Interaction of the RhoA Exchange Factor Net1 with Discs Large Homolog 1 Protects It from Proteasome-mediated Degradation and Potentiates Net1 Activity

Abstract

Net1 is a nuclear Rho guanine nucleotide exchange factor that is specific for the RhoA subfamily of small G proteins. Truncated forms of Net1 are transforming in NIH3T3 cells, and this activity requires cytoplasmic localization of Net1 as well as the presence of a COOH-terminal PDZ binding site. We have previously shown that Net1 interacts with PDZ domain-containing proteins within the Discs Large (Dlg) family and relocalizes them to the nucleus. In the present work, we demonstrate that Net1 binds directly to the first two PDZ domains of Dlg1 and that both PDZ domains are required for maximal interaction in cells. Furthermore, we show that Net1 is an unstable protein in MCF7 breast epithelial cells and that interaction with Dlg1 significantly enhances Net1 stability. Stabilization by Dlg1 significantly increases the ability of Net1 to stimulate RhoA activation in cells. The stability of endogenous Net1 is strongly enhanced by cell-cell contact, and this correlates with a dramatic increase in the interaction between Net1 and Dlg1. Importantly, disruption of E-cadherin-mediated cell contacts, either by depletion of external calcium or by treatment with transforming growth factor ␤, leads to a rapid loss of the interaction between Net1 and Dlg1 and a subsequent increase in the ubiquitylation of Net1. These results indicate that Net1 requires interaction with PDZ domain proteins, such as Dlg1, to protect it from proteasome-mediated degradation and to maximally stimulate RhoA and that this interaction is regulated by cell-cell contact.Rho family small G proteins control many aspects of cell physiology, including cytoskeletal organization, cell motility, and cell cycle progression (1, 2). They do so by acting as molecular switches, cycling between their active, GTPbound and inactive, GDP-bound states. Once activated, Rho proteins stimulate signaling in multiple pathways by binding to downstream effector proteins and modulating their activities. Currently, 21 mammalian Rho family GTPases have been identified, with the Rac1, Cdc42, and RhoA proteins being the most thoroughly characterized (3).Rho protein activation is controlled by a family of enzymes known as Rho guanine nucleotide exchange factors (Rho GEFs) 2 (4). Net1 (neuroepithelioma transforming gene 1) is a Rho GEF that was cloned as a transforming gene in a screen for novel oncogenes in NIH3T3 cells (5). Two isoforms of Net1 exist in cells, Net1 and Net1A, which are identical except for alternative NH 2 -terminal regulatory domains. Both isoforms of Net1 are nuclear proteins that display marked specificities for RhoA as compared with Rac1 or Cdc42 (6, 7). Correspondingly, overexpression of either Net1 isoform in cells profoundly stimulates actin stress fiber formation, which is a hallmark of RhoA activation (8). The mechanism by which Net1 stimulates cell proliferation and transformation is complex. We and others have shown that Net1 must be enzymatically active and localized to the cytoplasm to cause cell transformation (6,8). In addition, we have observed tha...

Differential regulation of the two RhoA-specific GEF isoforms Net1/Net1A by TGF-β and miR-24: role in epithelial-to-mesenchymal transition

“…In chicken embryos, it was shown that downregulation of Net1, triggered by an unknown factor, shut down basal RhoA activity and drove the onset of EMT (Nakaya et al, 2008). In addition, Net1 has been involved in the regulation of adherens junctions through its interaction with the polarity protein Dlg1 Carr et al, 2009). This interaction was dependent on the formation of E-cadherin-mediated cell contacts whose disruption caused dissociation of Net1 from Dlg1, leading to Net1 degradation by the proteasome (Carr et al, 2009).…”

“…Recently, the Net1 GEF has been implicated in the regulation of epithelial cell polarity and EMT (Nakaya et al, 2008;Carr et al, 2009). In chicken embryos, it was shown that downregulation of Net1, triggered by an unknown factor, shut down basal RhoA activity and drove the onset of EMT (Nakaya et al, 2008).…”

“…TGF-b targets for degradation many proteins involved in its pathway, such as Smad2/3, Smad7, ALK5, SnoN (Shi and Massague, 2003) and RhoA (Wang et al, 2003). Additionally, Net1 is an unstable protein targeted for degradation by the proteasome in MCF-7 breast cancer cells (Carr et al, 2009). In line with this, our experiments performed in the presence of the proteasome inhibitor MG-132 imply that the observed downregulation of Net1A protein after long-term TGF-b treatment is at least partially because of proteasomal degradation.…”

“…In addition, Net1 has been involved in the regulation of adherens junctions through its interaction with the polarity protein Dlg1 Carr et al, 2009). This interaction was dependent on the formation of E-cadherin-mediated cell contacts whose disruption caused dissociation of Net1 from Dlg1, leading to Net1 degradation by the proteasome (Carr et al, 2009). In line with these reports, we found that selective Net1A silencing disrupted E-cadherin-and ZO-1-mediated junctions and upregulated the mesenchymal markers N-cadherin and fibronectin as well as PAI-1 and the transcriptional repressor of E-cadherin, Slug.…”

“…Long-term downregulation of Net1 and Net1A by TGF-b is implicated in EMT Based on recent reports that implicate Net1 downregulation in the EMT process (Nakaya et al, 2008;Carr et al, 2009), we investigated the role of TGF-b-induced downregulation of Net1/Net1A in EMT. For this, we studied the integrity of E-cadherin-and zonula occludens-1 (ZO-1)-mediated junctions, the expression of the mesenchymal markers N-cadherin and fibronectin, as well as the actin cytoskeleton using fluorescence microscopy after selectively silencing Net1 or Net1A and stimulating cells or not with TGF-b for 48 to 72 h. As shown in Figure 6A, silencing of Net1A led to the disruption of E-cadherinsupported adherens junctions even in the absence of TGFb (Figure 6Ab), whereas silencing of Net1 had minor effect (Figure 6Ac).…”