Figure 6
From: Intramolecular hydrophobic interactions are critical mediators of STAT5 dimerization
F706 is required for constitutive activity of the Y665F mutation.
(a) STAT5A dimer interface (top view). The SH2 domains are depicted in ribbon (cyan, M1) or surface representation (grey, M2). The hydrophobic interface of M2 is highlighted in orange. Both CTSs are depicted as ribbon structures. Side chains of pY694, I699 and F706 are shown in stick representation. Magenta, carbon; red, oxygen; white, phosphorus. The F706 interface is magnified and residues F706, L663, Y665, F633, L666 and W631 are shown in stick representation. Mutation-site Y665 is highlighted in magenta. (b) Native PAGE analysis of WCLs of HeLa T-REx HA-EpoR cells stably expressing STAT5A-eYFP or STAT5A mutants, treated with sodium vanadate (1 mM, 1.5 h) and/or Epo (1 U/ml, 30 min) as indicated. The native gel was analyzed by fluorescence scanning for the detection of YFP-tagged proteins. WCLs were subjected to immunoblotting using antibodies against pY694/699-STAT5A/B and GFP. (c) Quantification of native PAGE experiments. Normalized STAT5A dimerization levels and normalized pSTAT5/STAT5A ratios relative to stimulated cells expressing wild-type STAT5A were plotted. The data shown represent geometric means ± 95% confidence intervals of n = 3 independent experiments statistically evaluated by a paired ratio t-test with ***p < 0.005, **p < 0.01 and *p < 0.05. (d) Tyrosine 694 phosphorylation kinetics of STAT5A-eYFP or the indicated STAT5A mutants stably expressed in HeLa T-REx HA-EpoR cells. Cells were pulse-stimulated with 1 U/ml Epo for 30 min and analyzed at the indicated time periods. WCLs were subjected to immunoblotting using antibodies against pY694/699-STAT5A/B and GFP.
