Association of the tensin N-terminal protein-tyrosine phosphatase domain with the α isoform of protein phosphatase-1 in focal adhesions
Association of the tensin N-terminal protein-tyrosine phosphatase domain with the α isoform of protein phosphatase-1 in focal adhesions
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DOI：
10.1074/jbc.m700944200
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发表时间：
2007-06-15
影响因子：
4.8
通讯作者：
Brautigan, David L.
中科院分区：
文献类型：
--
作者：
Eto, Masumi;Kirkbride, Jason;Brautigan, David L.
局灶性粘连将培养细胞附着在细胞外基质上，我们发现内源性蛋白磷酸酶-1α亚型(PP1α)定位于粘连中，分布于整个贴壁的成纤维细胞区域。然而，在迁移到擦伤伤口或在复制后扩散的成纤维细胞中，PP1α没有出现在靠近前沿的粘连中，而是被招募到其他粘连中，这些粘连在时间和空间上与张力蛋白的掺入一致。内源性张力蛋白和PP1α与异构体特异性PP1抗体从细胞裂解物中共沉淀。焦点黏附制剂与Lomant试剂的化学交联显示内源性PP1α和张力蛋白的分子接近，而焦点黏附蛋白和纽蛋白都不与PP1α交联和共沉淀，这表明粘连内存在明显的空间亚区。截短的张力蛋白的瞬时表达表明，仅包含蛋白酪氨酸磷酸酶结构域的N-末端360个残基就足以选择性地共沉淀共表达的PP1α。与成纤维细胞相比，人前列腺癌PC3细胞缺乏张力蛋白，而且粘连较少，主要是外周粘连。绿色荧光蛋白Tensin在这些癌细胞中的瞬时表达诱导粘连的形成，并将内源性PP1α招募到这些粘连中。因此，张力蛋白的蛋白酪氨酸磷酸酶结构域在细胞迁移过程中形成的有限空间粘连区域中与PP1α显示出异构体特异性的联系。
Focal adhesions attach cultured cells to the extracellular matrix, and we found endogenous protein phosphatase-1 alpha-isoform (PP1 alpha) localized in adhesions across the entire area of adherent fibroblasts. However, in fibroblasts migrating into a scrape wound or spreading after replating PP1 alpha did not appear in adhesions near the leading edge but was recruited into other adhesions coincident in time and space with incorporation of tensin. Endogenous tensin and PP1 alpha co-precipitated from cell lysates with isoform-specific PP1 antibodies. Chemical cross-linking of focal adhesion preparations with Lomant's reagent demonstrated molecular proximity of endogenous PP1 alpha and tensin, whereas neither focal adhesion kinase nor vinculin was cross-linked and co-precipitated with PP1 alpha, suggesting distinct spatial subdomains within adhesions. Transient expression of truncated tensin showed the N-terminal 360 residues, which comprise a protein-tyrosine phosphatase domain, alone were sufficient for isoform-selective co-precipitation of co-expressed PP1 alpha. Human prostate cancer PC3 cells are deficient in tensin relative to fibroblasts and have fewer, mostly peripheral adhesions. Transient expression of green fluorescent protein tensin in these cancer cells induced formation of adhesions and recruited endogenous PP1 alpha into those adhesions. Thus, the protein-tyrosine phosphatase domain of tensin exhibits isoform-specific association with PP1 alpha in a restricted spatial region of adhesions that are formed during cell migration.