Rapid recruitment of p120RasGAP and its associated protein, p190RhoGAP, to the cytoskeleton during integrin mediated cell-substrate interaction

Abstract

The interaction of cells with their substrate triggers cascades of signal transduction that result in profound changes in cell morphology. The nature of these signals and how they are integrated to orchestrate changes in cell shape are beginning to be elucidated. In particular, adhesive interactions between cells and their substrate, mediated by cell-surface integrins and extracellular matrix (ECM) proteins, appear to result in massive rearrangement of the cell cytoskeleton via the small Gprotein, Rho. Here we show that in mouse ®broblasts, the interaction between cells and their substrate results in the rapid recruitment to the cytoskeleton of RasGAP (p120RasGAP), its associated protein of 190 kilodaltons, the GTPase activating protein for RhoA (p190RhoGAP) and the focal adhesion kinase (p125FAK). Similar results were obtained when cells were plated on ECM proteins, such as ®bronectin, suggesting that the phenomenon is integrin mediated. These studies suggest that in ®bro-blasts, cell-substrate interaction triggered by integrin engagement result in the recruitment to the cytoskeleton of signaling molecules such as p120RasGAP, p190Rho-GAP and p125FAK and may be involved in the formation of membrane cytoskeleton-associated signaling complexes that are important in cytoarchitectural reorganization.

Integrin-mediated signalling through the MAP-kinase pathway

“…Similarly, Sydor et al [43] have shown that the Raf -Ras complex has an extremely short lifetime, after which Raf is released for downstream signalling. The resulting Ras species, denoted RasGTP Ã , is no longer capable of activating Raf and will undergo GTP hydrolysis catalysed by GTpase activating proteins (GAPs), which are recruited to focal adhesions after integrin-ligand binding [44,45]. In the case of high [Raf], Ras accumulates as RasGTP Ã , suggesting that the RafRasGTP reaction is much faster than the action of GAPs in recycling Ras.…”

Oncogenic Ras Leads to Rho Activation by Activating the Mitogen-activated Protein Kinase Pathway and Decreasing Rho-GTPase-activating Protein Activity

“…We found that expression of oncogenic Ras in LTR-H-Ras(A) cells induced the translocation of p190 Rho-GAP from the cytosol to a detergent-insoluble cytoskeletal fraction. Similarly, integrinmediated cell-substrate interaction has been shown to result in p190 Rho-GAP recruitment to the cytoskeleton at 1-2 h after plating NIH 3T3 cells on fibronectin, and this change in subcellular localization of Rho-GAP occurs at a time when Rho⅐GTP levels are elevated (11,36,44). In contrast to the stimulation of RhoA activity by prolonged integrin engagement, early integrin signaling within 10 -15 min after plating cells on fibronectin induces Src-dependent tyrosine phosphorylation of p190 Rho-GAP, increases Rho-GAP activity, and transiently inactivates RhoA (64).…”

“…7c, Membrane). To determine whether the decrease in cytosolic Rho-GAP may be the result of a shift into a cytoskeletal compartment, we extracted cells in Triton X-100-containing lysis buffer and examined the detergent-insoluble cytoskeletal fraction (44). Dexamethasone treatment of LTR-H-Ras(A) cells, but not of wild type NIH 3T3 cells, induced a significant increase in the amount of p190 Rho-GAP associated with the Triton-insoluble cytoskeletal fraction (Fig.…”

“…Triton X-100-insoluble cytoskeletal fractions were prepared as described previously (44). Cells were washed in phosphate-buffered saline and extracted in situ for 40 s at room temperature in 50 mM Na-Hepes, pH 6.4, 3 mM EGTA, 5 mM MgCl 2 , 0.5% Triton X-100.…”

Concise Review: Mechanotransduction via p190RhoGAP Regulates a Switch Between Cardiomyogenic and Endothelial Lineages in Adult Cardiac Progenitors

“…This function likely depends on the need to regulate the balance between cell-cell and cell-substratum interactions controlled by p120-Catenin, which can also regulate morphogenesis. Since p190RhoGAP directly interacts with focal adhesion kinase, regulates both RhoA activity and other multiple signaling pathways related to cell cycle [42], differentiation [13,14], morphogenesis [43], and determines cell shape [31,44], motility [45], and cell-cell interaction [41,46], it acts as an important conduit of information from the extracellular mechanical world [47] and a key decision maker in determining the coordinated control of temporally regulated phenotypic outcome.…”