Regulation of Ras-Dependent Signal Transduction Pathways

RAS通路是调控细胞增殖、分化和存活的重要信号转导通路。Raf丝氨酸/苏氨酸激酶家族成员是该途径的关键中间体，其功能是将信号从激活的RAS传递到下游的蛋白激酶MEK和ERK。在哺乳动物细胞中发现了三种Raf蛋白：Raf-1、A-Raf和B-Raf。正如在细胞信号转导中起中心作用的蛋白质所预期的那样，Raf激酶也参与了致癌转化和癌症。例如，Raf上游调节因子的突变或扩增，如受体酪氨酸激酶和RAS，经常通过Raf/MEK/ERK级联反应在含有这些等位基因的肿瘤中诱导信号失控。此外，结构上具有活性的Raf蛋白本身也可以引起细胞转化。特别是，在67%的恶性黑色素瘤以及许多结直肠癌、卵巢癌和乳头状甲状腺癌中观察到B-Raf家族成员的突变。在过去的这一财年，我们的研究确认CK2是一种上游蛋白激酶，有助于激活Raf。我们的研究还表明，B-Raf激酶的致癌潜力可以通过特定的磷酸化事件和蛋白质相互作用来改变。总之，这些发现确定了对具有结构性RAS或Raf依赖信号的肿瘤进行治疗干预的潜在靶点。RAS途径的另一个保守成分是KSR1，这是一种作为分子支架的蛋白质，促进了ERK级联信号的传递。在过去的一年里，我们的研究为KSR1在神经系统中的内源性支架作用提供了新的见解。这项工作证明了KSR1在海马区以生物化学的方式发挥作用，以支架ERK级联的组成部分，当ERK的膜部分通过依赖PKC的途径而不是通过cAMP/PKA依赖的途径被激活时，KSR1特异性地调节该级联。与这些发现一致的是，缺乏KSR1的小鼠被发现在联想学习和某些类型的突触可塑性方面存在缺陷。在本财年，我们还发现KSR1在凋亡细胞中经历了caspase依赖的切割，这种切割破坏了KSR1的支架功能，并产生了稳定的C末端片段(CTF)，可以抑制ERK的激活。这些发现表明，KSR1支架的裂解是半胱氨酸酶下调ERK生存信号促进细胞凋亡的另一种机制。

The Ras pathway is a critical signal transduction cascade involved in regulating cellular proliferation, differentiation, and survival. Members of the Raf serine/threonine kinase family are key intermediates in this pathway, functioning to relay signals from activated Ras to the downstream protein kinases, MEK and ERK. Three Raf proteins are found in mammalian cells, Raf-1, A-Raf and B-Raf. As might be expected for proteins so centrally involved in cell signaling, the Raf kinases also contribute to oncogenic transformation and cancer. For example, mutation or amplification of upstream regulators of Raf, such as receptor tyrosine kinases and Ras, frequently induces deregulated signaling through the Raf/MEK/ERK cascade in tumors harboring these alleles. Moreover, constitutively active Raf proteins can themselves cause cell transformation. In particular, mutation of the B-Raf family member is observed in 67% of malignant melanomas as well as in many colorectal, ovarian and papillary thyroid carcinomas. During this past fiscal year, our research identified CK2 as an upstream protein kinase that contributes to Raf activation. Our studies has also revealed that the oncogenic potential of the B-Raf kinase can be altered by specific phosphorylation events and protein interactions. Together, these findings identify potential targets for therapeutic intervention in tumors with constitutive Ras- or Raf-dependent signaling. Another conserved component of the Ras pathway is KSR1, a protein that acts as a molecular scaffold to facilitate signal transmission through the ERK cascade. During the past fiscal year, our studies provided novel insight into the endogenous scaffolding role of KSR1 within the nervous system. This work demonstrated that KSR1 functions biochemically in the hippocampus to scaffold the components of the ERK cascade, specifically regulating the cascade when a membrane fraction of ERK is activated via a PKC-dependent pathway but not via a cAMP/PKA-dependent pathway. Consistent with these findings, mice lacking KSR1 were found to have deficits in associative learning and certain typses of synaptic plasticity. In this fiscal year, we also discovered that KSR1 undergoes caspase-dependent cleavage in apoptotic cells and that cleavage destroys the scaffolding function of the KSR1 and generates a stable C-terminal fragment (CTF) that can inhibit ERK activation. These findings indicate that cleavage of the KSR1 scaffold is another mechanism whereby caspases down-regulate ERK survival signaling to promote cellular apoptosis.