK-SAPK CASCADE

生长信号在正常和恶性细胞中的转导机制 通过分析细胞质激酶变得更加清晰 被这些信号激活。对这种激活级联的刺激是一种 癌症形成的常见后果，也可能是原因。一个 这一级联反应的中心靶点出现在MAP激酶上，因为MAP激酶 通过大量的有丝分裂刺激激活，包括那些 在致癌转化中感到不安。目前的证据是MAP激酶 被另一种名为MEK的激酶磷酸化激活，而该MEK 可以被两种不同的激酶激活，RAF和MEK激酶。 尚未回答的问题是，这些单独的激酶如何调节细胞 转型和其他与增长相关的过程。到目前为止，基因工具 这将有助于表征MEK在MAP激酶中的作用 激活途径尚未被描述。我们建议对MEK进行调查 分子细节的生理学，并利用这些信息来开发活性 或MEK的抑制性突变体，以检验MEK参与的假设 在细胞转化至关重要的细胞过程中，转录 刺激性、刺激性等特点。 因此，我们建议： 1)用RAF和MEK激酶鉴定MEK的磷酸化位点。 2)鉴定和鉴定MEK的组成性活性突变体 活性不需要磷酸化。 3)鉴定和鉴定MEK和TO的显性抑制突变 用它们来表征通过MEK的信号转导。 4)用MAP激酶确定MEK的磷酸化位点。(该等 “反馈”磷酸化事件可能导致MAP的自动调节 通过自动调节环路激活的激酶)。 5)测试调节性基因截断后的MEK激酶激活 结构域，并测量MEK激酶作为一个 致癌基因。

Mechanisms of transduction of growth signals in normal and malignant cells are becoming clearer through analysis of cytoplasmic kinases that are activated by these signals. Stimulation of this kinase cascade is a frequent consequence, and possible also a cause, of cancer formation. A central target of this cascade appears to the MAP kinase, since MAP kinase is activated via numerous mitogenic stimuli including those that are perturbed in oncogenic transformation. Current evidence is that MAP kinase is activated by phosphorylation by another kinase, termed MEK, and that MEK can be activated by two distinct kinases, raf and MEK kinase. Unanswered is the question of how these individual kinases regulate cell transformation and other growth-related processes. To date, genetic tools that would be of use to characterize the role of MEK in the MAP kinase activation pathway have not been described. We propose to investigate MEK physiology in molecular detail, and use this information to develop active or inhibitory mutants of MEK to test the hypothesis that MEK participates in cellular processes vital for cell transformation, transcriptional stimulation, and other features of growth stimulation. Therefore, we propose: 1) To identify sites of phosphorylation on MEK by raf and MEK kinase. 2) To identify and characterize constitutive active mutants of MEK that do not require phosphorylation for activity. 3) To identify and characterize dominant inhibitory mutants of MEK and to use them to characterize signal transduction through MEK. 4) To identify sites of phosphorylation on MEK by MAP kinase. (Such "feedback" phosphorylation events may result in autoregulation of MAP kinase activity via an autoregulatory loop). 5) To test MEK kinase activation following truncation of a regulatory domain, and to measure the ability of MEK kinase to funcTIon as an oncogene.