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Substrate-Trapped Interactors of PHD3 and FIH Cluster in Distinct Signaling Pathways
2016-03-22, Rodriguez, Javier, Pilkington, Ruth, Garcia Munoz, Amaya, Nguyen, Lan K., Rauch, Nora, Kennedy, Susan A., Monsefi, Naser, Herrero, Ana, Taylor, Cormac T., Kriegsheim, Alexander von
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.
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Bistability in the Rac1, PAK, and RhoA Signaling Network Drives Actin Cytoskeleton Dynamics and Cell Motility Switches
2016-01-27, Byrne, Kate M., Monsefi, Naser, Dawson, John C., Degasperi, Andrea, Volinsky, Natalia, Dobrzyński, Maciej, Kida, Katarzyna, Kolch, Walter, Nguyen, Lan K., Kriegsheim, Alexander von, Kholodenko, Boris N., et al.
Dynamic interactions between RhoA and Rac1, members of the Rho small GTPase family, play a vital role in the control of cell migration. Using predictive mathematical modeling, mass spectrometry-based quantitation of network components, and experimental validation in MDA-MB-231 mesenchymal breast cancer cells, we show that a network containing Rac1, RhoA, and PAK family kinases can produce bistable, switch-like responses to a graded PAK inhibition. Using a small chemical inhibitor of PAK, we demonstrate that cellular RhoA and Rac1 activation levels respond in a history-dependent, bistable manner to PAK inhibition. Consequently, we show that downstream signaling, actin dynamics, and cell migration also behave in a bistable fashion, displaying switches and hysteresis in response to PAK inhibition. Our results demonstrate that PAK is a critical component in the Rac1-RhoA inhibitory crosstalk that governs bistable GTPase activity, cell morphology, and cell migration switches.
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HGF induces epithelial-to-mesenchymal transition by modulating the mammalian Hippo/MST2 and ISG15 pathways
2014-06-06, Farrell, Jennifer, Kelly, Ciara, Rauch, Jens, Kida, Katarzyna, Garcia Munoz, Amaya, Monsefi, Naser, Turriziani, Benedetta, Doherty, Carolanne, Mehta, J. P., Matallanas, David, Simpson, Jeremy C., Kolch, Walter, Kriegsheim, Alexander von
Epithelial to mesenchymal transition (EMT) is a fundamental cell differentiation/dedifferentiation process which is associated with dramatic morphological changes. Formerly polarized and immobile epithelial cells which form cell junctions and cobblestone-like cell sheets undergo a transition into highly motile, elongated, mesenchymal cells lacking cell-to-cell adhesions. To explore how the proteome is affected during EMT we profiled protein expression and tracked cell biological markers in Madin-Darby kidney epithelial cells undergoing hepatocyte growth factor (HGF) induced EMT. We were able to identify and quantify over 4000 proteins by mass spectrometry. Enrichment analysis of this revealed that expression of proteins associated with the ubiquitination machinery was induced, whereas expression of proteins regulating apoptotic pathways was suppressed. We show that both the mammalian Hippo/MST2 and the ISG15 pathways are regulated at the protein level by ubiquitin ligases. Inhibition of the Hippo pathway by overexpression of either ITCH or A-Raf promotes HGF-induced EMT. Conversely, ISG15 overexpression is sufficient to induce cell scattering and an elongated morphology without external stimuli. Thus, we demonstrate for the first time that the Hippo/MST2 and ISG15 pathways are regulated during growth-factor induced EMT.