Silveira L A, Smith J L, Tan J L, Spudich J A
Department of Biochemistry, Beckman Center, Stanford University Medical School, Stanford, CA 94305-5307, USA.
Proc Natl Acad Sci U S A. 1998 Oct 27;95(22):13000-5. doi: 10.1073/pnas.95.22.13000.
Dictyostelium myosin II is activated by phosphorylation of its regulatory light chain by myosin light chain kinase A (MLCK-A), an unconventional MLCK that is not regulated by Ca2+/calmodulin. MLCK-A is activated by autophosphorylation of threonine-289 outside of the catalytic domain and by phosphorylation of threonine-166 in the activation loop by an unidentified kinase, but the signals controlling these phosphorylations are unknown. Treatment of cells with Con A results in quantitative phosphorylation of the regulatory light chain by MLCK-A, providing an opportunity to study MLCK-A's activation mechanism. MLCK-A does not alter its cellular location upon treatment of cells with Con A, nor does it localize to the myosin-rich caps that form after treatment. However, MLCK-A activity rapidly increases 2- to 13-fold when Dictyostelium cells are exposed to Con A. This activation can occur in the absence of MLCK-A autophosphorylation. cGMP is a promising candidate for an intracellular messenger mediating Con A-triggered MLCK-A activation, as addition of cGMP to fresh Dictyostelium lysates increases MLCK-A activity 3- to 12-fold. The specific activity of MLCK-A in cGMP-treated lysates is 210-fold higher than that of recombinant MLCK-A, which is fully autophosphorylated, but lacks threonine-166 phosphorylation. Purified MLCK-A is not directly activated by cGMP, indicating that additional cellular factors, perhaps a kinase that phosphorylates threonine-166, are involved.
盘基网柄菌肌球蛋白II由肌球蛋白轻链激酶A（MLCK-A）对其调节轻链进行磷酸化而激活，MLCK-A是一种非常规的MLCK，不受Ca2+/钙调蛋白调节。MLCK-A通过催化结构域外的苏氨酸-289自身磷酸化以及由一种未知激酶对激活环中的苏氨酸-166进行磷酸化而被激活，但控制这些磷酸化的信号尚不清楚。用刀豆球蛋白A（Con A）处理细胞会导致MLCK-A对调节轻链进行定量磷酸化，这为研究MLCK-A的激活机制提供了一个机会。用Con A处理细胞后，MLCK-A的细胞定位没有改变，它也不会定位于处理后形成的富含肌球蛋白的帽状结构。然而，当盘基网柄菌细胞暴露于Con A时，MLCK-A的活性会迅速增加2至13倍。这种激活可以在没有MLCK-A自身磷酸化的情况下发生。环鸟苷酸（cGMP）是介导Con A触发的MLCK-A激活的细胞内信使的一个有前景的候选者，因为向新鲜的盘基网柄菌裂解物中添加cGMP会使MLCK-A的活性增加3至12倍。在经cGMP处理的裂解物中，MLCK-A的比活性比完全自身磷酸化但缺乏苏氨酸-166磷酸化的重组MLCK-A高210倍。纯化的MLCK-A不会被cGMP直接激活，这表明还涉及其他细胞因子，可能是一种对苏氨酸-166进行磷酸化的激酶。
