Hosoda Atsuko, Sato Naruki, Nagaoka Rie, Abe Hiroshi, Obinata Takashi
Department of Biology, Faculty of Science, Chiba University, Chiba 263-8522, Japan.
J Muscle Res Cell Motil. 2007;28(2-3):183-94. doi: 10.1007/s10974-007-9117-6. Epub 2007 Sep 7.
Cofilin plays a critical role in actin filament dynamics in a variety of eukaryotic cells. Its activity is regulated by phosphorylation/dephosphorylation of a Ser3 residue on the N-terminal side and/or its binding to a phosphoinositide, PIP(2). To clarify how cofilin activity is regulated in muscle cells, we generated analogues of the unphosphorylated form (A3-cofilin) and phosphorylated form (D3-cofilin) by converting the phosphorylation site (Ser3) of cofilin to Ala and Asp, respectively. These mutated proteins, as well as the cofilin having Ser3 residue (S3-cofilin), were produced in an E. coli expression system and conjugated with fluorescent dyes. In an in vitro functional assay, A3-cofilin retained the ability to bind to F-actin. Upon injection into cultured muscle cells, A3-cofilin and S3-cofilin promptly disrupted actin filaments in the cytoplasm, and many cytoplasmic rods containing both the exogenous cofilin and actin were generated, while D3-cofilin was simply diffused in the cytoplasm without affecting actin filaments. Several hours after the injection, however, the activity of A3-cofilin and S3-cofilin was suppressed: the actin-A3-cofilin (or S3-cofilin) rods disappeared, the cofilin diffused in the cytoplasm like D3-cofilin, and actin filaments reformed. Both GFP-fused A3-cofilin and S3-cofilin that were produced by cDNA transfection were also suppressed in the cytoplasm of muscle cells in culture. Thus, some mechanism(s) other than phosphorylation can suppress A3-cofilin activity. We observed that PIP(2) can bind to A3-cofilin just as to S3-cofilin and inhibits the interaction of A3-cofilin with actin. Our results suggest that the activity of A3-cofilin and also S3-cofilin can be regulated by PIP(2) in the cytoplasm of muscle cells.
丝切蛋白在多种真核细胞的肌动蛋白丝动力学中起着关键作用。其活性通过N端一侧Ser3残基的磷酸化/去磷酸化和/或其与磷酸肌醇PIP(2)的结合来调节。为了阐明丝切蛋白活性在肌肉细胞中是如何被调节的，我们通过分别将丝切蛋白的磷酸化位点（Ser3）转换为丙氨酸和天冬氨酸，生成了未磷酸化形式（A3-丝切蛋白）和磷酸化形式（D3-丝切蛋白）的类似物。这些突变蛋白以及具有Ser3残基的丝切蛋白（S3-丝切蛋白）在大肠杆菌表达系统中产生，并与荧光染料偶联。在体外功能测定中，A3-丝切蛋白保留了与F-肌动蛋白结合的能力。将其注射到培养的肌肉细胞中后，A3-丝切蛋白和S3-丝切蛋白迅速破坏细胞质中的肌动蛋白丝，并产生了许多同时含有外源性丝切蛋白和肌动蛋白的细胞质杆，而D3-丝切蛋白只是简单地扩散到细胞质中，不影响肌动蛋白丝。然而，注射数小时后，A3-丝切蛋白和S3-丝切蛋白的活性受到抑制：肌动蛋白-A3-丝切蛋白（或S3-丝切蛋白）杆消失，丝切蛋白像D3-丝切蛋白一样扩散到细胞质中，肌动蛋白丝重新形成。通过cDNA转染产生的绿色荧光蛋白融合的A3-丝切蛋白和S3-丝切蛋白在培养的肌肉细胞细胞质中也受到抑制。因此，除了磷酸化之外的某些机制可以抑制A3-丝切蛋白的活性。我们观察到PIP(2)可以像与S3-丝切蛋白结合一样与A3-丝切蛋白结合，并抑制A3-丝切蛋白与肌动蛋白的相互作用。我们的结果表明，A3-丝切蛋白以及S3-丝切蛋白的活性可以在肌肉细胞的细胞质中被PIP(2)调节。
