Regulation of Her2/neu by activation loop phosphorylation and ALG-2

申请人：罗恩·博斯，医学博士，毕业于康奈尔大学-洛克菲勒大学-纪念斯隆-凯特琳癌症中心联合举办的医学科学家培训项目。他的博士研究在斯隆·凯特林纪念馆进行，研究了诱导细胞凋亡的脂质第二信使途径。Bose博士于2002年完成内科住院医师资格，并进入约翰·霍普金斯大学的肿瘤内科奖学金。他的目标是通过应用蛋白质组学来研究乳腺癌的信号转导途径，从而进行基础和翻译研究。他发表了Her2/neu酪氨酸激酶信号的蛋白质组学研究(附录A)，并将这些结果与文献中已有的关于表皮生长因子受体(EGFR)信号的蛋白质组学研究进行了系统的比较。在这项提案中，他计划通过集中的机制实验，继续两个相关的蛋白质组学发现，即Her2/neu激活环酪氨酸877的磷酸化和Her2/neu诱导的凋亡相关基因2(ALG2)的磷酸化。激活环磷酸化和ALG-2磷酸化都可能是Her2/neu功能的调节机制，并可能有助于在二聚化发生后控制受体酪氨酸激酶。激活环是激活域的一个主要结构特征，已被证明调节胰岛素受体激酶和Src的活性。ALG-2与参与受体内化的蛋白质相互作用。Her2/neu的内化和降解是抗Her2/neu抗体曲妥珠单抗(Herceptin)的主要作用机制。在这项提议中，具体目的将测试：1)重组的Her2/neu激动域是否在体外能够在激活环上自我磷酸化，以及这一过程与最近的激活域二聚化模型的相互作用；2)激活环位点(Y877F)的突变是否影响下游信号转导和曲妥珠单抗诱导的Her2/neu内化；3)ALG-2及其相互作用蛋白Alix/AIP1是否影响曲妥珠单抗诱导的Her2/neu内化和降解。这项研究将在约翰·霍普金斯大学肿瘤学系进行。相关性：HER2/neu和EGFR是参与多种人类癌症的蛋白激酶。对它们的调节进行仔细的、机械性的研究将导致更好地应用当前的靶向治疗，并可能设计出一种用于癌症治疗的新类型的激酶抑制剂。

DESCRIPTION (provided by applicant): The candidate, Ron Bose, M.D., Ph.D., is a graduate of the Medical Scientist Training Program held jointly at Cornell University- Rockefeller University- Memorial Sloan-Kettering Cancer Center. His Ph.D. research, conducted at Memorial Sloan Kettering, studied a lipid second messenger pathway which induced apoptosis. Dr. Bose completed residency in Internal Medicine in 2002 and entered Medical Oncology fellowship at Johns Hopkins University. His goals are to pursue basic and translational research by applying proteomics to study signal transduction pathways in breast cancer. He has published a proteomic study of Her2/neu tyrosine kinase signaling (Appendix A) and systematically compared these results to proteomic studies of Epidermal growth factor receptor (EGFR) signaling already in the literature. In this proposal, he plans to pursue two related proteomic findings, Her2/neu activation loop phosphorylation on tyrosine 877 and Her2/neu induced phosphorylation of Apoptosis-linked gene 2 (ALG2), with focused mechanistic experiments. Both activation loop phosphorylation and ALG-2 phosphorylation may be regulatory mechanisms for Her2/neu function and may help control the receptor tyrosine kinase after dimerization has occurred. The activation loop is a major structural feature of kinase domains and has been shown to regulate the activity of insulin receptor kinase and Src. ALG-2 interacts with proteins involved in receptor internalization. Internalization and degradation of Her2/neu is a major mechanism of action of the anti-Her2/neu antibody, trastuzumab (Herceptin). In this proposal, the specific aims will test: 1) if recombinant Her2/neu kinase domain can in vitro autophosphorylate itself on the activation loop and the interaction of this process with a recent model of kinase domain dimerization, 2) if mutation of the activation loop site (Y877F) affects downstream signaling and trastuzumab-induced Her2/neu internalization, 3) if ALG-2 and its interacting protein Alix/AIP1 affect trastuzumab induced Her2/neu internalization and degradation. This research will be performed in the Department of Oncology, Johns Hopkins University. Relevance: Her2/neu and EGFR are protein kinases involved in multiple human cancers. Careful, mechanistic study of their regulation will result in better application of current targeted therapies and possible design of a new class of kinase inhibitors for the treatment of cancer.