Now showing 1 - 5 of 5
  • Publication
    Substrate-Trapped Interactors of PHD3 and FIH Cluster in Distinct Signaling Pathways
    Amino acid hydroxylation is a post-translational modification that regulates intra- and inter-molecular protein-protein interactions. The modifications are regulated by a family of 2-oxoglutarate- (2OG) dependent enzymes and, although the biochemistry is well understood, until now only a few substrates have been described for these enzymes. Using quantitative interaction proteomics, we screened for substrates of the proline hydroxylase PHD3 and the asparagine hydroxylase FIH, which regulate the HIF-mediated hypoxic response. We were able to identify hundreds of potential substrates. Enrichment analysis revealed that the potential substrates of both hydroxylases cluster in the same pathways but frequently modify different nodes of signaling networks. We confirm that two proteins identified in our screen, MAPK6 (Erk3) and RIPK4, are indeed hydroxylated in a FIH- or PHD3-dependent mechanism. We further determined that FIH-dependent hydroxylation regulates RIPK4-dependent Wnt signaling, and that PHD3-dependent hydroxylation of MAPK6 protects the protein from proteasomal degradation.
      292Scopus© Citations 67
  • Publication
    A Compendium of Co-regulated Protein Complexes in Breast Cancer Reveals Collateral Loss Events
    Protein complexes are responsible for the bulk of activities within the cell, but how their behavior and abundance varies across tumors remains poorly understood. By combining proteomic profiles of breast tumors with a large-scale protein-protein interaction network, we have identified a set of 285 high-confidence protein complexes whose subunits have highly correlated protein abundance across tumor samples. We used this set to identify complexes that are reproducibly under- or overexpressed in specific breast cancer subtypes. We found that mutation or deletion of one subunit of a co-regulated complex was often associated with a collateral reduction in protein expression of additional complex members. This collateral loss phenomenon was typically evident from proteomic, but not transcriptomic, profiles, suggesting post-transcriptional control. Mutation of the tumor suppressor E-cadherin (CDH1) was associated with a collateral loss of members of the adherens junction complex, an effect we validated using an engineered model of E-cadherin loss. Ryan et al. develop an approach to identify co-regulated protein complexes from breast tumor proteomic profiles and demonstrate that genomic loss of one subunit is often associated with a reduction in the protein expression of an entire complex.
      17Scopus© Citations 35
  • Publication
    ADAM10: a new player in breast cancer progression?
    Background: The ADAM proteases are best known for their role in shedding the extracellular domain of transmembrane proteins. Among the transmembrane proteins shed by ADAM10 are notch, HER2, E-cadherin, CD44, L1 and the EGFR ligands, EGF and betacellulin. As cleavage of several of these proteins has been implicated in cancer formation and progression, we hypothesised that ADAM10 is also involved in these processes. Methods: ADAM10 expression was decreased by RNA interference and the effects of this on cell numbers, invasion and migration were determined. We also examined the effect of ADAM10 inhibition on breast cancer cell line invasion and migration. Results: Using the triple-negative (TN) breast cancer cell lines, BT20, MDA-MB-231 and the non-TN cell line MDA-MB-453, knockdown of ADAM10 expression significantly decreased in vitro migration (Po0.01; for each cell line). Similarly, treatment with the ADAM10-selective inhibitor GI254023X reduced migration in the three cell lines (for BT20, Po0.001; for MDA-MB-231, P ¼ 0.005; for MDA-MB-453, P ¼ 0.023). In contrast, neither knockdown of ADAM10 nor treatment with the ADAM10-selective inhibitor GI254023X significantly affected cell numbers. Using extracts of primary breast cancers, higher levels of ADAM10 were found more frequently in high-grade vs low-grade tumours (Po0.001) and in oestrogen receptor (ER)-negative compared with ERpositive tumours (P ¼ 0.005). Analysis of pooled publicly available data sets found that high levels of ADAM10 mRNA were associated with adverse outcome in patients with the basal subtype of breast cancer. Conclusions: Based on our combined cell line and breast cancer extract data, we conclude that ADAM10 is likely to be involved in breast cancer progression, especially in the basal subtype.
      282Scopus© Citations 63
  • Publication
    Extensive rewiring of the EGFR network in colorectal cancer cells expressing transforming levels of KRAS G13D
    Protein-protein-interaction networks (PPINs) organize fundamental biological processes, but how oncogenic mutations impact these interactions and their functions at a network-level scale is poorly understood. Here, we analyze how a common oncogenic KRAS mutation (KRASG13D) affects PPIN structure and function of the Epidermal Growth Factor Receptor (EGFR) network in colorectal cancer (CRC) cells. Mapping >6000 PPIs shows that this network is extensively rewired in cells expressing transforming levels of KRASG13D (mtKRAS). The factors driving PPIN rewiring are multifactorial including changes in protein expression and phosphorylation. Mathematical modelling also suggests that the binding dynamics of low and high affinity KRAS interactors contribute to rewiring. PPIN rewiring substantially alters the composition of protein complexes, signal flow, transcriptional regulation, and cellular phenotype. These changes are validated by targeted and global experimental analysis. Importantly, genetic alterations in the most extensively rewired PPIN nodes occur frequently in CRC and are prognostic of poor patient outcomes.
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  • Publication
    Vascular Endothelial Growth Factor (VEGF) Promotes Assembly of the p130Cas Interactome to Drive Endothelial Chemotactic Signaling and Angiogenesis
    (American Society for Biochemistry and Molecular Biology, 2016-12-22) ; ; ; ; ;
    p130Cas is a polyvalent adapter protein essential for cardiovascular development, and with a key role in cell movement. In order to identify the pathways by which p130Cas exerts its biological functions in endothelial cells we mapped the p130Cas interactome and its dynamic changes in response to VEGF using high-resolution mass spectrometry and reconstruction of protein interaction (PPI) networks with the aid of multiple PPI databases. VEGF enriched the p130Cas interactome in proteins involved in actin cytoskeletal dynamics and cell movement, including actin-binding proteins, small GTPases and regulators or binders of GTPases. Detailed studies showed that p130Cas association of the GTPase-binding scaffold protein, IQGAP1, plays a key role in VEGF chemotactic signaling, endothelial polarization, VEGF-induced cell migration, and endothelial tube formation. These findings indicate a cardinal role for assembly of the p130Cas interactome in mediating the cell migratory response to VEGF in angiogenesis, and provide a basis for further studies of p130Cas in cell movement.
      304Scopus© Citations 21