期刊
JOURNAL OF PROTEOME RESEARCH
卷 5, 期 3, 页码 581-588出版社
AMER CHEMICAL SOC
DOI: 10.1021/pr050362b
关键词
phosphoproteomics; SILAC; Eph signaling; mass spectrometry
资金
- NCRR NIH HHS [S10 RR017990-01] Funding Source: Medline
- NINDS NIH HHS [R21 NS044184-02, P30 NS050276, R21 NS044184, R21 NS44184] Funding Source: Medline
Eph-related receptor tyrosine kinases (RTK) have been implicated in several biological functions including synaptic plasticity, axon guidance, and morphogenesis, yet the details of the signal transduction pathways that produce these specific biological functions after ligand-receptor interaction remain unclear. We used Stable Isotope Labeling by Amino Acids in Cell Culture (SILAC) in combination with LC-MS/MS to characterize cellular signaling following stimulation by ephrinB1-Fc of NG-108 cells that overexpress EphB2 receptors. Because tyrosine phosphorylation functions as a key regulatory event in RTK signaling, we used anti-phosphotyrosine immunoprecipitation (pY IP) of cell lysates to isolate potential participants in the EphB2 pathway. Our SILAC experiments identified 127 unique proteins, 40 of which demonstrated increased abundance in pY lPs from ephrinBl-Fc stimulated cells as compared with unstimulated cells. Six proteins demonstrated decreased abundance, and 81 did not change significantly in relative abundance. Western blotting analysis of five proteins after pY IP verified their SILAC results. On the basis of previously published work and use of PathwayAssist software, we proposed an interaction network downstream of EphB2 for the proteins with changed ratios.
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