Regulation of MGMT by Phosphorylation

描述（由申请人提供）：O 6-甲基鸟嘌呤-DNA甲基转移酶（MGMT）是一种非常有前途的靶点，可用于改善烷化剂治疗脑肿瘤和其他癌症的疗效。最近，我们的实验室提供了第一个数据，人类MGMT蛋白是磷酸化的酪氨酸和丝氨酸/苏氨酸和磷酸化抑制其活性。两个细胞蛋白激酶，磷酸化这些氨基酸的MGMT蛋白质的特点。该提案的总体目标是确定蛋白磷酸化在MGMT功能中的作用，并评估其对改善胶质瘤治疗的意义。我们推测，MGMT的多位点磷酸化控制其催化活性和蛋白质在人脑胶质瘤中的稳定性。我们推测肿瘤MGMT的净磷酸化状态深刻地影响其功能，进而影响胶质瘤对烷化剂的敏感性。这个为期4年的项目的具体目标是：（1）鉴定人MGMT的体内磷酸化位点，并确定每个磷酸化对MGMT DNA修复活性的贡献，（2）表征MGMT蛋白通过泛素蛋白水解途径的磷酸化依赖性翻转，以及（3）产生针对MGMT蛋白的磷酸特异性抗体，评估胶质瘤标本、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相关联。磷酸化对MGMT的DNA结合结构域的结构影响，以及MGMT对O 6-甲基鸟嘌呤和O 6-苯甲基鸟嘌呤的活性随之发生的改变，也将作为第一个特定目标的一部分进行检查。将通过使用磷酸化特异性抗体测定细胞周期进展期间MGMT磷酸化的变化。这些原创性研究有望为MGMT的生理调节提供新的关键信息，并可能为改善胶质瘤治疗和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.