Localization of two phosphorylation sites adjacent to a region important for polymerization on the tail of Dictyostelium myosin
Localization of two phosphorylation sites adjacent to a region important for polymerization on the tail of Dictyostelium myosin
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与盘基网柄菌肌球蛋白尾部聚合重要区域相邻的两个磷酸化位点的定位
DOI：
10.1002/j.1460-2075.1984.tb02289.x
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发表时间：
1984
期刊：
影响因子：
--
通讯作者：
G. Gerisch
中科院分区：
文献类型：
--
作者：
K. Pagh;H. Maruta;M. Claviez;G. Gerisch
已知盘基网柄藻的肌球蛋白重链的磷酸化在细胞的趋化刺激后被抑制。去磷酸化对肌球蛋白组装及其肌动蛋白激活的ATP酶活性的影响提出了一个问题，即磷酸化位点相对于负责聚合和肌动蛋白结合的位点位于何处。使用在电子显微镜中定位其结合位点的七种单克隆抗体，两个磷酸化位点，即，苏氨酸残基被来自D.通过胰凝乳蛋白酶片段的免疫印迹进行定位。两个抗体结合到末端的五分之一的尾巴，并识别38 kd的磷酸化胰凝乳蛋白酶片段。通过双向电泳分离该片段的非磷酸化形式和单磷酸化形式和双磷酸化形式。低分子量抗体标记。重量多肽表明两个磷酸化位点均位于距离尾末端至少32kD处。由于聚合，在低离子强度下不溶的非磷酸化片段被证明是尾部的内部裂解产物。该片段的一段必要的聚合被映射相邻的磷酸化位点。
Phosphorylation of the myosin heavy chains of Dictyostelium discoideum is known to be inhibited following chemotactic stimulation of the cells. Effects of dephosphorylation on the assembly of myosin and on its actin‐activated ATPase activity raised the question of where the phosphorylated sites are located with respect to sites responsible for polymerization and actin binding. Using seven monoclonal antibodies the binding sites of which were mapped in the electron microscope, two phosphorylation sites, i.e., threonine residues that were phosphorylated by a kinase from D. discoideum, were localized by immunoblotting of chymotryptic fragments. Two of the antibodies bound to the terminal one fifth of the tail and recognized a phosphorylated chymotryptic fragment of 38 kd. The non‐phosphorylated form and single and double phosphorylated forms of this fragment were separated by two‐dimensional electrophoresis. Antibody labeling of lower mol. wt. polypeptides indicated that both phosphorylation sites were located at least 32 kd from the end of the tail. A non‐phosphorylated fragment, that was insoluble at low ionic strength due to polymerization, proved to be an internal cleavage product of the tail. A segment of this fragment necessary for polymerization was mapped adjacent to the phosphorylation sites.