Regulation of MGMT by Phosphorylation

通过磷酸化调节 MGMT

• 批准号: 6541045
• 负责人: KALKUNTE S SRIVENUGOPAL
• 金额: $ 21.91万
• 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/6541045
• 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）是提高烷基化剂在治疗脑肿瘤和其他癌症方面的疗效的高度有希望的靶标。最近，我们的实验室提供了第一个数据，即人类MGMT蛋白在酪氨酸和丝氨酸/苏胺上被磷酸化，并且磷酸化抑制其活性。还表征了两种磷酸化这些氨基酸的细胞蛋白激酶。该提案的总体目的是确定蛋白质磷酸化在MGMT功能中的作用，并评估其改善神经胶质瘤治疗的重要性。我们假设MGMT的多站点磷酸化控制其催化活性和人神经胶质瘤中的蛋白质稳定性。我们假设肿瘤MGMT的净磷酸化状态会深刻影响其功能，进而影响神经胶质瘤对烷基化剂的敏感性。 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针对MGMT蛋白，评估神经胶质瘤标本，细胞系中磷酸-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，以及随之而来的MGMT活性对O6-甲基圭氨酸的变化以及O6-苯唑烷氨酸的结构变化也将作为第一个特定目的的一部分。细胞周期进程中MGMT磷酸化的变化将通过使用磷酸特异性抗体确定。这些原始研究承诺将产生有关MGMT生理调节的新颖和关键信息，并可能为改善神经胶质瘤治疗和MGMT靶向癌症疗法提供新的方向。