Regulation of MGMT by Phosphorylation

通过磷酸化调节 MGMT

• 批准号: 6800109
• 负责人: KALKUNTE S SRIVENUGOPAL
• 金额: $ 22.2万
• 依托单位: TEXAS TECH UNIVERSITY HEALTH SCIS CENTER
• 依托单位国家: 美国
• 财政年份: 2002
• 资助国家: 美国
• 起止时间: 2002-09-16 至 2006-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6800109
• 关键词: DNA repair; biological signal transduction; cell line; clinical research; enzyme activity; human tissue; matrix assisted laser desorption ionization; methylguanine DNA methyltransferase; monoclonal antibody; phosphorylation; proteolysis; ubiquitin

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.

描述（由申请人提供）：O6-甲基鸟嘌呤-DNA甲基转移酶（MGMT）是提高烷化剂在脑肿瘤和其他癌症治疗中的功效的非常有前景的靶标。最近，我们实验室首次提供了人MGMT蛋白在酪氨酸和丝氨酸/苏氨酸处被磷酸化并且磷酸化抑制其活性的数据。还对 MGMT 蛋白中这些氨基酸进行磷酸化的两种细胞蛋白激酶进行了表征。该提案的总体目标是确定蛋白磷酸化在 MGMT 功能中的作用，并评估其对改善神经胶质瘤治疗的重要性。我们假设 MGMT 的多位点磷酸化控制其催化活性和人类神经胶质瘤中的蛋白质稳定性。我们假设肿瘤 MGMT 的净磷酸化状态深刻影响其功能，进而影响神经胶质瘤对烷化剂的敏感性。这个为期 4 年的项目要追求的具体目标是 (1) 确定人 MGMT 的体内磷酸化位点并定义每种磷酸化对 MGMT DNA 修复活性的贡献，(2) 表征磷酸化依赖性转折通过泛素蛋白水解途径对 MGMT 蛋白进行修饰，以及 (3) 生成针对 MGMT 蛋白的磷酸化特异性抗体，评估磷酸化 MGMT 的水平神经胶质瘤标本、细胞系，并将其与 MGMT 的催化活性和药物敏感性相关联。作为第一个具体目标的一部分，还将检查磷酸化对 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-苄基鸟嘌呤活性的变化。细胞周期进展期间 MGMT 磷酸化的变化将通过使用磷酸特异性抗体来确定。这些原创研究有望提供有关 MGMT 生理调节的新颖且关键的信息，并可能为改进神经胶质瘤治疗和 MGMT 靶向癌症治疗提供新方向。