Wang Z Y, Brzeska H, Baines I C, Korn E D
Laboratory of Cell Biology, NHLBI, National Institutes of Health, Bethesda, Maryland 20892, USA.
J Biol Chem. 1995 Nov 17;270(46):27969-76. doi: 10.1074/jbc.270.46.27969.
The actin-activated Mg(2+)-ATPase and in vitro motility activities of the three Acanthamoeba myosin I isozymes depend upon phosphorylation of their single heavy chains by myosin I heavy chain kinase. Previously, the kinase had been shown to be activated by autophosphorylation, which is enhanced by acidic phospholipids, or simply by binding to purified plasma membranes in the absence of significant autophosphorylation. In this paper, we show that the rate of phosphorylation of myosin I by unphosphorylated kinase is approximately 20-fold faster when both the myosin I and the kinase are bound to acidic phospholipid vesicles than when both are soluble. This activation is not due to an increase in the local concentrations of vesicle-bound kinase and myosin I. Thus, acidic phospholipids, like membranes, can activate myosin I heavy chain kinase in the absence of significant autophosphorylation, i.e. membrane proteins are not required. Kinetic studies show that both binding of kinase to phospholipid vesicles and autophosphorylation of kinase in the absence of phospholipid increase the Vmax relative to soluble, unphosphorylated kinase with either an increase in the apparent Km (when myosin I is the substrate) or no significant change in Km (when a synthetic peptide is the substrate). Kinetic data showed that autophosphorylation of phospholipid-bound kinase is both intermolecular and intervesicular, and that phosphorylation of phospholipid-bound myosin I by phospholipid-bound kinase is also intervesicular even when the kinase and myosin are bound to the same vesicles. The relevance of these results to the activation of myosin I heavy chain kinase and phosphorylation of myosin I isozymes in situ are discussed.
三种棘阿米巴肌球蛋白I同工酶的肌动蛋白激活的Mg(2+)-ATP酶及体外运动活性取决于肌球蛋白I重链激酶对其单条重链的磷酸化作用。此前已表明，该激酶可通过自身磷酸化被激活，酸性磷脂可增强这种自身磷酸化，或者在无显著自身磷酸化的情况下，仅通过与纯化的质膜结合即可激活。在本文中，我们发现，当肌球蛋白I和激酶都结合到酸性磷脂囊泡上时，未磷酸化的激酶对肌球蛋白I的磷酸化速率比二者均为可溶状态时快约20倍。这种激活并非由于囊泡结合的激酶和肌球蛋白I局部浓度的增加。因此，酸性磷脂与膜一样，在无显著自身磷酸化的情况下即可激活肌球蛋白I重链激酶，即不需要膜蛋白。动力学研究表明，激酶与磷脂囊泡的结合以及在无磷脂情况下激酶的自身磷酸化，相对于可溶的未磷酸化激酶而言，均会增加Vmax，此时表观Km增加（当肌球蛋白I为底物时）或Km无显著变化（当合成肽为底物时）。动力学数据表明，磷脂结合的激酶的自身磷酸化是分子间和囊泡间的，并且即使激酶和肌球蛋白结合到同一囊泡上，磷脂结合的激酶对磷脂结合的肌球蛋白I的磷酸化也是囊泡间的。本文讨论了这些结果与肌球蛋白I重链激酶的激活及肌球蛋白I同工酶原位磷酸化的相关性。
