Regulation Of Myosins And Myosin Kinases (PAKs)

(1)去年，我们发现由棘阿米巴或鸡平滑肌球蛋白II的头部、颈部和轻链以及棘阿米巴或鸡的平滑肌球蛋白II的尾部组成的嵌合体的肌动蛋白激活的镁ATPase活性比未磷酸化的野生型Dictyostelialmyosin II高约25倍，并且它们的活性不受调节轻链或棘阿米巴嵌合体尾部的磷酸化的影响。嵌合体使得细丝与母体肌球蛋白的细丝基本上无法区分，肌球蛋白是尾巴的来源。今年，我们量化了这些嵌合肌球蛋白支持胞质分裂、ConA封顶、运动性、趋化性以及分化和发育的能力。只有当嵌合体在DictyostelialHeavy Chain Null细胞中表达时，才不能支持分化和发育。(2)Nidulans AspergillusNidulans的单一肌球蛋白MYOA是该真菌生存所必需的。我们发现，MYOA突变体的MgATPase活性不超过野生型MYOA的1%，体外运动活性太低，无法测量，基本上支持正常的真菌形态，仅对真菌生长速度和菌丝伸长造成轻微损害，这意味着MYOA的基本功能是结构性的，而不是催化的。这项工作是与德克萨斯大学安德森癌症中心的格雷戈里·梅博士合作完成的。(3)测定了p21激活蛋白激酶(PAK)棘阿米巴肌球蛋白I重链激酶(PAK)的钙调蛋白结合区和自身调节区。(4)我们继续寻找棘阿米巴肌球蛋白II的尾部结构变化，这是由于尾部调节丝氨酸的磷酸化造成的。(5)通过研究肌球蛋白在该区域的点突变、缺失和嵌合体的体内外性质，我们正在研究含有TEDS位点的肌球蛋白头部表面环的功能。

(1) Last year we found that chimeras consisting of the head, neck and light chains of Dictyostelium myosin II and the tails of either Acanthamoeba or chicken smooth muscle myosin II had about 25-fold more actin-activated MgATPase activity than unphosphorylated wild-type Dictyostelium myosin II and that their activities were unaffected by phosphorylation of either the regulatory light chain or the tail of the Acanthamoeba chimera. The chimeras made filaments essentially indistinguishable from the filaments of the parent myosin that was the source of the tail. This year we have quantified the ability of these chimeric myosins to support cytokinesis, ConA-capping, motility, chemotaxis and differentiation and development. Only differentiation and development fail to be supported when the chimeras are expressed in Dictyostelium heavy chain null cells. (2) MYOA, the single myosin I of Aspergillus nidulans, is essential for viability of this fungus. We find that MYOA mutants with no more than 1% of the MgATPase activity of wild type MYOA and in vitro motility activity too low to measure support essentially normal fungal morphology with only slight impairment of fungal growth rate and hyphal elongation implying that the essential function of MYOA is structural and not catalytic. This work was done in collaboration with Dr. Gregory May, University of Texas, Anderson Cancer Center. (3) The calmodulin-binding and autoregulatory regions of Acanthamoeba myosin I heavy chain kinase, a p21-activated kinase (PAK), have been determined. (4) We continue to look for structural changes in the tail of Acanthamoeba myosin II resulting from phosphorylation of the regulatory serines at the end of the tail. (5) We are investigating the function of the surface loop on the head of myosin that contains the TEDS-site by characterizing the properties in vivo and in vitro of myosins with point mutants, deletions and chimeras in this region.