Function and Regulation of Scaffold Proteins in Signal Transduction

Ras激活后，KSR1支架从细胞质转移到质膜，并协调一个大的多蛋白复合物的组装，该复合物的功能是调节ERK级联信号的强度和持续时间。在过去的财政年度，我们已经在MEK1和MEK2富含脯氨酸的序列中发现了一个疏水基序列，这是与KSR1支架组成性结合所必需的，并发现KSR1与B-Raf和MEK形成三元复合物，以响应生长因子处理，增强B-Raf介导的MEK激活。引人注目的是，我们还发现，活性ERK与KSR1支架的对接允许ERK在反馈位点磷酸化KSR1和B-Raf，这些反馈事件通过促进B-Raf /KSR1/MEK复合物的解离并导致KSR1从质膜释放来减弱ERK级联信号。今年，我们实验室还采用蛋白质组学方法进一步研究了两种哺乳动物KSR支架KSR1和KSR2的功能特性。这些研究表明，KSR1和KSR2都与ERK级联的核心激酶组分相互作用，并在促进rtk介导的ERK信号传导方面具有共同的功能。值得注意的是，这些研究还发现蛋白磷酸酶钙调磷酸酶选择性地与KSR2相互作用，KSR2独特地参与Ca2+介导的ERK信号传导。在Ca2+水平升高的情况下，我们发现钙调磷酸酶在特定位点使KSR2去磷酸化，从而调节KSR2的膜定位和支架活性。此外，我们发现KSR2的缺失会在表达KSR2的两种不同细胞系中损害Ca2+介导的ERK激活和信号传导。-细胞和NG108神经母细胞瘤细胞。这些发现确定了KSR2是Ca2+调控的ERK支架，并揭示了Ca2+影响Ras到ERK通路信号传导的新机制。

The KSR1 scaffold translocates from the cytosol to the plasma membrane upon Ras activation and coordinates the assembly of a large multiprotein complex that functions to regulate the intensity and duration of ERK cascade signaling. In the past fiscal year, we have identified a hydrophobic motif in the proline-rich sequence of MEK1 and MEK2 that is required for constitutive binding to the KSR1 scaffold and find that KSR1 forms a ternary complex with B-Raf and MEK in response to growth factor treatment that enhances B-Raf-mediated MEK activation. Strikingly, we have also found that docking of active ERK to the KSR1 scaffold allows ERK to phosphorylate KSR1 and B-Raf on feedback sites and that these feedback events attenuate ERK cascade signaling by promoting the dissociation of the B-RAF/KSR1/MEK complex and causing KSR1 to be released from the plasma membrane. This year our laboratory has also taken a proteomic approach to further investigate the functional properties of the two mammalian KSR scaffolds, KSR1 and KSR2. These studies have revealed that both KSR1 and KSR2 interact with the core kinase components of the ERK cascade and have a common function in promoting RTK-mediated ERK signaling. Remarkably, these studies also found that the protein phosphatase calcineurin selectively interacts with KSR2 and that KSR2 uniquely contributes to Ca2+-mediated ERK signaling. In response to increased Ca2+ levels, we found that calcineurin dephosphorylates KSR2 on specific sites and, as a a result, regulates the membrane-localization and scaffolding activity of KSR2. Moreover, we found that depletion of KSR2 impairs Ca2+-mediated ERK activation and signaling in two different cell lines that express KSR2, INS1 pancreatic β-cells and NG108 neuroblastoma cells. These findings identify KSR2 as a Ca2+-regulated ERK scaffold and reveal a new mechanism whereby Ca2+ impacts Ras to ERK pathway signaling.