Regulation Of Myosins And Myosin Kinases (PAKs)

(1)去年我们发现，由头、颈、轻链和棘阿米巴或鸡平滑肌肌球蛋白II尾部组成的嵌合体，其肌动蛋白激活的MgATPase活性比未磷酸化的野生型肌动蛋白激活的MgATPase活性高25倍，并且它们的活性不受棘阿米巴嵌合体调节轻链或尾部磷酸化的影响。嵌合体产生的细丝与作为尾巴来源的亲本肌凝蛋白的细丝基本无法区分。今年，我们量化了这些嵌合肌球蛋白支持细胞分裂、cona封顶、运动性、趋化性、分化和发育的能力。当嵌合体在盘基骨柱重链零细胞中表达时，只支持分化和发育。(2)细粒曲霉（Aspergillus nidulans）的单个肌球蛋白I MYOA对该真菌的生存至关重要。我们发现，MYOA突变体的MgATPase活性不超过野生型MYOA的1%，其体外运动活性低到无法测量，基本上支持正常的真菌形态，真菌生长速度和菌丝伸长只有轻微的损害，这表明MYOA的基本功能是结构性的，而不是催化性的。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)已经确定了棘阿米巴肌球蛋白I重链激酶（p21活化激酶（PAK））的钙调素结合区和自调节区。(4)我们继续寻找棘阿米巴肌球蛋白II尾部由于尾部调控丝氨酸磷酸化而引起的结构变化。(5)我们正在研究肌凝蛋白头部含有ted位点的表面环的功能，通过表征该区域具有点突变、缺失和嵌合的肌凝蛋白在体内和体外的特性。

(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.