Regulation Of Myosins And Myosin Kinases (PAKs)

(1)去年，我们发现由网囊藻肌球蛋白II的头、颈和轻链以及阿米巴或鸡平滑肌肌球蛋白II的尾部组成的嵌合体比未磷酸化的野生型网囊藻肌球蛋白II具有大约25倍的肌动蛋白激活的MgATPase活性，并且它们的活性不受调节轻链或阿米巴嵌合体尾部磷酸化的影响。嵌合体制造的细丝基本上与作为尾巴来源的母体肌球蛋白的细丝无法区分。今年，我们量化了这些嵌合肌球蛋白支持胞质分裂、ConA加帽、运动性、趋化性以及分化和发育的能力。当嵌合体在网骨藻重链缺失细胞中表达时，仅分化和发育不能得到支持。(2)MYOA是构巢曲霉的单一肌球蛋白I，是该真菌生存所必需的。我们发现MYOA突变体的MgATP酶活性不超过野生型MYOA的1%，并且体外运动活性太低而无法测量，从而支持基本上正常的真菌形态，仅轻微损害真菌生长速率和菌丝伸长，这意味着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)已确定阿米巴肌球蛋白I重链激酶（p21激活的激酶（PAK））的钙调蛋白结合区和自身调节区。(4)我们继续寻找结构变化的尾部阿米巴肌球蛋白II导致磷酸化的监管丝氨酸在尾部。(5)我们正在研究肌球蛋白的头部，包含TEDS网站的表面环的功能，通过表征在体内和体外的肌球蛋白与点突变，缺失和嵌合体在这个区域。

(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.