Rac activity and cell migration

Post-translational modifications of Rac1 during cell migration

To gain further insight into the regulation of Rac during cell migration, we performed a screen for proteins that interact with Rac following treatment of cells with a motility-inducing factor, Hepatocyte Growth Factor (HGF). This revealed the small ubiquitin-like modifier (SUMO) E3-ligase, PIAS3, as a novel Rac interacting protein. PIAS3 interacts better with GTP-bound Rac and is required for increased Rac activation and optimal cell migration in response to HGF. Subsequently we demonstrated that Rac1 can be conjugated to SUMO-1 in response to HGF and that the GTP-bound form of Rac is a better substrate for SUMOylation. Furthermore, we identified non-consensus sites within the polybasic region of Rac1 as the main location for SUMO conjugation. We demonstrated that PIAS3-mediated SUMOylation of Rac1 controls Rac1-GTP levels and the ability of Rac1 to stimulate lamellipodia, cell migration and invasion (Castillo-Lluva et al. Nat Cell Biol. 2010; 12:1078).

Rac1 activity is also regulated through ubiquitylation and subsequent degradation. However, the E3 ubiquitin ligase responsible for Rac1 degradation following activation by a migration stimulus was unknown. Recently, we identified this to be the tumour suppressor HACE1. We showed that HACE1 and Rac1 interaction is enhanced by HGF signalling and that HACE1 catalyses the poly-ubiquitylation of Rac1 at lysine 147 following its activation by HGF, resulting in its proteasomal degradation. HACE1-depletion is accompanied by increased total Rac1 levels and accumulation of Rac1 in membrane ruffles. Moreover, HACE1-depletion enhances cell migration independently of growth factor stimulation, which may have significance for malignant conversion. These findings identified HACE1 as an antagonist of cell migration through its ability to degrade active Rac1 (Castillo-Lluva et al. Oncogene 2012). Jointly the above two studies suggest that SUMOylation and ubiquitylation of Rac1 act coordinately to fine-tune Rac1 activity in migrating cells, promoting Rac activity at sites where the cell membrane is advancing, while antagonizing Rac1 at sites where membrane protrusion needs to cease.

Role of the Rac activator Tiam1 in cell-cell adhesion.

To better our understanding of the contribution of Tiam1-Rac signalling to tumourigenesis, we further investigated its function at cell-cell adhesions. A screen we performed for Tiam1 interacting proteins identified β2-syntrophin as one of its binding partners. β2-syntrophin is a component of the dystroglycan adhesion complex. Our study (Mack et al. Nat Cell Biol. 2012; 14: 1169) unearthed a novel role for this complex in regulating the assembly of adherens junctions (AJ) and tight junctions (TJ) and the generation of apicobasal polarity through controlling Tiam1-Rac signalling. The mechanism we uncovered entails the generation of a Rac activity gradient in the membrane region encompassing these junctions, with lower Rac activity at apical TJ and higher Rac activity sub-apically. This gradient depends upon the ability of β2-syntrophin to stimulate Tiam1-Rac signalling at the sub-apical end and of the polarity determinant Par3 to inhibit Tiam1-Rac signalling at the apical end. By targeting constitutively active Rac to TJs which disrupts the gradient, we demonstrated that the gradient of Rac activity is required for optimal TJ assembly and the generation of apicobasal polarity. Finally, we found that reduced membrane β2-syntrophin correlates with human prostate cancer progression. We conclude that β2-syntrophin and Par-3 finely-tune Rac activity along cell-cell junctions controlling TJ assembly and the establishment of apicobasal polarity. Furthermore, we propose that deregulation of β2-syntrophin, Par-3, or Tiam1, would disrupt the Rac activity gradient, and in turn disrupt TJs and apicobasal polarity, thereby promoting tumourigenesis.

 

 

Figure 1. Multiple mechanisms exist to regulate Rac activity. The Rac GTPase cycles between inactive GDP-bound and active GTP-bound states. Rac activation is facilitated by the action of GEFs (such as Tiam1), which promote GDP dissociation from Rac and allow GTP to bind instead. Through the association with GAPs the intrinsic GTPase activity of Rac is accelerated thereby inactivating Rac. Through association with RhoGDIs Rac can be sequestered in its inactive state. Activated Rac can also be removed through ubiquitylation-induced degradation (mediated by HACE1 following a migration stimulus) or it can be maintained following its modification by SUMO (mediated by PIAS3).

Figure 2. Model depicting the differential localisations of Par-3 and β2-syntrophin and their differential effects on Tiam1-Rac activity at cell-cell junctions