Semiosis del Constructo Social de Interculturalidad

C3 transferase.

Vinculin is a major structural protein located at the cytoplasmic face of cell-substrate and cell-cell interfaces where it has important roles in adhesion (reviewed by Jockusch et al., 1995). Vinculin is a 90 kDa protein which interacts in vivo with many cytoskeietal proteins including actin, a-actinin, paxillin, and talin as well as acidic phospholipids. These ligands are known to bind different determinants in a conformation-regulated manner. Ptdlns(4,5)?2 causes vinculin to adopt an open conformation which allows the binding of talin and F-actin, two important components of focal adhesions (Gilmore and Burridge, 1996). Microinjection of serum starved fibroblasts with monoclonal antibodies directed against PtdIns(4,5)P2 prior to serum stimulation prevented the formation of stress

fibres or focal adhesions (Gilmore and Burridge, 1996). The requirement for PtdIns(4,5)P2 synthesis in the formation of focal adhesions and stress fibres is consistent with the increases in cellular PtdIns(4,5)P2 seen in the adhesion of fibroblasts to fibronectin (Chong et at., 1994; McNamee et al., 1993) and platelet aggregation (Hinchliffe et al., 1996). Recent evidence suggests that the Rho family of small GTPases may directly regulate phosphoinositide pathways which are important in the control of the actin cytoskeleton.

1 . 1 0 .1 Rho proteins and the actin cytos kel eto n

To date, some thirteen distinct Rho-related proteins have been described: RhoA-E and G, Racl, 2, and E, Cdc42Hs, TCIO, and Rndl and 2. Like other GTPases, the Rho proteins cycle between GTP-bound (active) and GDP-bound (inactive) states, and function as molecular switches controlling diverse cellular processes. Activation of Rho proteins occurs by exchange of bound GDP for GTP, a reaction catalysed by a group of activating molecules called guanine nucleotide exchange factors (GEFs). The GTP-bound protein remains active until the intrinsic GTPase activity hydrolyses the nucleotide to the GDP form. Certain proteins can influence activity by either stimulating the rate of hydrolysis (GTPase-activating proteins, GAPs), or by stabilising the inactive state (guanine nucleotide dissociation inhibitors, GDIs). Together these modulatory proteins play important roles in regulating the large number of biological processes controlled by Rho GTPases. At present, only Rac and Rho have been implicated in phosphoinositide pathways involving PtdlnsPKs and for reasons of space, only these will be discussed here (for review see Hall, 1998; Tapon and Hall, 1997; Van Aelst and D ’Souza Schorey, 1997).

Rho proteins control the actin cytoskeleton in all eukaryotic cells. Activation of Rho in fibroblasts by growth factors has been shown to cause the formation of stress fibres and the clustering of integrins and associated proteins into focal adhesion complexes.

Activation of Rac causes the formation of lamellipodial extensions and membrane ruffles by promoting de novo actin polymerisation at the cell periphery. Actin filaments in stress fibres and lamellipodia are associated with integrin adhesion complexes whose dynamic assembly and disassembly provides a mechanism which generates the traction and force required for cell migration.

Small GTPase targets often bind cognate GTPases in a GTP-dependent manner and consequently, affinity purification and yeast two hybrid screening have been used to identify potential regulator and effector proteins which interact with Rac and Rho. These interacting proteins include protein kinases (Amano et al., 1996; Ishizaki et al., 1996; Matsui et al., 1996; Watanabe et al., 1996). Several GEFs and GAPs have also been found to physically interact with Rac and Rho (Van Aelst and D'Souza-Schorey, 1997, for review). Although two hybrid screens have not yielded any interacting Pl-kinases, Rac and Rho have been shown to bind PtdlnsPK and PI 3-kinase activities in vitro (see Figure

1.7; Ren et al., 1996; Tolias etal., 1995).

The involvement of a PtdlnsPK in a Rho pathway was first suggested when it was shown that RhoA was able to stimulate a PtdlnsPK activity in fibroblast lysates in a GTP- dependent manner (Chong et a i, 1994). It has since been found that RhoA binds a 68 kDa type I PtdlnsPK (Ren et a i, 1996). Interestingly, this interaction was independent of GTP, data reminiscent of the results obtained with the Rac-associated PtdlnsPK (Tolias et a i, 1995). However, these results are contrary to those shown in Figure 1.7 and the data obtained from fibroblast lysates (Chong et a i, 1994). The discrepancy between these results is probably due to differences in experimental conditions. However, the ability of RhoA to stimulate GTP-dependent PtdlnsPK activity in lysates but not in precipitated complexes argues for an additional GTP-dependent component (or components) of a complex which mediates the stimulatory interaction with RhoA and Racl but not the constitutive physical interaction with these proteins. The GTP-dependent activity shown in Figure 1.7 is likely to be due to the low salt concentration (50 mM) used in the washing buffer in order to maintain weaker interactions. The hypothesis of a complex is consistent with recent findings (Tolias et a i, 1998) and the fact that no PtdlnsPK has been isolated by two hybrid, a method which only detects binary interactions. The link between Rac and P td ln sP K was strengthened when it was shown that thrombin-stimulated actin polymerisation in platelets was accompanied by an increase in PtdIns(4,5)P2 and that the effects of thrombin could be emulated by GTP-Rac 1 but not RhoA (Hartwig et a i, 1995). This has led the authors to suggest a model in which PtdlnsPK is activated by Rac on thrombin stimulation and leads to an increase in PtdIns(4,5)P2. The PtdIns(4,5)P2

generated leads to the dissociation of actin-capping proteins from actin filaments resulting in rapid polymerisation. The use of intact cells or semi-intact (ie, permeabilised) systems may be important when studying the function of the PtdlnsPK I-Racl/RhoA complex

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