Xia Donglan, Stull James T, Kamm Kristine E
Department of Physiology, University of Texas Southwestern Medical Center, 5323 Harry Hines Boulevard, Dallas, TX 75390-9040, USA.
Exp Cell Res. 2005 Apr 1;304(2):506-17. doi: 10.1016/j.yexcr.2004.11.025. Epub 2004 Dec 30.
Myosin II plays important roles in many contractile-like cell functions, including cell migration, adhesion, and retraction. Myosin II is activated by regulatory light chain (RLC) phosphorylation whereas RLC dephosphorylation by myosin light chain phosphatase containing a myosin phosphatase targeting subunit (MYPT1) leads to myosin inactivation. HeLa cells contain MYPT1 in addition to a newly identified human variant 2 containing an internal deletion. RLC dephosphorylation, cell migration, and adhesion were inhibited when either or both MYPT1 isoforms were knocked down by RNA interference. RLC was highly phosphorylated (60%) when both isoforms were suppressed by siRNA treatment relative to control cells (10%) with serum-starvation and ROCK inhibition. Prominent stress fibers and focal adhesions were associated with the enhanced RLC phosphorylation. The reintroduction of MYPT1 or variant 2 in siRNA-treated cells decreased stress fibers and focal adhesions. MYPT1 knockdown also led to an increase of F-actin relative to G-actin in HeLa cells. The myosin inhibitor blebbistatin did not inhibit this effect, indicating MYPT1 likely affects actin assembly independent of RLC phosphorylation. Proper expression of MYPT1 or variant 2 is critical for RLC phosphorylation and actin assembly, thus maintaining normal cellular functions by simultaneously controlling cytoskeletal architecture and actomyosin activation.
肌球蛋白II在许多类似收缩的细胞功能中发挥重要作用，包括细胞迁移、黏附和收缩。肌球蛋白II通过调节性轻链（RLC）磷酸化被激活，而含有肌球蛋白磷酸酶靶向亚基（MYPT1）的肌球蛋白轻链磷酸酶使RLC去磷酸化会导致肌球蛋白失活。除了新鉴定出的含有内部缺失的人类变体2外，HeLa细胞还含有MYPT1。当通过RNA干扰敲低MYPT1的一种或两种异构体时，RLC去磷酸化、细胞迁移和黏附均受到抑制。相对于血清饥饿和ROCK抑制的对照细胞（10%），当两种异构体均被siRNA处理抑制时，RLC高度磷酸化（60%）。明显的应力纤维和黏着斑与增强的RLC磷酸化有关。在siRNA处理的细胞中重新引入MYPT1或变体2可减少应力纤维和黏着斑。敲低MYPT1还导致HeLa细胞中F-肌动蛋白相对于G-肌动蛋白增加。肌球蛋白抑制剂blebbistatin并未抑制这种作用，表明MYPT1可能独立于RLC磷酸化影响肌动蛋白组装。MYPT1或变体2的正确表达对于RLC磷酸化和肌动蛋白组装至关重要，从而通过同时控制细胞骨架结构和肌动球蛋白激活来维持正常细胞功能。
