Eto Masumi, Kirkbride Jason, Elliott Elizabeth, Lo Su Hao, Brautigan David L
Center for Cell Signaling, University of Virginia School of Medicine, Charlottesville, Virginia 22908, USA.
J Biol Chem. 2007 Jun 15;282(24):17806-15. doi: 10.1074/jbc.M700944200. Epub 2007 Apr 15.
Focal adhesions attach cultured cells to the extracellular matrix, and we found endogenous protein phosphatase-1alpha isoform (PP1alpha) localized in adhesions across the entire area of adherent fibroblasts. However, in fibroblasts migrating into a scrape wound or spreading after replating PP1alpha 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 PP1alpha 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 PP1alpha and tensin, whereas neither focal adhesion kinase nor vinculin was cross-linked and co-precipitated with PP1alpha, 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 PP1alpha. 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 PP1alpha into those adhesions. Thus, the protein-tyrosine phosphatase domain of tensin exhibits isoform-specific association with PP1alpha in a restricted spatial region of adhesions that are formed during cell migration.
粘着斑将培养的细胞附着于细胞外基质，我们发现内源性蛋白磷酸酶-1α同工型（PP1α）定位于贴壁成纤维细胞整个区域的粘着斑中。然而，在迁移至刮伤创口或重铺板后铺展的成纤维细胞中，PP1α在前缘附近的粘着斑中未出现，而是在与张力蛋白整合在时间和空间上一致的其他粘着斑中被募集。内源性张力蛋白和PP1α从细胞裂解物中与同工型特异性PP1抗体共同沉淀。用洛曼试剂对粘着斑制剂进行化学交联显示内源性PP1α和张力蛋白在分子水平上接近，而粘着斑激酶和纽蛋白均未与PP1α交联和共同沉淀，这表明粘着斑内存在不同的空间亚结构域。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端360个残基就足以实现共表达的PP1α的同工型选择性共沉淀。相对于成纤维细胞，人前列腺癌PC3细胞缺乏张力蛋白，且粘着斑较少，大多位于周边。在这些癌细胞中瞬时表达绿色荧光蛋白张力蛋白可诱导粘着斑形成，并将内源性PP1α募集到这些粘着斑中。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粘着斑的受限空间区域内与PP1α表现出同工型特异性结合。
