NSP-Cas protein structures reveal a promiscuous interaction module in cell signaling

Program of Apoptosis and Cell Death Research, Cancer Center, Sanford-Burnham Medical Research Institute, La Jolla, California, USA.
Nature Structural & Molecular Biology (Impact Factor: 13.31). 11/2011; 18(12):1381-7. DOI: 10.1038/nsmb.2152
Source: PubMed


Members of the novel SH2-containing protein (NSP) and Crk-associated substrate (Cas) protein families form multidomain signaling platforms that mediate cell migration and invasion through a collection of distinct signaling motifs. Members of each family interact via their respective C-terminal domains, but the mechanism of this association has remained enigmatic. Here we present the crystal structures of the C-terminal domain from the NSP protein BCAR3 and the complex of NSP3 with p130Cas. BCAR3 adopts the Cdc25-homology fold of Ras GTPase exchange factors, but it has a 'closed' conformation incapable of enzymatic activity. The structure of the NSP3-p130Cas complex reveals that this closed conformation is instrumental for interaction of NSP proteins with a focal adhesion-targeting domain present in Cas proteins. This enzyme-to-adaptor conversion enables high-affinity, yet promiscuous, interactions between NSP and Cas proteins and represents an unprecedented mechanistic paradigm linking cellular signaling networks.

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    • "Overexpression of BCAR3 results in a bypass of estrogen dependence for proliferation leading to resistance to anti-estrogens [5] [6]. BCAR3 belongs to the Novel SH2-containing Protein (NSP) 1–3 family and their crystal structures show similarities in molecular structure, containing an N-terminal SH2 domain, a proline/serine-rich (P/S) domain and a C-terminal GEF domain [7]. Despite similarities in overall structural features among the NSP family, there are important differences in expression patterns. "
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    ABSTRACT: Breast cancer anti-estrogen resistance 3 (BCAR3) is an SH2-containing signal transducer and is implicated in tumorigenesis of breast cancer cells. In this study, we found that BCAR3 mediates the induction of ERK activation and DNA synthesis by insulin, but not by IGF-1. Specifically, the SH2 domain of BCAR3 is involved in insulin-stimulated DNA synthesis. Differential tyrosine-phosphorylated patterns of the BCAR3 immune complex were detected in insulin and IGF-1 signaling, suggesting that BCAR3 is a distinct target molecule of insulin and IGF-1 signaling. Moreover, microinjection of BCAR3 inhibitory materials inhibited membrane ruffling induced by insulin, while this did not affect insulin-mediated GLUT4 translocation. Taken together, these results demonstrated that BCAR3 plays an important role in the signaling pathways of insulin leading to cell cycle progression and cytoskeleton reorganization, but not GLUT4 translocation.
    Full-text · Article · Nov 2013 · Biochemical and Biophysical Research Communications
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    • "BCAR3 shares structural and sequence homology with the Cdc25 family of Rac GEFs [24]. However, a recent report has shown that the C-terminal GEF-like domain adopts a “closed” conformation that is prohibitive for catalytic activity [25]. While it is therefore unlikely that BCAR3 functions as a GEF, it is possible that the Rac1 GEF DOCK180/ELMO becomes activated through BCAR3-dependent augmentation of Cas/Crk coupling [12], [13], [14], [26], [27] (Fig. 8). "
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    ABSTRACT: Metastatic breast cancer is incurable. In order to improve patient survival, it is critical to develop a better understanding of the molecular mechanisms that regulate metastasis and the underlying process of cell motility. Here, we focus on the role of the adaptor molecule Breast Cancer Antiestrogen Resistance 3 (BCAR3) in cellular processes that contribute to cell motility, including protrusion, adhesion remodeling, and contractility. Previous work from our group showed that elevated BCAR3 protein levels enhance cell migration, while depletion of BCAR3 reduces the migratory and invasive capacities of breast cancer cells. In the current study, we show that BCAR3 is necessary for membrane protrusiveness, Rac1 activity, and adhesion disassembly in invasive breast cancer cells. We further demonstrate that, in the absence of BCAR3, RhoA-dependent signaling pathways appear to predominate, as evidenced by an increase in RhoA activity, ROCK-mediated phosphorylation of myosin light chain II, and large ROCK/mDia1-dependent focal adhesions. Taken together, these data establish that BCAR3 functions as a positive regulator of cytoskeletal remodeling and adhesion turnover in invasive breast cancer cells through its ability to influence the balance between Rac1 and RhoA signaling. Considering that BCAR3 protein levels are elevated in advanced breast cancer cell lines and enhance breast cancer cell motility, we propose that BCAR3 functions in the transition to advanced disease by triggering intracellular signaling events that are essential to the metastatic process.
    Full-text · Article · Aug 2013 · PLoS ONE
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    • "Previous studies have shown that this residue is critical for NEDD9 targeting by the proteasome [19] and plays a role in NEDD9-mediated cell spreading [20]. Leucine (L) 751 of NEDD9 that mediates binding to BCAR3/AND34/NSP2 [21] is conserved in p130Cas and this site has recently been confirmed to mediate the same interaction for p130Cas [22]. By contrast S296 that is phosphorylated by Aurora kinase [23] is unique to the NEDD9 sequence. "
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    ABSTRACT: The focal adhesion docking protein NEDD9/HEF1/Cas-L regulates cell migration and cancer invasion. NEDD9 is a member of the Cas family of proteins that share conserved overall protein-protein interaction domain structure, including a substrate domain that is characterized by extensive tyrosine (Y) phosphorylation. Previous studies have suggested that phosphorylation of Y253 in the substrate domain of the Cas family protein p130Cas is specifically required for p130Cas function in cell migration. While it is clear that tyrosine phosphorylation of the NEDD9 substrate domain is similarly required for the regulation of cell motility, whether individual NEDD9 tyrosine residues have discrete function in regulating motility has not previously been reported. In the present study we have used a global sequence alignment of Cas family proteins to identify a putative NEDD9 equivalent of p130Cas Y253. We find that NEDD9 Y189 aligns with p130Cas Y253 and that it is conserved among NEDD9 vertebrate orthologues. Expression of NEDD9 in which Y189 is mutated to phenylalanine results in increased rates of cell migration and is correlated with increased disassembly of GFP.NEDD9 focal adhesions. Conversely, mutation to Y189D significantly inhibits cell migration. Our previous data has suggested that NEDD9 stabilizes focal adhesions and the present data therefore suggests that phosphorylation of Y189 NEDD9 is required for this function. These findings indicate that the individual tyrosine residues of the NEDD9 substrate domain may serve discrete functional roles. Given the important role of this protein in promoting cancer invasion, greater understanding of the function of the individual tyrosine residues is important for the future design of approaches to target NEDD9 to arrest cancer cell invasion.
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