Net1 (Neuroepithelial Cell Transforming Gene 1 Protein)
Rho family small GTPases control multiple cell functions, including organization of the actin cytoskeletal, cell motility and invasion, and cell cycle progression. They act as molecular switches, cycling between their active GTP-bound and inactive GDP-bound states. When bound to GTP, Rho GTPases initiate intracellular signaling by binding to downstream proteins known as effectors (Jaffe and Hall 2005). The activation state of Rho GTPases is controlled by three families of proteins, known as GDP exchange factors (Rho GEFs), GTPase activating proteins (Rho GAPs), and guanine nucleotide dissociation inhibitors (Rho GDIs). Rho GEFs activate Rho proteins by stimulating the release of GDP, thereby allowing the binding of GTP (Rossman et al. 2005; Meller et al. 2005). Rho GAPs accelerate the intrinsic GTPase activity of Rho proteins to hydrolyze GTP to GDP (Tcherkezian and Lamarche-Vane 2007). Rho GDIs sequester inactive, GDP-bound Rho GTPases in the cytosol, thereby maintaining and stabilizing a ready pool of Rho proteins for subsequent activation (DerMardirossian and Bokoch 2005). Within this regulatory network, it is the Rho GEFs that are primarily responsible for translating upstream signaling events into Rho protein activation.
During the mid-1990s a number of groups used NIH 3T3 cell focus formation assays to identify candidate oncogenes. In this type of screen, 3T3 cells are made to express a cDNA expression library derived from cancer cells and then tested for subsequent loss of contact inhibition. This can be observed through the formation of cell foci, which are essentially masses of transformed cells. The neuroepithelial transforming gene 1 (Net1) was discovered in this way using a cDNA library derived from neuroepithelioma cells (Chan et al. 1996). The Net1 cDNA initially cloned from this screen lacked the coding sequence for the first 145 amino acids of full length Net1, which was subsequently found to be critical to its transforming activity. NIH 3T3 cells expressing truncated Net1 exhibited anchorage-independent growth in soft agar assays and were tumorigenic when injected into nude mice. Thus, Net1 fit the requirements for a putative oncogene.
Regulation of Net1 Activity
Net1 isoform transcription is precisely regulated by alternative promoters which allows for their differential expression (Dutertre et al. 2010). For example, in MCF7 breast cancer cells estrogen potently stimulates Net1 transcription and weakly downregulates Net1A transcription. On the other hand, progesterone stimulates the transcription of both Net1 and Net1A in MCF7 and T47D breast cancer cells (Dutertre et al. 2010) (Richer et al. 2002). TGFβ has been reported to stimulate Net1A expression in HaCaT human keratinocytes and ARPE-19 retinal pigment epithelial cells by Smad2/3- and Erk1/2-dependent pathways (Shen et al. 2001; Papadimitriou et al. 2011; Lee et al. 2010). Moreover, Net1 isoform mRNAs are subject to downregulation by miR-24 in keratinocytes and miR-22 in K562 leukemia cells (Papadimitriou et al. 2011; Ahmad et al. 2014). Other studies have not differentiated between Net1 isoforms when assessing Net1 expression. For example, IL-2 stimulation increases Net1 expression in Kit 225 human lymphocytes (Mzali et al. 2005). Both TNFα and LPA stimulate Net1 expression in AGS gastric cancer cells (Leyden et al. 2006; Murray et al. 2008). RANKL stimulates Net1 expression in RAW264.7 mouse macrophages (Brazier et al. 2006). Sonic hedgehog stimulates Net1 expression in myocytes during limb bud development (Hu et al. 2012).
Once in the cytoplasm Net1A activity is negatively regulated by phosphorylation and proteasome-mediated degradation. Net1 is phosphorylated by the Rac and Cdc42-regulated kinase Pak1, which inhibits its catalytic activity (Alberts et al. 2005). Presumably Net1A is also phosphorylated by Pak1, as these phosphorylation sites are conserved between isoforms. Pak1 phosphorylates Net1 on serines 152, 153, and 538 in vitro, and on serine 152 in cells. Serines 152 and 153 are contained within an amino-terminal extension of the DH domain that is restricted to a subset of RhoA-specific GEFs (Alberts and Treisman 1998). Substitution of serines 152 and 153 with the phosphorylation mimetic glutamate inhibits the ability of Net1 to catalyze GDP exchange on RhoA in vitro and blocks the ability of expressed Net1ΔN to cause actin stress fiber formation in cells. Similarly, overexpression of constitutively active Pak1 blocks actin stress fiber formation caused by coexpression of wild type Net1, but not by a Net1 mutant containing alanine substitutions at serines 152 and 153 (Alberts et al. 2005). Net1A is also subject to phosphorylation on serine 46 by AMPK, which inhibits its ability to stimulate invadopodia formation (Schaffer et al. 2015).
