Regulation of MGMT by Phosphorylation

描述（申请人提供）：o6 -甲基鸟嘌呤- dna甲基转移酶（MGMT）是一个非常有前途的靶点，可以提高烷基化剂治疗脑肿瘤和其他癌症的疗效。最近，我们的实验室首次提供了人类MGMT蛋白在酪氨酸和丝氨酸/苏氨酸位点磷酸化的数据，并且磷酸化抑制了其活性。两种细胞蛋白激酶磷酸化MGMT蛋白中的这些氨基酸。本提案的总体目标是明确蛋白磷酸化在MGMT功能中的作用，并评估其对改善胶质瘤治疗的意义。我们假设MGMT的多位点磷酸化控制其在人类胶质瘤中的催化活性和蛋白质稳定性。我们假设肿瘤MGMT的净磷酸化状态深刻影响其功能，进而影响胶质瘤对烷基化剂的敏感性。在这个为期4年的项目中，具体目标是(1)确定人MGMT的体内磷酸化位点，并确定每种磷酸化对MGMT DNA修复活性的贡献；(2)通过泛素蛋白水解途径表征MGMT蛋白磷酸化依赖性的转化；(3)生成针对MGMT蛋白的磷酸化特异性抗体，评估胶质瘤标本、细胞系、细胞系、细胞系中磷酸化-MGMT的水平。并将其与MGMT的催化活性和药物敏感性联系起来。磷酸化对MGMT DNA结合域的结构影响，以及随之而来的MGMT对o6 -甲基鸟嘌呤和o6 -苄基鸟嘌呤活性的改变，也将作为第一个Specific Aim的一部分进行研究。在细胞周期进程中MGMT磷酸化的变化将通过使用磷酸化特异性抗体来确定。这些原创性的研究有望为MGMT的生理调控提供新的关键信息，并可能为改进胶质瘤治疗和MGMT靶向癌症治疗提供新的方向。

DESCRIPTION (provided by applicant): O6-Methylguanine-DNA methyltransferase (MGMT) is a highly promising target for improving the efficacy of alkylating agents in the treatment of brain tumors and other cancers. Recently, our laboratory provided the first data that human MGMT protein is phosphorylated at tyrosine and serine/threonines and that phosphorylation inhibits its activity. Two cellular protein kinases that phosphorylate these amino acids in the MGMT protein were also characterized. The overall objective of this proposal is to define the role of protein phosphorylation in MGMT function and to assess its significance to improve glioma therapy. We hypothesize that multisite phosphorylation of MGMT controls its catalytic activity, and protein stability in human gliomas. We postulate that the net phosphorylation state of tumor MGMT profoundly affects its function and, in turn the sensitivity of gliomas to alkylating agents. The Specific Aims to be pursued in this 4-year project are (1) to identify the in vivo phosphorylation sites of human MGMT and define the contribution of each phosphorylation to MGMT's DNA repair activity, (2) to characterize the phosphorylation-dependent turn-over of the MGMT protein through the ubiquitin-proteolytic pathway, and (3) to generate phospho-specific antibodies against the MGMT protein, assess the levels of phospho-MGMT in glioma specimens, cell lines, and to correlate it with the catalytic activity of MGMT and drug sensitivity. The structural impact of phosphorylations on the DNA binding domain of MGMT, and the consequent alterations in MGMT activity towards O6-methylguanine, and O6-benzylguanine will also be examined as a part of the first Specific Aim. Changes in MGMT phosphorylation during the cell cycle progression will be determined by using the phospho-specific antibodies. These original studies promise to yield novel and critical information on the physiological regulation of MGMT, and are likely to provide new directions for improved glioma therapy and MGMT-targeted cancer therapies.