Abstract
Receptor tyrosine kinases (RTK) are proteins that undergo dimerization and/or multimerization and autophosphorylation in response to ligand stimulation. Members of the RTK family are receptors for a series of growth factors that, upon stimulation, are able to start signaling events that promote cell growth and differentiation. A class of RTKs, the Eph receptors (EphRs), are found in a variety of cell types and play important roles in patterning the central and peripheral nervous systems, as well as in synapse and neural crest formation. Interaction of Eph receptors with their ephrin ligands activates signal transduction pathways that lead to cytoskeletal remodeling through formation of many stable or transient protein-protein interactions. However, these intracellular signal transduction pathways that lead to cytoskeletal remodeling are not well understood. Here, we combined Blue Native PAGE (BN-PAGE) and mass spectrometry (MS) to analyze protein-protein interactions as a result of ephrin stimulation. We analyzed both lysates and phosphotyrosine immunoprecipitate (pY99-IP) of unstimulated and ephrin-stimulated cells. Our experiments allowed us to characterize many constitutive homo- and hetero-protein complexes from the cell lysate. Furthermore, BN-PAGE and MS of the pY99-IPs from both unstimulated and stimulated cells allowed us to analyze protein-protein interactions that resulted upon ephrin stimulation. Combination of BN-PAGE and MS also has the potential for the analysis of stable and transient protein-protein interactions in other ligand-stimulated RTK-dependent signal transduction pathways.
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Keywords
- Focal Adhesion Kinase
- Ligand Stimulation
- Receptor Tyrosine Kinase Family
- Blue Native Page
- Tyrosine Kinase Substrate
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.
References
Adams, R. H. (2003). “Molecular control of arterial-venous blood vessel identity.” J Anat 202(1): 105-12.
Aebersold, R. and M. Mann (2003). “Mass spectrometry-based proteomics.” Nature 422(6928): 198-207.
Aivaliotis, M., M. Karas, et al. (2006). “High throughput two-dimensional blue-native electro-phoresis: a tool for functional proteomics of cytoplasmatic protein complexes from Chlorobium tepidum.” Photosynth Res 88(2): 143-57.
Blagoev, B., I. Kratchmarova, et al. (2003). “A proteomics strategy to elucidate functional protein-protein interactions applied to EGF signaling.” Nat Biotechnol 21(3): 315-8.
Bruckner, K., E. B. Pasquale, et al. (1997). “Tyrosine phosphorylation of transmembrane ligands for Eph receptors.” Science 275(5306): 1640-3.
Camacho-Carvajal, M. M., B. Wollscheid, et al. (2004). “Two-dimensional Blue native/SDS gel electrophoresis of multi-protein complexes from whole cellular lysates: a proteomics approach.” Mol Cell Proteomics 3(2): 176-82.
Carter, N., T. Nakamoto, et al. (2002). “EphrinA1-induced cytoskeletal re-organization requires FAK and p130(cas).” Nat Cell Biol 4(8): 565-73.
Cheng, N., D. M. Brantley, et al. (2002). “The ephrins and Eph receptors in angiogenesis.” Cytokine Growth Factor Rev 13(1): 75-85.
Cowan, C. A. and M. Henkemeyer (2001). “The SH2/SH3 adaptor Grb4 transduces B-ephrin reverse signals.” Nature 413(6852): 174-9.
Dalva, M. B., M. A. Takasu, et al. (2000). “EphB receptors interact with NMDA receptors and regulate excitatory synapse formation.” Cell 103(6): 945-56.
Darie, C. C., M. L. Biniossek, et al. (2005). “Isolation and structural characterization of the Ndh complex from mesophyll and bundle sheath chloroplasts of Zea mays.” Febs J 272(11): 2705-16.
Darie, C. C., W. G. Janssen, et al. (2007). “Purified trout egg vitelline envelope proteins VEbeta and VEgamma polymerize into homomeric fibrils from dimers in vitro.” Biochim Biophys Acta doi:10.1016/j.bbapap.2007.10.011
Darie, C. C. and T. A. Neubert (2008). “Analysis of protein-protein interactions in EphB2-NG108 cells as a result of ephrinB1 stimulation.” In preparation.
DeLaBarre, B. and A. T. Brunger (2003). “Complete structure of p97/valosin-containing protein reveals communication between nucleotide domains.” Nat Struct Biol 10(10): 856-63.
Gale, N. W., S. J. Holland, et al. (1996). “Eph receptors and ligands comprise two major speci-ficity subclasses and are reciprocally compartmentalized during embryogenesis.” Neuron 17 (1): 9-19.
Gavin, A. C., M. Bosche, et al. (2002). “Functional organization of the yeast proteome by sys-tematic analysis of protein complexes.” Nature 415(6868): 141-7.
Gygi, S. P., B. Rist, et al. (1999). “Quantitative analysis of complex protein mixtures using isotope-coded affinity tags.” Nat Biotechnol 17(10): 994-9.
Ho, Y., A. Gruhler, et al. (2002). “Systematic identification of protein complexes in Saccharomyces cerevisiae by mass spectrometry.” Nature 415(6868): 180-3.
Holland, S. J., N. W. Gale, et al. (1996). “Bidirectional signalling through the EPH-family recep-tor Nuk and its transmembrane ligands.” Nature 383(6602): 722-5.
