PGEX-mDIA was provided by S. column are shown on the left as control for equal loading. Open in a separate window Open in a separate window Open in a separate window Figure 4. ICAP-1 inhibits and binds Rac1. (A) COS cells transiently transfected with control vector or mycCICAP-1 were suspended by trypsin treatment, kept in suspension 2 h 30 min in serum-free DME (Su), and plated on fibronectin (5 g/ml) for 1 h (Ad). Rac1 activation level was evaluated as described in Materials and methods using a GST-PAK-CD fusion protein that selectively binds to GTP-Rac1. The bound GTP-Rac1 was analyzed by Western blotting with a polyclonal antibody to Rac1. To probe for Rac1 and ICAP-1 expression, total cell lysates were blotted with the corresponding antibodies. (B) The amount of GTP-bound and total Rac1 (A) was quantified by densitometric analysis; the activation level of Rac1 is expressed as a ratio between the values of GTP-bound and total Rac1. Comparable results were obtained in three independent experiments. (C) Total lysate of COS cells transfected with myc-Rac1 was loaded on Sepharose coupled with MBPCICAP-1 fusion protein or MBP as control. The proteins eluted with glycine-HCl, pH 3, buffer (fractions numbers 1-2-3-4) were analyzed by Western blotting with a monoclonal antibody against Rac1. In the experimental condition used to analyze Sav1 the activation level of Cdc42 and Rac1 during cell spreading, we were not able to detect any significant activation of RhoA, as measured by pull-down assay with the Rho effector protein mDia (Kimura et al., 2000), in accordance with previous reports (Ren et al., 1999; Arthur et al., 2000). Given that the adhesive stimulus was not sufficient to induce RhoA activation, we then activated RhoA by either treating COS cells with LPA or transfecting them with pCEFL-GST-DH/PH coding for MLS0315771 a GST fusion protein containing the DH/PH domains of the GEF protein Dbl. However, in both of these conditions ICAP-1 expression did not affect RhoA activity (unpublished data; Fig. 5 A). Open in a separate window Figure 5. ICAP-1 does neither inhibit nor bind RhoA. (A) RhoA activity assay was performed as described in Materials and methods using COS cells either untransfected or transfected as indicated. The same blot was reprobed for ICAP-1 expression with a polyclonal antibody against ICAP-1. (B) Total protein extract of COS cells was loaded on Sepharose MLS0315771 coupled with MBPCICAP-1 fusion protein or MBP as control. The eluted proteins were analyzed by Western blotting with a monoclonal antibody against RhoA. The same filter was stripped and tested for MLS0315771 the presence of Rac1 using a specific monoclonal antibody (B’). Total extract is shown as control. To better understand the inhibitory action of ICAP-1 on Cdc42 and Rac1, we performed affinity chromatography experiments to test the ability of these proteins to interact with each other. COS cell extracts were incubated with Sepharose coupled to purified MBPCICAP-1 fusion protein. As shown in Fig. 3 C and Fig. 4 C, Cdc42 and Rac1 bind to MBPCICAP-1 but not to MBP carrier protein. On the other hand, affinity chromatography experiments showed that RhoA did not appreciably bind to MBPCICAP-1 (Fig. 5 B). Moreover, we tested the binding of transfected molecules unrelated to GTPases, such as melusin MLS0315771 (Brancaccio et al., 1999), or endogenous COS proteins such as p125Fak and paxillin. None of these proteins bind to ICAP-1 (unpublished data), demonstrating the specificity of the interaction with Cdc42 and Rac1 GTPases. Rho family GTPases are posttranslationally modified in eukaryotic cells by covalent binding of an isoprenyl chain that allows association with cellular membranes to exert their biological activity. To test whether this modification affects binding to ICAP-1, Cdc42-containing extracts were prepared from membrane and cytosol fractions.
