Rac proteins belong to the larger family of Rho GTPases that act as molecular switches. When bound to GTP, they are in an active conformation. Hydrolysis of GTP to GDP causes an inactivating conformational change. Racs are post-translationally modified by the removal of C-terminal amino acid residues, followed by the attachment of a lipid moiety in the form of a geranyl-geranyl group 1 .

Because of this modification, mature Rac proteins are lipophilic and are only present in the cytosol when bound to the chaperone and transport protein RhoGDI. RhoGDI regulates the movement of Rac between the cytosol and membranes. Moreover, a recent study showed that Rho proteins compete for binding to a limited pool of RhoGDI, which, once it has bound them, provides stabilization and protection from degradation 2 .

There are only three distinct RhoGDI proteins, of which RhoGDIα is the most ubiquitously expressed. The structure of RhoGDI bound to small GTPases has been elucidated. Rac makes contact with both N-and C-terminal regions in RhoGDI, with its hydrophobic tail fitting into a pocket in the C-terminal part of RhoGDI. However, the exact mechanism by which binding and release of Racs from RhoGDI is mediated is not well known 3 4 .

RhoGDI can be phosphorylated on serine S101 and S174 by the serine/threonine kinase Pak 5 and PKCα also phosphorylates RhoGDIα, on residue S96 6 . Interestingly, constitutively activated oncogenic Src, which has a deregulated tyrosine kinase activity, was shown to phosphorylate RhoGDIα on tyrosine residue Y156 7 . In the current study we report that a normal cellular tyrosine kinase which is member of the Src family, Fer, is able to specifically tyrosine phosphorylate RhoGDIα and that this can regulate the binding to Rac1.

The BCR/ABL oncogene encodes a chimeric protein that contains most of the Abl tyrosine kinase. Proteomic analysis of cells transfected with Bcr/Abl 8 reported changes in phosphorylation of RhoGDI. To investigate if the deregulated Bcr/Abl tyrosine kinase, similar to oncogenic Src 7 , is able to modify RhoGDIα, we performed an immunocomplex kinase assay with cells expressing Bcr/Abl or with Fer, a non-receptor tyrosine kinase related to Abl, in the presence of added recombinant GST-RhoGDIα or control GST. As shown in Figure 1A, left panel, the Bcr/Abl protein exhibited strong autophosphorylation activity, as expected. However, there was very little phosphorylated RhoGDIα. In contrast, there was a prominent signal of phosphorylated RhoGDIα in the sample expressing Fer (Figure 1A right panel).

To examine whether Fer and RhoGDIα interact in cells, we transfected COS-1 cells with pEGFP-Fer (wild type, WT) or kinase dead D742R mutant pEGFP-Fer (mutant, MT), with or without RhoGDIα and investigated possible protein complex formation between these by co-immunoprecipitation. As shown in Figure 1B, left panel, Fer-WT clearly co-immunoprecipitated with RhoGDIα, which was phosphorylated on tyrosine, as judged by Western blotting using phosphotyrosine antibodies. In the converse experiment (Figure 1B, right panel, Fer IP) we confirmed the interaction between Fer-WT and RhoGDIα. In the presence of the Fer mutant lacking tyrosine kinase activity, no tyrosine phosphorylated RhoGDIα was detected, indicating that Fer tyrosine kinase activity is directly or indirectly needed to generate tyrosine phosphorylated RhoGDIα. Interestingly, the kinase-defective Fer protein exhibited a reduced ability to form a complex with RhoGDIα (compare lanes in IP Fer, IB for RhoGDIα), suggesting that the binding of these two proteins is facilitated by tyrosine phosphorylation. This is in agreement with the finding that the isolated Fer SH2 domain binds to RhoGDIα on far-Western blots (not shown). Importantly, we also were able to co-immunoprecipitate endogenous Fer with endogenous RhoGDIα in a lysate of the mast cell line RBL-2H3. This indicates that the complex formation between RhoGDIα and Fer also takes place without overexpression (Figure 1C).

Although reactive oxygen species (ROS) were originally identified as a bactericidal mechanism, a role for H₂O₂ as secondary messenger has now been well-established. Many groups suggest that Src may be a primary target of ROS, with some studies reporting activation of Src kinase activity upon ROS exposure 9 . Since constitutively activated oncogenic Src had been shown to phosphorylate RhoGDIα 10 11 , we tested if exposure of c-Src to H₂O₂ would activate it to phosphorylate RhoGDIα. As shown in Figure 2A, in the presence of c-Src and after H₂O₂ exposure, we could detect tyrosine phosphorylated RhoGDIα. Sangrar et al 12 showed that ROS also induce Fer tyrosine autophosphorylation as well as trans-phosphorylation activity. As shown in Figure 2B, we confirmed Fer activation by H₂O₂ in different types of cells by immunoprecipitation and Western blotting with anti-phosphotyrosine antibodies. In addition, when we co-transfected Fer with RhoGDIα and treated the cells with H₂O₂, increased RhoGDIα tyrosine phosphorylation was detected (Figure 2C). The tyrosine phosphorylated RhoGDIα was visible as two discrete bands, indicating this protein is subject to processive phosphorylation.

