IQGAP1 in tumorigenesis

在本财年，我们完成了以下工作： 1.分泌的信号蛋白Wnt5a促进Wnt-受体-肌动蛋白-肌球蛋白极性(WRAMP)结构的形成，该结构协调后缘的回缩。我们证明了WRAMP的组装需要IQGAP1与黑色素瘤细胞中的跨膜蛋白黑色素瘤细胞黏附分子(MCAM)之间的相互作用。一旦组装，WRAMP就协调了几个促进膜回缩的过程，包括从细胞后部的内质网释放钙离子。Ca~(2+)既激活了在后缘裂解蛋白质的钙蛋白酶，又促进了肌动球蛋白的收缩，导致了膜的回缩。这些发现表明，IQGAP1在迁移细胞的后缘起作用。 2.证实支架蛋白IQGAP1与人雌激素受体ER和ER直接结合。对多个蛋白质短片段的研究表明，ER与IQGAP1的IQ结构域结合，而ER的铰链区负责结合IQGAP1。此外，IQGAP1和ER从细胞中共沉淀，这种结合受雌二醇的调节。内源性IQGAP1的敲除减弱了雌二醇诱导雌激素反应基因PS2、孕激素受体和细胞周期蛋白D1转录的能力。这些数据表明，IQGAP1与ER结合并调节其转录功能，提示IQGAP1可能是乳腺癌患者治疗的靶点。 3.肝细胞生长因子(HGF)激活受体酪氨酸激酶c-MET，调节细胞骨架和细胞间的接触。HGF刺激增强了某些细胞类型的屏障功能，如内皮细胞。我们发现IQGAP1或rac1的siRNA敲除减弱了HGF诱导的内皮屏障的增加。对其作用机制的研究表明，HGF可诱导RAC-1依赖的IQGAP1、EB1和Cortactin复合体的形成。Cortactin促进肌动蛋白聚合，该复合体是增强屏障功能所需的皮质肌动蛋白重塑和外周微管生长所必需的。这些发现表明，IQGAP1作为肌动蛋白和微管之间的支架，促进HGF下游增强的屏障功能。 4.在某些组织中，HGF促进细胞间黏附的解体和细胞的迁移。例如，HGF诱导DU145前列腺癌和HT29结肠癌细胞的集落逃逸。我们证明，这种作用是由IQGAP1、E-钙粘蛋白和PAK6(p-21激活的6)之间的复合体介导的，PAK6是一种经常在癌症中过度表达的激酶。IQGAP1促进PAK6的激活，然后PAK6磷酸化细胞-细胞连接的一个成分-连环蛋白，以介导连接的分解。这些数据表明，IQGAP1和PAK6之间的相互作用可能有助于肿瘤的转移。

During the fiscal year we accomplished the following: 1. The secreted signaling protein Wnt5a promotes the formation of the Wnt-receptor-actin-myosin-polarity (WRAMP) structure, which coordinates retraction at the trailing edge. We demonstrated that WRAMP assembly requires an interaction between IQGAP1 and the transmembrane protein melanoma cell adhesion molecule (MCAM) in melanoma cells. Once assembled, WRAMP coordinates several processes that promote membrane retraction, including Ca2+ release from the endoplasmic reticulum at the rear of the cell. Ca2+ both activates the protease calpain, which cleaves proteins at the trailing edge, and facilitates actomyosin contraction, leading to membrane retraction. These findings indicate that IQGAP1 operates at the trailing edge of migrating cells. 2. Demonstrated that the scaffold protein IQGAP1 directly binds the human estrogen receptors ERα and ERβ. Investigation with multiple short fragments of the proteins showed that ERα binds to the IQ domain of IQGAP1, whereas the hinge region of ERα is responsible for binding IQGAP1. In addition, IQGAP1 and ERα co-immunoprecipitated from cells, and the association was modulated by estradiol.. Knockdown of endogenous IQGAP1 attenuated the ability of estradiol to induce transcription of the estrogen-responsive genes pS2, progesterone receptor, and cyclin D1. These data reveal that IQGAP1 binds to ERα and modulates its transcriptional function, suggesting that IQGAP1 might be a target for therapy in patients with breast carcinoma. 3. Hepatocyte growth factor (HGF) activates the receptor tyrosine kinase, c-MET, which regulates the cytoskeleton and cell-cell contacts. HGF stimulation strengthens barrier function in some cell types, e.g., endothelial cells. We showed that siRNA knockdown of IQGAP1 or Rac1 attenuated HGF-induced increase in endothelial barrier. Investigation into the mechanism revealed that HGF induced the formation of a Rac-1 dependent complex containing IQGAP1, EB1 and cortactin. Cortactin promotes actin polymerization, and the complex is necessary for cortical actin remodeling and peripheral microtubule growth that are required for strengthening barrier functions. These findings indicate that IQGAP1 functions as a scaffold between actin and microtubules to promote enhanced barrier functions downstream of HGF. 4. In some tissues, HGF promotes disassembly of cell-cell adhesions and cell migration. For example, HGF induces colony escape of DU145 prostate cancer and HT29 colon cancer cells. We demonstrated that this effect is mediated by a complex between IQGAP1, E-cadherin and PAK6 (p-21 activated 6), a kinase that is often overexpressed in cancers. IQGAP1 promotes activation of PAK6, which then phosphorylates β-catenin, a component of cell-cell junctions, to mediate junction disassembly. These data suggest that the interaction between IQGAP1 and PAK6 may contribute to cancer metastasis.