Holland, S. J., E. Peles, et al. (1998). “Cell-contact-dependent signalling in axon growth and guidance: Eph receptor tyrosine kinases and receptor protein tyrosine phosphatase beta.” Curr Opin Neurobiol 8(1): 117-27.
Ito, T., K. Tashiro, et al. (2000). “Toward a protein-protein interaction map of the budding yeast: A comprehensive system to examine two-hybrid interactions in all possible combinations be-tween the yeast proteins.” Proc Natl Acad Sci U S A 97(3): 1143-7.
Kobayashi, H., N. Tanaka, et al. (2005). “Hrs, a mammalian master molecule in vesicular trans-port and protein sorting, suppresses the degradation of ESCRT proteins signal transducing adaptor molecule 1 and 2.” J Biol Chem 280(11): 10468-77.
Litscher, E. S., W. G. Janssen, et al. (2008). “Purified mouse egg zona pellucida glycoproteins polymerize into homomeric fibrils under non-denaturing conditions.” J Cell Physiol 214(1): 153-7.
Lu, L., M. Komada, et al. (1998). “Human Hrs, a tyrosine kinase substrate in growth factor-stimulated cells: cDNA cloning and mapping of the gene to chromosome 17.” Gene 213(1-2): 125-32.
McNaught, K. S., C. W. Olanow, et al. (2001). “Failure of the ubiquitin-proteasome system in Parkinson’s disease.” Nat Rev Neurosci 2(8): 589-94.
Miao, H., E. Burnett, et al. (2000). “Activation of EphA2 kinase suppresses integrin function and causes focal-adhesion-kinase dephosphorylation.” Nat Cell Biol 2(2): 62-9.
Ong, S. E., B. Blagoev, et al. (2002). “Stable isotope labeling by amino acids in cell culture, SILAC, as a simple and accurate approach to expression proteomics.” Mol Cell Proteomics 1 (5): 376-86.
Ong, S. E., L. J. Foster, et al. (2003). “Mass spectrometric-based approaches in quantitative pro-teomics.” Methods 29(2): 124-30.
Polte, T. R. and S. K. Hanks (1995). “Interaction between focal adhesion kinase and Crk-associated tyrosine kinase substrate p130Cas.” Proc Natl Acad Sci U S A 92(23): 10678-82.
Reifschneider, N. H., S. Goto, et al. (2006). “Defining the mitochondrial proteomes from five rat organs in a physiologically significant context using 2D blue-native/SDS-PAGE.” J Pro-teome Res 5(5): 1117-32.
Robinson, V., A. Smith, et al. (1997). “Roles of Eph receptors and ephrins in neural crest path-finding.” Cell Tissue Res 290(2): 265-74.
Schagger, H., W. A. Cramer, et al. (1994). “Analysis of molecular masses and oligomeric states of protein complexes by blue native electrophoresis and isolation of membrane protein com-plexes by two-dimensional native electrophoresis.” Anal Biochem 217(2): 220-30.
Schagger, H. and G. von Jagow (1991). “Blue native electrophoresis for isolation of membrane protein complexes in enzymatically active form.” Anal Biochem 199(2): 223-31.
Schwikowski, B., P. Uetz, et al. (2000). “A network of protein-protein interactions in yeast.” Nat Biotechnol 18(12): 1257-61.
Steen, H., B. Kuster, et al. (2002). “Tyrosine phosphorylation mapping of the epidermal growth factor receptor signaling pathway.” J Biol Chem 277(2): 1031-9.
Uetz, P., L. Giot, et al. (2000). “A comprehensive analysis of protein-protein interactions in Sac-charomyces cerevisiae.” Nature 403(6770): 623-7.
Vearing, C. J. and M. Lackmann (2005). “Eph receptor signalling; dimerisation just isn’t enough.” Growth Factors 23(1): 67-76.
von Mering, C., R. Krause, et al. (2002). “Comparative assessment of large-scale data sets of protein-protein interactions.” Nature 417(6887): 399-403.
Wang, Y., R. Li, et al. (2006). “Proteomic analysis reveals novel molecules involved in insulin sig-naling pathway.” J Proteome Res 5(4): 846-55.
Wilkinson, D. G. (2001). “Multiple roles of EPH receptors and ephrins in neural development.” Nat Rev Neurosci 2(3): 155-64.
Yancopoulos, G. D., M. Klagsbrun, et al. (1998). “Vasculogenesis, angiogenesis, and growth factors: ephrins enter the fray at the border.” Cell 93(5): 661-4.
Zhang, G., D. S. Spellman, et al. (2006). “Quantitative phosphotyrosine proteomics of EphB2 signaling by stable isotope labeling with amino acids in cell culture (SILAC).” J Proteome Res 5(3): 581-8.
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Darie, C.C. et al. (2008). Blue Native PAGE and Mass Spectrometry Analysis of Ephrin Stimulation-Dependent Protein-Protein Interactions in NG108-EphB2 Cells. In: Popescu, C., Zamfir, A.D., Dinca, N. (eds) Applications of Mass Spectrometry in Life Safety. NATO Science for Peace and Security Series A: Chemistry and Biology. Springer, Dordrecht. https://doi.org/10.1007/978-1-4020-8811-7_1
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