The cellular activity of Net1A is tightly regulated by ubiquitylation (Carr et al. 2009; Papadimitriou et al. 2011). Net1 isoforms contain a C-terminal PDZ domain-binding site that mediates interaction with proteins within the Dlg1 family (Garcia-Mata et al. 2007; Carr et al. 2009). Interaction of Net1A with Dlg1 prevents its ubiquitylation in MCF7 breast cancer cells and increases its half-life from 25 min to 105 h. This effect is specific for Dlg1, since coexpression of the Net1-interacting PDZ domain protein Magi1b does not stabilize Net1A (Carr et al. 2009). The truncation mutant Net1ΔN is stable when expressed in MCF7 cells, indicating that the amino-terminus of Net1A is important for regulating its degradation (Carr et al. 2009). Interestingly, interaction of endogenous Net1A with Dlg1 in these cells is dependent on the formation of E-cadherin-mediated cell contacts, since disruption of these contacts causes a rapid and dramatic increase in Net1A ubiquitylation. Net1A is also degraded following integrin ligation, as treatment with the proteasome inhibitor MG132 prolongs cytoplasmic accumulation of Net1A following cell replating on collagen (Carr et al. 2012).
Regulation of Actin Cytoskeletal Organization by Net1
Net1 has been shown to act as a GEF for RhoA and RhoB, but not Rac1 or Cdc42 (Alberts and Treisman 1998; Srougi and Burridge 2011). When overexpressed in mouse fibroblasts, Net1 stimulates the formation of actin stress fibers, which is a hallmark of RhoA activation (Alberts and Treisman 1998). The N-terminal truncation mutant Net1ΔN is far more efficient at stimulating stress fiber formation than full length Net1 or Net1A, which is due to the enhanced cytoplasmic localization of Net1ΔN (Qin et al. 2005). Net1 isoforms have also been reported to mediate actin stress fiber formation in human keratinocytes and retinal pigment epithelial cells following stimulation with TGFβ (Shen et al. 2001; Papadimitriou et al. 2011; Lee et al. 2010). In addition, Net1A promotes actin polymerization and focal adhesion maturation in MCF7 cells during adhesion (Carr et al. 2012). Similar to other RhoA-subfamily GEFs, the ability of Net1 isoforms to stimulate actin stress fiber formation is dependent on downstream activation of the RhoA effector kinases ROCK1 and ROCK2 (Tran et al. 2000).
Additional Physiological Roles of Net1
Net1 plays important roles during development and differentiation. For example, in chicken epiblasts Net1A expression is required for RhoA activation at the basal surface and maintenance of the integrity of the basement membrane (Nakaya et al. 2008). During gastrulation, epiblasts undergo an epithelial to mesenchymal transition (EMT) that is accompanied by loss of Net1A expression on the basal membrane, and enforced expression of RhoA or Net1A in these cells prevents basement membrane breakdown and EMT (Nakaya et al. 2008). Similarly, overexpression of xNet1A in Xenopus embryos inhibits gastrulation movements (Miyakoshi et al. 2004). Long-term treatment of HaCaT keratinocytes with TGFβ, which causes EMT, also downregulates Net1A expression (Papadimitriou et al. 2011). These data indicate that cells must downregulate Net1A for EMT to occur. Alternatively, Net1A expression is strongly upregulated during mouse osteoclast differentiation in vitro, and inhibition of Net1A expression prevents cell fusion that is required for osteoclast formation (Brazier et al. 2006). All of these processes are associated with dramatic changes in actin cytoskeletal organization or altered cell motility, both of which are RhoA-regulated events.
Recently it has been shown that Net1 expression is required for mouse mammary gland development during puberty (Zuo et al. 2014). Mice with a Net1 deletion are viable and have offspring at normal Mendelian ratios, but experience a delay in mammary gland development. This is characterized by reduced invasion of milk ducts into the mammary fat pad and reduced branching complexity of the ductal tree. This is due to blunted RhoA signaling to the actomyosin contractile apparatus and reduced expression of estrogen receptor α. Although the ductal tree eventually fills the fat pad, ductal branching remains incomplete (Zuo et al. 2014).
