Analysis of the regulatory phosphorylation site in Acanthamoeba myosin IC by using site-directed mutagenesis.
Analysis of the regulatory phosphorylation site in Acanthamoeba myosin IC by using site-directed mutagenesis.
复制标题
使用定点诱变分析棘阿米巴肌球蛋白 IC 中的调节磷酸化位点。
DOI：
10.1073/pnas.95.26.15200
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发表时间：
1998
影响因子：
11.1
通讯作者：
J. Hammer
中科院分区：
文献类型：
--
作者：
Z. Wang;F. Wang;J. Sellers;E. Korn;J. Hammer
肌动蛋白激活的ATP酶活性的阿米巴肌球蛋白IC的刺激15- 20倍的磷酸化的Ser-329的重链。在大多数肌球蛋白中，谷氨酸或天冬氨酸占据这个位置，它位于形成肌动球蛋白界面的一部分的表面环内。为了研究在该位点明显需要负电荷，我们将Ser-329突变为丙氨酸、天冬酰胺、天冬氨酸或谷氨酸，并在杆状病毒感染的昆虫细胞中共表达Flag标记的野生型或突变体重链和轻链。重组野生型肌球蛋白IC与从阿米巴中纯化的肌球蛋白IC是不可区分的，这是通过（i）其肌动蛋白激活的ATP酶活性对重链磷酸化的依赖性，（ii）其ATP酶活性对F-肌动蛋白浓度的不寻常的三相依赖性，（iii）其对ATP的Km，以及（iv）其移位肌动蛋白丝的能力来确定的。Ala和Asn突变体具有与未磷酸化的野生型肌球蛋白IC相同的低肌动蛋白激活的ATP酶活性。与磷酸化野生型蛋白一样，Glu突变体的活性是未磷酸化野生型蛋白的16倍，而Asp突变体的活性是未磷酸化野生型的8倍。野生型和突变体蛋白对ATP的Km相同。未磷酸化的野生型蛋白质和Ala和Asn突变体不能易位肌动蛋白丝，而Glu突变体易位丝以相同的速度，和50%的速度，作为磷酸化的野生型蛋白质的Asp突变体。这些结果表明，酸性氨基酸可以提供所需的肌动蛋白依赖性活动的阿米巴肌球蛋白IC在体外的表面环中的负电荷，并表明，提供这种电荷的侧链的长度是很重要的。
The actin-activated ATPase activity of Acanthamoeba myosin IC is stimulated 15- to 20-fold by phosphorylation of Ser-329 in the heavy chain. In most myosins, either glutamate or aspartate occupies this position, which lies within a surface loop that forms part of the actomyosin interface. To investigate the apparent need for a negative charge at this site, we mutated Ser-329 to alanine, asparagine, aspartate, or glutamate and coexpressed the Flag-tagged wild-type or mutant heavy chain and light chain in baculovirus-infected insect cells. Recombinant wild-type myosin IC was indistinguishable from myosin IC purified from Acanthamoeba as determined by (i) the dependence of its actin-activated ATPase activity on heavy-chain phosphorylation, (ii) the unusual triphasic dependence of its ATPase activity on the concentration of F-actin, (iii) its Km for ATP, and (iv) its ability to translocate actin filaments. The Ala and Asn mutants had the same low actin-activated ATPase activity as unphosphorylated wild-type myosin IC. The Glu mutant, like the phosphorylated wild-type protein, was 16-fold more active than unphosphorylated wild type, and the Asp mutant was 8-fold more active. The wild-type and mutant proteins had the same Km for ATP. Unphosphorylated wild-type protein and the Ala and Asn mutants were unable to translocate actin filaments, whereas the Glu mutant translocated filaments at the same velocity, and the Asp mutant at 50% the velocity, as phosphorylated wild-type proteins. These results demonstrate that an acidic amino acid can supply the negative charge in the surface loop required for the actin-dependent activities of Acanthamoeba myosin IC in vitro and indicate that the length of the side chain that delivers this charge is important.
运动蛋白2：肌球蛋白。
DOI：
--
发表时间：
1995
期刊：
Protein profile.
影响因子：
--
作者：
Sellers,JR;Goodson,HV
通讯作者：
Goodson,HV
肌动球蛋白亚片段 1 复合物的结构：使用电子显微镜和 X 射线晶体学数据的搜索结果。
DOI：
10.1073/pnas.94.16.8533
发表时间：
1997
影响因子：
11.1
作者：
Mendelson,R;Morris,EP
通讯作者：
Morris,EP
寡聚组织的催化后果：低 ATP 浓度下“束缚”的重肌球蛋白的动力学证据。
DOI：
10.1073/pnas.81.17.5345
发表时间：
1984
影响因子：
11.1
作者：
Hackney,DD;Clark,PK
通讯作者：
Clark,PK