Activated R-ras, Rac1, PI 3-kinase and PKCepsilon can each restore cell spreading inhibited by isolated integrin beta1 cytoplasmic domains.
Activated R-ras, Rac1, PI 3-kinase and PKCepsilon can each restore cell spreading inhibited by isolated integrin beta1 cytoplasmic domains.
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DOI：
10.1083/jcb.151.7.1549
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发表时间：
2000-12-25
影响因子：
7.8
通讯作者：
LaFlamme, S E
中科院分区：
文献类型：
--
作者：
Berrier, A L;Mastrangelo, A M;Downward, J;Ginsberg, M;LaFlamme, S E
许多细胞类型与细胞外基质蛋白的附着触发细胞铺展，这是一个加强细胞粘附的过程，并且是许多粘附依赖性过程（包括细胞迁移、存活和增殖）的先决条件。细胞铺展需要具有完整β胞质结构域的整合素，推测其将整合素与肌动蛋白细胞骨架连接并激活促进细胞铺展的信号传导途径。已知几种信号蛋白调节细胞铺展，包括R-Ras、PI 3-激酶、PKCε和Rac 1;然而，尚不清楚它们是否通过涉及整合素β胞质结构域的机制来调节细胞铺展。为了研究整合素β胞质结构域调节细胞铺展的机制，我们通过表达tac-β1（一种整合素功能的显性负性抑制剂）来抑制细胞在胶原蛋白I或纤维蛋白原上的铺展，并检查是否可以通过V38 R-Ras、p110α-CAAX、myr-PKCε或L 61 Rac 1的共表达来恢复细胞铺展。这些活化的信号蛋白中的每一种都能够恢复细胞扩散，如通过表达tac-β1的细胞面积的增加所测定的。R-Ras和Rac 1以GTP依赖的方式挽救细胞扩散，而PKCε需要完整的激酶结构域。重要的是，这些信号传导蛋白中的每一种都需要介导粘附的整合素上的完整β胞质结构域，以便恢复细胞铺展。此外，LY 294002抑制V38 R-Ras对细胞铺展的拯救，表明V38 R-Ras恢复细胞铺展需要PI 3-激酶活性。相反，L 61 Rac 1和myr-PKCε各自独立于PI 3-激酶活性增加细胞铺展。此外，Rac 1的显性负性突变体N17 Rac 1消除了细胞铺展，并抑制了p110α-CAAX和myr-PKCε增加细胞铺展的能力。这些研究表明R-Ras、PI 3-kinase、Rac 1和PKCε需要整合素β胞质结构域的功能才能调节细胞铺展，Rac 1在PI 3-kinase和PKCε参与细胞铺展的整合素β胞质结构域功能通路中处于下游。
Attachment of many cell types to extracellular matrix proteins triggers cell spreading, a process that strengthens cell adhesion and is a prerequisite for many adhesion-dependent processes including cell migration, survival, and proliferation. Cell spreading requires integrins with intact β cytoplasmic domains, presumably to connect integrins with the actin cytoskeleton and to activate signaling pathways that promote cell spreading. Several signaling proteins are known to regulate cell spreading, including R-Ras, PI 3-kinase, PKCε and Rac1; however, it is not known whether they do so through a mechanism involving integrin β cytoplasmic domains. To study the mechanisms whereby cell spreading is regulated by integrin β cytoplasmic domains, we inhibited cell spreading on collagen I or fibrinogen by expressing tac-β1, a dominant-negative inhibitor of integrin function, and examined whether cell spreading could be restored by the coexpression of either V38R-Ras, p110α-CAAX, myr-PKCε, or L61Rac1. Each of these activated signaling proteins was able to restore cell spreading as assayed by an increase in the area of cells expressing tac-β1. R-Ras and Rac1 rescued cell spreading in a GTP-dependent manner, whereas PKCε required an intact kinase domain. Importantly, each of these signaling proteins required intact β cytoplasmic domains on the integrins mediating adhesion in order to restore cell spreading. In addition, the rescue of cell spreading by V38R-Ras was inhibited by LY294002, suggesting that PI 3-kinase activity is required for V38R-Ras to restore cell spreading. In contrast, L61Rac1 and myr-PKCε each increased cell spreading independent of PI 3-kinase activity. Additionally, the dominant-negative mutant of Rac1, N17Rac1, abrogated cell spreading and inhibited the ability of p110α-CAAX and myr-PKCε to increase cell spreading. These studies suggest that R-Ras, PI 3-kinase, Rac1 and PKCε require the function of integrin β cytoplasmic domains to regulate cell spreading and that Rac1 is downstream of PI 3-kinase and PKCε in a pathway involving integrin β cytoplasmic domain function in cell spreading.