Mechanisms of MEK/ERK growth arrest signaling

描述（由申请人提供）：该项目的目标是确定MEK/ERK信号的机制差异，MEK/ERK信号在异常Ras和/或Raf信号的反应中介导生长停滞和细胞增殖。Ras/Raf/MEK/ERK通路的异常激活是许多癌症的中心特征。然而，矛盾的是，该通路的持续激活会导致正常细胞和某些癌症类型的细胞周期停滞和衰老。已经提出，生长停滞是Ras/ raf介导的癌变的防御机制，克服这种“生长停滞屏障”是肿瘤进展的必要步骤。我们对这一在MEK/ERK水平上控制的致癌过程中的关键事件的了解目前相当有限。我们已经开始使用正常和Ras/ raf应答的肿瘤细胞作为模型来解决MEK/ERK信号传导的潜在机制。第一个有趣的发现是，ERK不仅可以利用其“规范”激酶活性，还可以利用尚未确定的非催化功能来介导生长停滞。我们最近报道了致癌Raf信号诱导的生长停滞被ERK1/2耗尽所消除，但将催化失能的突变体ERK引入ERK1/2耗尽的细胞可以选择性地恢复生长停滞现象。基于MEK/ERK与特定蛋白相互作用介导生长停滞信号的假设，我们进行了串联亲和纯化，并确定了mortalin是MEK/ERK生长停滞信号的潜在负调控因子。我们的初步研究表明，mortalin与非活性MEK结合，而不是活性MEK，并且mortalin的缺失增加了基础和raf诱导的MEK/ERK活性。此外，mortalin的缺失促进生长抑制信号，而mortalin的过表达则发挥相反的作用。这些初步研究表明，MEK/ERK利用一种独特的信号机制介导生长停滞，其中mortalin可能通过其与该途径的物理相互作用发挥负调控作用，而ERK调节的机制需要其非催化功能。为了验证这些假设，我们建议(i)通过在正常和K-Rasor B-Raf突变的肿瘤细胞中进行功能的获得或丧失研究，并通过比较肿瘤组织标本中死亡蛋白的表达水平与改变的MEK/ERK活性，来确定死亡蛋白在Raf/MEK/ERK介导的生长停滞信号的调节中的作用；（ii）确定mortalin是否通过差异隔离MEK1或MEK2来调节Raf/MEK/ERK通路；（iii）确定非催化性ERK功能的分子机制。这项研究将增强我们对MEK/ERK信号阻断Ras/ raf驱动的致癌作用的具体机制的认识。

DESCRIPTION (provided by applicant): The goal of this project is to determine the mechanistic differences in MEK/ERK signaling that mediates growth arrest versus cell proliferation in response to aberrant Ras and/or Raf signals. Aberrant activation of the Ras/Raf/MEK/ERK pathway is a central feature in many cancers. However, paradoxically, sustained activation of the pathway induces cell cycle arrest and senescence in normal cells and certain cancer types. It has been proposed that growth arrest acts as a defense mechanism against Ras/Raf-mediated carcinogenesis and that overcoming this "growth arrest barrier" is a necessary step in tumor progression. Our understanding of this key event in carcinogenesis, controlled at the level of MEK/ERK, is currently quite limited. We have begun to address the underlying mechanisms of MEK/ERK signaling using normal and Ras/Raf-responsive tumor cells as models. The first intriguing finding is that ERK can mediate growth arrest by utilizing not only its "canonical" kinase activity but also, as yet unidentified, non-catalytic functions. We recently reported that oncogenic Raf signal-induced growth arrest is abrogated by ERK1/2 depletion but introduction of catalytically disabled mutant ERK into ERK1/2- depleted cells can selectively restore the growth arrest phenomenon. Based upon a hypothesis that MEK/ERK would interact with specific proteins to mediate growth arrest signaling, we conducted tandem affinity purification and identified mortalin as a potential negative regulator of MEK/ERK-growth arrest signaling. Our preliminary studies show that mortalin binds to inactive, but not active, MEK and that mortalin depletion increases basal as well as Raf-induced MEK/ERK activity. In addition, mortalin depletion promotes growth inhibitory signaling whereas mortalin overexpression exerts the opposite effects. These preliminary studies suggest that MEK/ERK utilizes a unique signaling mechanism to mediate growth arrest, for which mortalin has a negative-regulatory role possibly via its physical interaction with the pathway and ERK modulates a mechanism that requires its non-catalytic function. To test these hypotheses, we propose to (i) determine the role of mortalin in the regulation of Raf/MEK/ERK-mediated growth arrest signaling by gain or loss of function studies in normal and K-Rasor B-Raf mutated tumor cells and by comparing mortalin expression levels with altered MEK/ERK activity in tumor tissue specimens; (ii) determine whether mortalin regulates the Raf/MEK/ERK pathway by differentially sequestering MEK1 or MEK2; and (iii) determine molecular mechanisms underlying the non-catalytic ERK functions. This study will enhance our knowledge of the specific mechanisms of MEK/ERK signaling that interrupts Ras/Raf-driven carcinogenesis. PUBLIC HEALTH RELEVANCE: Activation of the MEK/ERK pathway induces terminal growth arrest in certain types of cancer lines derived from small cell lung carcinoma, medullary thyroid carcinoma, prostate carcinoma, pheochromocytoma, glioma, and gastrointestinal carcinoid. Exploration of the possibility that these cancer types can be effectively suppressed by modulating the pathway via mortalin into the direction of growth arrest may allow development of new therapeutic strategies to control them.