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. Author manuscript; available in PMC: 2010 Jul 7.
Published in final edited form as: Cancer Cell. 2009 Jul 7;16(1):9–20. doi: 10.1016/j.ccr.2009.04.009

Figure 5. Multiple growth factors can induce S897 phosphorylation of EphA2, which is required for EphA2 localization to the leading edge and for cell polarization.

Figure 5

A–B. Characterization of a rabbit polyclonal antibody against S897 phospho-peptide. Serum-starved HEK 293 cells that express vector, WT-EphA2, or S897A-EphA2 were stimulated with FBS in the absence and presence of ephrin-A1-Fc for 10 min. Total cell lysates (A) and EphA kinase precipitates (B) were analyzed by immunoblotting with polyclonal anti-pS897-EphA2 and total EphA2.

C. PP2A treatment causes dephosphorylation of serum-induced pS897-EphA2. Serum-stimulated U373 cell lysates were subjected to PP2A phosphatase assay followed by immunoblot with polyclonal anti-pS897-EphA2.

D–E. Multiple growth factors induce S897 phosphorylation of EphA2 and migration. (D) Serum-starved U373 cells were stimulated with 10 ng/ml of EGF, bFGF, PDGF, HGF, 10% FBS, or 10 μM LPA alone or in combination with Fc or ephrin-A1-Fc for 10 min. Total cell lysates were analyzed as in A. (E) U373 cell migration toward same concentrations of growth factors and LPA or 5%FBS. Numbers represent mean ± S.D. from 6 random fields. The numbers over solid bars show the percentage of inhibition by ephrin-A1.

F. Inhibition of FGFR is not sufficient for inhibiting serum-induced pS897-EphA2. Serum-starved U373 cells were pretreated with 10 μM SU5402 or 100 nM 1-(2-Amino-6-(3,5-dimethoxyphenyl)pyrido[2,3-d]pyrimidin-7-yl)-3-tert-butyl urea (BU) for 30 min. Cells were then stimulated with FBS for 10 min. Cell lysates were analyzed by immunoblotting with polyclonal anti-pS897-EphA2 and pS473-Akt.

G. Phospho-S897-EphA2 is localized to the leading edge and the tips of actin stress fibers. Serum-starved cells were treated and processed for immunofluorescent staining as described in Fig. 1K. Scale bar, 25 μm in top and bottom row, 10 μm in the zoomed images.

H. Cells expressing mutant S897A-EphA2 show defective cell polarization, which was correlated with mislocalization of the mutant proteins to cell-cell junctions. Scale bar, 25 μm.