The role of tissue transglutaminase in cancer cell migration

描述(由申请人提供)：细胞迁移对细胞形态发生和组织修复以及许多其他重要的生物学结果是必不可少的，并与各种发育障碍和疾病状态密切相关。尤其令人感兴趣的是潜在的细胞迁移调节事件，因为它们与细胞侵袭、转移和癌症进展有关。组织转谷氨酰胺酶(TTG)是一种与GTP结合的蛋白，它还具有酶促转氨酶活性，使其能够使蛋白质交联，已被证明在细胞迁移和侵袭过程中发挥着重要作用。作为表皮生长因子(EGF)受体下游的信号蛋白，tTG被激活，并被募集到细胞的前沿。除了在细胞迁移和侵袭中发挥作用外，tTG还被证明在RAS驱动的转化中起重要作用。这一应用的主要焦点是更多地了解tTG的激活和定位到迁移细胞的前沿如何参与人类癌细胞表现出的增强的细胞迁移和侵袭活性，并有助于RAS介导的细胞转化。为实现这一目标，将开展以下调查工作。1)确定负责将tTG定位于活跃迁移细胞前沿的信号机制。通过RAS向JNK传递的EGF受体信号对tTG的激活和定位具有重要意义。这一目标将涉及确定JNK调节这些效应的底物。特别是，我将确定tTG或Hsp70是否可以作为JNK的底物，或者其他已知底物(即巴西林)是否对调节tTG的激活和定位很重要。2)确定热休克蛋白70(Hsp70)的伴侣功能如何帮助将tTG靶向细胞前沿。这一系列研究源于我们最近的发现，即Hsp70和tTG沿着细胞的前沿相互作用和共定位，这种定位在Hsp70、杨梅素、亚甲蓝和VER 155008三磷酸腺苷水解酶活性抑制剂的存在下被阻断。这一目标将涉及识别Hsp70的客户蛋白，这些蛋白有助于将tTG靶向领先的 单元格的边缘。3)确定tTG如何与RAS协同促进细胞转化。通过RAS的EGF信号导致tTG被激活并定位到前沿，RAS需要tTG才能发挥其转化潜能。为此，我将确定tTG促进的RAS转化是由于tTG增强RAS活性或激活其下游效应因子的结果，还是由于tTG激活了RAS非依赖性信号事件，与RAS信号协同推动恶性转化。这些研究应该有助于深入了解tTG激活和定位与细胞前沿结合的意义，以及tTG如何促进细胞迁移和侵袭以及细胞转化。

DESCRIPTION (provided by applicant): Cell migration is essential for cellular morphogenesis and tissue repair, as well as a number of other important biological outcomes, and has a critical involvement in various developmental disorders and disease states. Of particular interest have been the regulatory events underlying cell migration as they relate to cell invasiveness, metastasis, and cancer progression. Tissue transglutaminase (tTG), a GTP- binding protein which also possesses an enzymatic transamidation activity that allows it to cross-link proteins, has been shown to play an important role in cell migration and invasion. As a signaling protein downstream of the epidermal growth factor (EGF) receptor, tTG becomes activated and is recruited to the leading edges of cells in response to EGF. In addition to its role in cell migratin and invasion, tTG has been shown to be important for Ras-driven transformation. The primary focus of this application is to learn more about how the activation and localization of tTG to the leading edges of migrating cells participates in the enhanced cell migration and invasive activity exhibited by human cancer cells and contributes to Ras- mediated cellular transformation. The following lines of investigation will be carried out to achieve this goal. 1) Determine the signalig mechanisms responsible for localizing tTG to the leading edges of actively migrating cells. EGF receptor signaling through Ras to JNK is important for tTG activation and localization. This aim will involve identifying the substrates of JNK that mediate these effects. In particular, I will determine whether tTG or Hsp70 can serve as a substrate for JNK or whether other known substrates (i.e. paxillin) are important for regulating tTG activation and localization. 2) Determie how the chaperonin function of heat shock protein 70 (Hsp70) helps target tTG to the leading edges of cells. This line of study stems from our recent findings that Hsp70 and tTG interact and co-localize along the leading edges of cells and this localization is blocked in the presence of inhibitors of the ATP hydrolytic activity of Hsp70, myricetin, methylene blue, and VER 155008. This aim will involve identifying the client proteins of Hsp70 which help target tTG to the leading edges of cells. 3) Determine how tTG works with Ras to promote cellular transformation. EGF signaling through Ras results in activation and localization of tTG to leading edges and Ras needs tTG for its transforming potential. In this aim, I will determine whether tTG- promoted Ras transformation occurs as a result of tTG's ability to enhance Ras activity or the activation of one of its downstream effectors, or whether tTG activates a Ras-independent signaling event that synergizes with Ras signaling to drive malignant transformation. These studies should provide insight into the significance of coupling tTG activation and localization to the leading edges of cells and how tTG is contributing to cell migration and invasion as well as cellular transformation.