Cell cycle-dependent phosphorylation of mammalian protein phosphatase 1 by cdc2 kinase
Cell cycle-dependent phosphorylation of mammalian protein phosphatase 1 by cdc2 kinase
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DOI：
10.1073/pnas.94.6.2168
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发表时间：
1997-03-18
影响因子：
11.1
通讯作者：
Nairn, AC
中科院分区：
文献类型：
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作者：
Kwon, YG;Lee, SY;Nairn, AC
蛋白磷酸酶1（PP-1）已知是真核细胞周期进程的关键组分，在体外，我们先前的研究表明cdc 2激酶磷酸化PP-1中的Thr-320（T320），并且这导致酶活性的抑制。已经制备了特异性识别在T320磷酸化的PP-1C α的抗体。细胞同步化研究显示，在多种细胞类型中，PP-1的T320仅在有丝分裂早期至中期被磷酸化至高水平。PP-1的T320磷酸化可被细胞周期蛋白依赖性蛋白激酶抑制剂olomoucine抑制，而被PP-1/PP-2A抑制剂calyculin A增加。磷酸化T320抗体的免疫荧光显微镜显示，在NIH 3 T3细胞中，PP-1的磷酸化从前期的基础水平开始增加，并在中期达到峰值，免疫染色表明磷酸-PP-1仅定位于非染色体区域。此外，在有丝分裂细胞的细胞分级研究中，磷酸-PP-1仅在可溶性级分中可检测到，这些观察结果表明，cdc 2激酶在有丝分裂早期至中期的磷酸化和PP-1的抑制，1活性可能有助于蛋白质磷酸化状态的增加，这对正常细胞分裂的启动至关重要。
Protein phosphatase 1 (PP-1) is known to be a critical component of eukaryotic cell cycle progression, In vitro, our previous studies showed that cdc2 kinase phosphorylates Thr-320 (T320) in PP-1, and that this leads to inhibition of enzyme activity, To examine directly the phosphorylation of PP-1 in intact mammalian cells, an antibody has been prepared that specifically recognizes PP-1C alpha phosphorylated at T320, Cell synchronization studies revealed in a variety of cell types that T320 of PP-1 was phosphorylated to high levels only during early to mid-mitosis. The phosphorylation of T320 of PP-1 mas reduced by the cyclin-dependent protein kinase inhibitor, olomoucine, and increased by the PP-1/PP-2A inhibitor, calyculin A, Immunofluorescence microscopy using phospho-T320 antibody indicated that in NIH 3T3 cells the phosphorylation of PP-1 began to increase from basal levels in prophase and to peak at metaphase, Immunostaining indicated that phospho-PP-1 was localized exclusively to nonchromosomal regions, Furthermore, in cell fractionation studies of mitotic cells, phospho-PP-1 was detectable only in the soluble fraction, These observations suggest that phosphorylation by cdc2 kinase in early to mid-mitosis and inhibition of PP-1 activity is likely to contribute to the increased state of phosphorylation of proteins that is critical to the initiation of normal cell division.