PHOSPHORYLATION OF INTERMEDIATE FILAMENTS DURING MITOSIS

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了 有丝分裂过程中培养细胞的磷酸化 是被更改的。用波形蛋白类型的细丝进行的实验表明， 在有丝分裂细胞中的磷酸化是丝氨酸专一性的。建议数 实验将使用培养的小鼠(L-929)、仓鼠(BHK21)和人 (A-431)细胞已被双重胸苷阻断以产生 非有丝分裂细胞和有丝分裂细胞从 胸腺嘧啶核苷阻断。磷酸化的中间丝蛋白将是 从这些有丝分裂和非有丝分裂细胞中分离出来的特定片段或 由化学和有限的酶裂解产生的结构域。这些域 片断将被绘制成肽图，以确定 在有丝分裂时被特殊修饰的蛋白质。实验将是 用完整的细胞和体外实验确定一种独特的波形蛋白 蛋白激酶活性在有丝分裂细胞中表达。体内和体内 体外磷酸化实验也将试图确定 有丝分裂特异的磷酸化是一个cAMP不依赖的过程。 实验将在从有丝分裂中分离出来的波形蛋白细丝上进行 和非有丝分裂细胞，并用纯化的蛋白进行体外磷酸化 确定丝氨酸结合部位的可用性是否发生变化 有丝分裂期间的花丝。最后，定量多肽图谱 将进行实验以确定相对占有率 有丝分裂和非有丝分裂中波形蛋白丝氨酸位点的磷酸 细胞。这些研究的目标是确定一种特定的 中间纤维磷酸化的修饰代表着一种 细胞骨架组织中的调节机制在控制中起重要作用 细胞增殖率。长期目标是理解这一点 机制是参与维持转化或肿瘤的 表型。

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.