Mechanisms of MEK/ERK growth arrest signaling

描述(由申请人提供)：本项目的目标是确定MEK/ERK信号在调节细胞生长停滞和细胞增殖对RAS和/或Raf信号异常的反应中的机制差异。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，该通路的持续激活导致正常细胞和某些癌症类型的细胞周期停滞和衰老。有人提出，生长抑制是对抗RAS/Raf介导的致癌的一种防御机制，克服这一“生长抑制障碍”是肿瘤进展过程中的必要步骤。目前，我们对这一控制在MEK/ERK水平上的致癌关键事件的了解相当有限。我们已经开始使用正常和RAS/Raf反应的肿瘤细胞作为模型来研究MEK/ERK信号的潜在机制。第一个耐人寻味的发现是，ERK不仅通过利用其“典型”的激酶活性，而且还通过利用其非催化功能来调节生长停滞。我们最近报道，致癌的Raf信号诱导的生长停滞可以通过ERK1/2缺失而被取消，但将催化失活的突变体ERK引入ERK1/2缺失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与非活性的MEK结合，而mortalin的缺失增加了基础的和Raf诱导的MEK/ERK的活性。此外，mortalin的缺失促进了生长抑制信号的传递，而mortalin的过度表达则起到相反的作用。这些初步研究表明，MEK/ERK利用一种独特的信号机制来调节生长停滞，其中mortalin可能通过与该途径的物理相互作用发挥负调节作用，而ERK调节一种需要其非催化功能的机制。为了验证这些假说，我们建议(I)通过对正常和K-Rasor B-Raf突变的肿瘤细胞进行功能获得或丧失研究，并通过比较肿瘤组织标本中mortalin的表达水平和改变的MEK/ERK活性，来确定mortalin在调节Raf/MEK/ERK介导的生长抑制信号中的作用；(Ii)确定mortalin是否通过不同地隔离MEK1或MEK2来调节Raf/MEK/ERK通路；以及(Iii)确定非催化ERK功能的分子机制。这项研究将加深我们对MEK/ERK信号阻断RAS/Raf致癌的具体机制的了解。

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.