Net1 plays a role in DNA damage signaling (Fig. 3b). Net1 is dephosphorylated on the negative regulatory site serine 152 following exposure of Hela cells to ionizing radiation (IR) (Guerra et al. 2008). In addition, RNAi-mediated knockdown of Net1 expression prevents RhoA activation and sensitizes these cells to IR-induced apoptosis. RhoA activation by Net1 may occur exclusively in the nucleus (Dubash et al. 2011). Net1 knockdown also blocks activation of p38 MAPK and its downstream substrate MAPKAP2, both of which are required for cell survival following IR exposure (Guerra et al. 2008). Net1, along with the Rho GEF Ect2, has also been shown to stimulate RhoB activation after IR exposure in MCF7 cells, an event which promotes cell death (Srougi and Burridge 2011). However, whether Net1 promotes cell survival or death after IR is controversial, since another group found that Net1 knockdown protected MCF7 cells from IR-mediated cell death (Oh and Frost 2014). In this study it was also found that inhibition of Net1 expression reduced ATM activation and H2AX phosphorylation. Surprisingly, overexpression of wild type or catalytically inactive Net1A also suppressed ATM activation. Expression of constitutively active RhoA or RhoB did not affect ATM. These data suggest that Net1A controls DNA damage signaling in a Rho GTPase independent manner.
Net1 and Cancer
Net1 was originally identified as a potential oncogene in mouse fibroblasts (Chan et al. 1996), and subsequent work suggests that it may play an important role in human cancer. For example, Net1 transcripts are overexpressed in human gastric and hepatocellular cancers, as well as gliomas (Leyden et al. 2006; Shen et al. 2008; Tu et al. 2010). In gastric cancer this may be due to alternative polyadenylation which shortens the 3′ UTR of Net1, resulting in enhanced expression (Lai et al. 2015). In estrogen receptor positive breast cancer patients, coexpression of Net1 protein with alpha6beta4 integrin is predictive of decreased distant metastasis-free survival (Gilcrease et al. 2009). This study did not distinguish between Net1 isoforms. In a retrospective analysis of breast cancer patients, high expression of Net1 mRNA, but not Net1A, was prognostic for decreased metastasis-free survival (Dutertre et al. 2010).
Net1 isoforms appear to contribute to cancer cell function in an isoform-specific manner, such that the longer Net1 isoform primarily contributes to proliferation while the shorter Net1A isoform contributes to motility. For example, siRNA knockdown of both Net1 isoforms in AGS gastric cancer cells inhibits their proliferation as well as their ability to invade a Matrigel extracellular matrix (Leyden et al. 2006; Murray et al. 2008). However, only knockdown of Net1, but not Net1A, inhibits MCF7 cell proliferation (Dutertre et al. 2010). This may be a due to a role for the Net1 isoform in controlling mitotic progression (Menon et al. 2013). On the other hand, knockdown of Net1A, but not Net1, inhibits motility and invasion of human breast cancer cells (Carr et al. 2013). Accordingly, Net1A, but not Net1, interacts with focal adhesion kinase (FAK) during cell adhesion and colocalizes with FAK in an actomyosin-dependent manner. During Matrigel invasion, Net1A is required for amoeboid, RhoA-dependent, but not integrin-driven, mesenchymal invasion (Carr et al. 2013). Overexpression of Net1A also drives invadopodia formation, which is inhibited by phosphorylation on serine 46 by AMPK (Schaffer et al. 2015). Taken together these data suggest that Net1 isoforms may contribute to human cancer. Future studies in mouse models of cancer will be required to assess whether the functions of each isoform identified in cancer cells in vitro will apply to tumorigenesis or metastasis in vivo.
Net1 is a Rho GEF that is specific for the RhoA subfamily of small G proteins. Two isoforms exist in most cells which exhibit differential subcellular distributions and may play distinct roles in the cell. The activities of Net1 proteins are tightly regulated by phosphorylation and ubiquitylation. Net1 proteins play important roles in development and also contribute to cellular differentiation. Net1 proteins may also be aberrantly expressed in human cancers and contribute to cancer initiation and/or progression.
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