PHOSPHORYLATION OF INTERMEDIATE FILAMENTS DURING MITOSIS

有丝分裂期间中间丝的磷酸化

• 批准号: 3285431
• 负责人: ROBERT M EVANS
• 金额: $ 9.93万
• 依托单位: UNIVERSITY OF COLORADO DENVER
• 依托单位国家: 美国
• 财政年份: 1984
• 资助国家: 美国
• 起止时间: 1984-12-01 至 1988-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3285431
• 关键词: autoradiography; cell growth regulation; chemical binding; cyclic AMP; cytoskeleton; flow cytometry; gel electrophoresis; human tissue; phosphorylation; protein kinase; radiotracer; serine

Intermediate filaments are a major component of the cytoskeleton of animal cells. Preliminary evidence indicates that intermediate filaments of vimentin, desmin, and cytokeratin types undergo an increased phosphorylation in cultured cells during mitosis when filament organization is altered. Experiments with vimentin-type filaments show that the change in phosphorylation in mitotic cells is serine site specific. The proposed experiments will use cultured mouse (L-929), hamster (BHK21) and human (A-431) cells that have been double thymidine blocked to produce non-mitotic cells, and cells mitotically selected after release from the thymidine block. Phosphorylated intermediate filament proteins will be isolated from these mitotic and non-mitotic cells and specific fragments or domains produced by chemical and limited enzymatic cleavage. These domain fragments will be peptide mapped to determine the functional area of the protein that is specifically modified at mitosis. Experiments will be conducted with intact cells and in vitro to determine if a unique vimentin protein kinase activity is expressed in mitotic cells. In vivo and in vitro phosphorylation experiments will also attempt to determine if the mitosis-specific phosphorylation is a cAMP-independent process. Experiments will be performed on vimentin filaments isolated from mitotic and non-mitotic cells and phosphorylated in vitro with purified protein kinase to determine if there is a change in serine site availability in filaments during mitosis. Finally, quantitative peptide mapping experiments will be carried out to determine the relative occupancy of phosphate at serine sites on vimentin isolated from mitotic and non-mitotic cells. The goal of these studies is to determine if a specific modification of intermediate filament phosphorylation represents a regulatory mechanism in cytoskeletal organization important in the control of cell proliferation. The long term objective is to understand if this mechanism is involved in the maintenance of the transformed or neoplastic phenotype.

中间丝是动物细胞骨架的主要组成部分 细胞 初步证据表明， 波形蛋白、结蛋白和细胞角蛋白类型经历增加的 在有丝分裂过程中，当细丝组织时， 被改变了 波形蛋白型纤维的实验表明， 有丝分裂细胞中的磷酸化是丝氨酸位点特异性的。 拟议 实验将使用培养的小鼠（L-929）、仓鼠（BHK 21）和人 （A-431）已被双胸苷阻断以产生 非有丝分裂细胞，以及从细胞膜释放后有丝分裂选择的细胞。 胸苷阻滞 磷酸化的中间丝蛋白将被 分离自这些有丝分裂和非有丝分裂细胞和特定片段， 通过化学和有限的酶促裂解产生的结构域。 这些域 将对片段进行肽图谱分析，以确定 在有丝分裂时被特殊修饰的蛋白质。 实验将 用完整的细胞和体外进行，以确定是否一个独特的波形蛋白 蛋白激酶活性在有丝分裂细胞中表达。 体内和 体外磷酸化实验也将试图确定 有丝分裂特异性磷酸化是不依赖cAMP的过程。 实验将在从有丝分裂细胞中分离的波形蛋白细丝上进行。 和非有丝分裂细胞，并在体外用纯化的蛋白质磷酸化 激酶，以确定是否存在丝氨酸位点可用性的变化， 丝在有丝分裂期间。 最后，定量肽图谱 将进行实验以确定 在分离自有丝分裂和非有丝分裂的波形蛋白上丝氨酸位点的磷酸盐 细胞 这些研究的目的是确定一种特定的 中间丝磷酸化的修饰代表了 细胞骨架组织中的调节机制在控制 细胞增殖。 长期目标是了解， 机制涉及维持转化或肿瘤性 表型