Assembly of MYPT1 with protein phosphatase-1 in fibroblasts redirects localization and reorganizes the actin cytoskeleton
Assembly of MYPT1 with protein phosphatase-1 in fibroblasts redirects localization and reorganizes the actin cytoskeleton
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DOI：
10.1002/cm.20088
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发表时间：
2005-10-01
影响因子：
--
通讯作者：
Brautigan, DL
中科院分区：
文献类型：
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作者：
Eto, M;Kirkbride, JA;Brautigan, DL
肌动蛋白结合蛋白被一种特殊形式的丝氨酸/苏氨酸磷酸酶-1(PP1)去磷酸化，调节平滑肌收缩和非肌肉细胞的形态和运动。这种肌球蛋白和Ezrin/Radioxin/moesin(ERM)靶向的磷酸酶由Delta亚基PP1催化亚基和一个称为肌球蛋白磷酸酶靶向(MYPT1)的主要调节亚基组成。我们通过瞬时表达表位标记的MYPT1(myc-MYPT1)和HA标记的PP1在活的大鼠胚胎成纤维细胞(REF52细胞)中重建了肌球蛋白/ERM磷酸酶。免疫荧光显微镜显示，单独表达的野生型myc-MYPT1主要聚集在细胞核中，而如果与HA-PP1共表达，它定位于胞浆并沉积在细胞骨架肌丝上。MYPT1的F38A突变消除了PP1结合，使myc-MYPT1即使在与HA-PP1共表达时也有核定位。因此，这两个亚基的表达是肌球蛋白/ERM磷酸酶原位形成和介导肌丝定位所必需的。结果表明，在活细胞中几乎没有内源性PP1可用于与异位调节亚基相互作用或交换。我们的结论是，MYPT1的C末端结构域与肌球蛋白的结合不足以推翻成纤维细胞的核输入，但PP1与myc-MYPT1的结合中和了核输入。全长的mye-MYPT1+HA-PP1只引起肌动蛋白细胞骨架的细微变化，而myc-MYPT1(1-300)与HA-PP1的共表达分散了应力纤维，而mycMYPT1(1-498)破坏了细胞骨架，并产生了看起来像神经元的伸展细胞。根据这些反应，我们得出结论：MYPT1的C末端作为一个自抑制结构域发挥作用，并且MYPT1的一个中心结构域可以介导肌动蛋白细胞骨架的广泛重组。
Dephosphorylation of actin-binding proteins by a specialized form of protein Ser/Thr phosphatase type-1 (PP1) regulates smooth muscle contraction and morphology and motility of nonmuscle cells. This myosin and ezrin/radixin/moesin (ERM)-targeted phosphatase comprises the delta isoform PP1 catalytic subunit plus a primary regulatory subunit called myosin phosphatase targeting (MYPT1). We reconstructed myosin/ERM phosphatase in living rat embryo fibroblasts (REF52 cells) by transient expression of epitope-tagged MYPT1 (myc-MYPT1) plus HA-tagged PP1 Unexpectedly, wild-type myc-MYPT1 expressed alone accumulated predominantly in the nucleus, as visualized by immunofluorescent microscopy, whereas if coexpressed with HA-PP1, it was localized in the cytosol and deposited on cytoskeleton myofilaments. The F38A mutation of MYPT1 that eliminates PP1 binding gave nuclear localization of myc-MYPT1, even when coexpressed with HA-PP1. Thus, expression of both subunits was necessary to form myosin/ERM phosphatase in situ and mediate myofilament localization. The results indicate there is little endogenous PP1 available for interaction or interchange with ectopic regulatory subunits in living cells. We concluded that myosin binding by the C-terminal domain of MYPT1 is not sufficient to override nuclear import in fibroblasts, but the binding of PP1 to myc-MYPT1 neutralizes nuclear import. Full-length mye-MYPT1 plus HA-PP1 induced only subtle changes in organization of the actin cytoskeleton, however coexpression of myc-MYPT1(1-300) with HA-PP1 dispersed stress fibers without major alteration in morphology and mycMYPT1(1-498) disrupted the cytoskeleton and produced radically extended cells that appeared like neurons. Based on these responses, we conclude that the MYPT1 C-terminus functions as an auto-inhibitory domain, and a central domain in MYPT1 can mediate extensive reorganization of the actin cytoskeleton.