Tyrosine phosphorylation of signal transduction proteins in the absence of an oncogenic, constitutively activated tyrosine kinase may be difficult to detect due to the transient nature of the phosphorylation and the activity of phosphatases. Wang et al. reported that pervanadate treatment allows detection of phosphorylation on RhoGDIα because this inhibits protein-tyrosine phosphatases and traps proteins in their tyrosine-phosphorylated form 13 . Next, we examined if pervanadate would further activate Fer and increase RhoGDIα tyrosine phosphorylation. As shown in Figure 3A, when cells co-expressing RhoGDIα and Fer were exposed to pervanadate for 5 minutes, a marked increase in tyrosine phosphorylation was seen both in Fer and RhoGDIα proteins (Figure 3A). The N-terminal regulatory domain of RhoGDI binds to the switch region of Rac whereas the C-terminal end adopts a conformation that accommodates the hydrophobic tail of Rac 6 . To examine which domain interacts with Fer, we expressed the N- and C-terminus of RhoGDIα as Xpress-tagged proteins, together with Fer, and immunoprecipitated each fragment of RhoGDIα with Xpress antibodies. We could not detect a clear interaction of the N-terminal end with Fer (data not shown). As shown in Figure 3B (RhoGDIα-CT + Fer), in immunoprecipitates with the C-terminal end of RhoGDIα and in the presence of pervanadate, we recovered tyrosine-phosphorylated Fer. Moreover, this domain of RhoGDIα also was tyrosine phosphorylated (Figure 3B). This indicates that Fer interacts with and tyrosine phosphorylates residues in the part of RhoGDIα that includes the domain that binds to the C-terminal end of Rac. We were also able to demonstrate that endogenous RhoGDIα was phosphorylated on tyrosine in response to pervanadate, which activates Fer in RBL-2H3 cells (Figure 3C).

We used purified proteins to examine if tyrosine phosphorylation of RhoGDIα could regulate its ability to bind Rac. Fer was expressed as a GST fusion protein in baculovirus. In Figure 4A (left panel), we first incubated GST-RhoGDIα with Rac to allow these proteins to form a complex. Then GST-Fer or control GST was added with a kinase reaction buffer and the kinase reaction was initiated. As shown in the top panel (pY20 blot), both autophosphorylation of Fer and transphosphorylation of RhoGDIα occurred as expected. However, there was no evidence that Rac was tyrosine phosphorylated (not shown). Rac protein was recovered in immunoprecipitates from both Fer-treated and non-Fer treated samples, indicating that the presence or absence of Fer does not affect preformed Rac-RhoGDIα complexes. A different result was obtained when Fer was first incubated with RhoGDIα and a kinase reaction was done. When Rac was subsequently added and allowed to bind, we found that it was not co-immunoprecipitated with RhoGDIα in the Fer-treated sample (Figure 4B). The lack of binding of Rac to RhoGDIα could be because tyrosine-phosphorylated RhoGDIα has decreased affinity for Rac, or because the binding of Fer to RhoGDIα prevented subsequent binding of Rac to RhoGDIα. Interestingly, this effect is similar to that reported for oncogenic Src: when Src phosphorylated RhoGDIα, the affinity of RhoGDIα for Rac was also significantly reduced 7 .

Tyrosine phosphorylation as a mechanism to regulate RhoGDIα has not been widely investigated, although it was recently reported that pervanadate treatment of a pancreatic islet beta cell line caused the appearance of phosphotyrosine on RhoGDIα and was accompanied by lack of Cdc42 binding 13 . There is also increasing evidence that H₂O₂ regulates the balance of tyrosine phosphorylation and de-phosphorylation in cells. The current study shows that non-oncogenic tyrosine kinases are likely to play a role in regulating the binding of Rac GTPases to RhoGDIα and through this, the cellular pool of Rac.

RhoGDI: Rho GDP-dissociation inhibitor; ROS: reactive oxygen species

FF performed experiments, brought in new ideas and wrote part of the manuscript; S-M K performed experiments and wrote part of the manuscript; LH made the baculovirus Fer protein; PS provided critical reagents; JG designed experiments and provided critical input; NH provided ideas and wrote the manuscript. All authors read and approved the final manuscript.