Abstract
While the affinities and specificities of SH2 domain-phosphotyrosine interactions have been well characterized, spatio-temporal changes in phosphosite availability in response to signals, and their impact on recruitment of SH2-containing proteins in vivo, are not well understood. To address this issue, we used three complementary experimental approaches to monitor phosphorylation and SH2 binding in human A431 cells stimulated with epidermal growth factor (EGF): 1) phospho-specific mass spectrometry; 2) far-Western blotting; and 3) live cell single-molecule imaging of SH2 membrane recruitment. Far-Western and MS analyses identified both well-established and previously undocumented EGF-dependent tyrosine phosphorylation and binding events, as well as dynamic changes in binding patterns over time. In comparing SH2 binding site phosphorylation with SH2 domain membrane recruitment in living cells, we found in vivo binding to be much slower. Delayed SH2 domain recruitment correlated with clustering of SH2 domain binding sites on the membrane, consistent with membrane retention via SH2 rebinding.
Highlights
Receptor tyrosine kinases (RTK) and tyrosine kinase-associated receptors play an essential role in transducing extracellular signals into the cell
Anti-pY blots of the same lysates showed a rapid increase in total phosphorylation upon Epidermal Growth Factor (EGF) treatment, dominated by a major band corresponding to EGFR (Figure 2A, top panel)
These major bands were (1) EGFR, a ~ 195 kDa band whose phosphorylation increased rapidly following EGF treatment, dipped slightly and remained relatively constant; (2 and 3) the focal adhesion protein p130CAS, a doublet at 150 and 115 kDa whose phosphorylation was high in unstimulated cells, decreased rapidly upon EGF treatment, and rebounded at later time points; (4) the scaffolding protein GAB1, a single band at 130 kDa that was phosphorylated with kinetics similar to EGFR, rapidly returned to near basal levels; and (5) the scaffold/adaptor SHCA, a relatively weak band at 71 kDa which displayed kinetics similar to that of EGFR (Figure 2A, Figure 2—figure supplement 1, Supplementary file 2)
Summary
Receptor tyrosine kinases (RTK) and tyrosine kinase-associated receptors play an essential role in transducing extracellular signals into the cell. These proteins function as central signaling nodes for a diverse set of normal biological processes including proliferation, differentiation, immune cell activation, neuronal development, angiogenesis, and cell migration. 120 SH2 domains have been identified in 110 different proteins (Liu et al, 2006; Tinti et al, 2013) The central role these proteins play in cellular signaling has made them popular targets for study. Much less is known about the role that spatial and temporal changes in protein
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
