Dosé Andréa C, Ananthanarayanan Shobana, Moore Judy E, Corsa Amoreena C, Burnside Beth, Yengo Christopher M
Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, California 94720, USA.
Biochemistry. 2008 Feb 26;47(8):2485-96. doi: 10.1021/bi7021574. Epub 2008 Jan 30.
Myosin IIIA is unique among myosin proteins in that it contains an N-terminal kinase domain capable of autophosphorylating sites on the motor domain. A construct of myosin IIIA lacking the kinase domain localizes more efficiently to the stereocilia tips and alters the morphology of the tips in inner ear hair cells. Therefore, we performed a kinetic analysis of myosin IIIA without the kinase domain (MIII DeltaK) and compared these results with our reported analysis of myosin IIIA containing the kinase domain (MIII). The steady-state kinetic properties of MIII DeltaK indicate that it has a 2-fold higher maximum actin-activated ATPase rate (kcat = 1.5 +/- 0.1 s-1) and a 5-fold tighter actin affinity (KATPase = 6.0 +/- 1.4 microM, and KActin = 1.4 +/- 0.4 microM) compared to MIII. The rate of ATP binding to the motor domain is enhanced in MIII DeltaK (K1k+2 approximately 0.10 +/- 0.01 microM-1.s-1) to a level similar to the rate of binding to MIII in the presence of actin. The rate of ATP hydrolysis in the absence of actin is slow and may be rate limiting. Actin-activated phosphate release is identical with and without the kinase domain. The transition between actomyosin.ADP states, which is rate limiting in MIII, is enhanced in MIII DeltaK. MIII DeltaK accumulates more efficiently at the tips of filopodia in HeLa cells. Our results suggest a model in which the activity and concentration of myosin IIIA localized to the tips of actin bundles mediates the morphology of the tips in sensory cells.
肌球蛋白IIIA在肌球蛋白蛋白中独具特色，因为它含有一个N端激酶结构域，该结构域能够对运动结构域上的位点进行自磷酸化。缺少激酶结构域的肌球蛋白IIIA构建体更有效地定位于静纤毛尖端，并改变内耳毛细胞尖端的形态。因此，我们对缺少激酶结构域的肌球蛋白IIIA（MIII DeltaK）进行了动力学分析，并将这些结果与我们之前报道的含有激酶结构域的肌球蛋白IIIA（MIII）的分析结果进行了比较。MIII DeltaK的稳态动力学特性表明，与MIII相比，它的最大肌动蛋白激活ATP酶速率高2倍（kcat = 1.5 +/- 0.1 s-1），肌动蛋白亲和力高5倍（KATPase = 6.0 +/- 1.4 microM，KActin = 1.4 +/- 0.4 microM）。在MIII DeltaK中，ATP与运动结构域的结合速率增强（K1k+2约为0.10 +/- 0.01 microM-1.s-1），达到与在肌动蛋白存在下与MIII结合速率相似的水平。在没有肌动蛋白的情况下，ATP水解速率很慢，可能是限速步骤。有无激酶结构域时，肌动蛋白激活的磷酸释放是相同的。在MIII中起限速作用的肌动球蛋白.ADP状态之间的转变在MIII DeltaK中得到增强。MIII DeltaK在HeLa细胞的丝状伪足尖端更有效地积累。我们的结果提出了一个模型，其中定位于肌动蛋白束尖端的肌球蛋白IIIA的活性和浓度介导了感觉细胞中尖端的形态。
