Fig 3.

Identification of tyrosine residues in HIP1 required for EGFR-mediated phosphorylation of HIP1. (A) Schematic of HIP1 Y-to-F mutations. The diagram also displays the locations of all 17 tyrosine residues (diamond-headed stalks) within the domains of HIP1. The key mutants that were not phosphorylated by wild-type RTKs are illustrated in the magnified area (HIP1 4xYF and 7xYF). Abbreviations are as described in the legend to Fig. 2. HPM, HIP1 phosphorylation motif. (B and C) HEK293T cells were cotransfected with EGFR-V5 and either Myc-tagged wild-type HIP1 or Myc-tagged HIP1 Y-to-F point mutants. Immunoprecipitation was performed with anti-Myc beads, and phosphorylation was detected with antiphosphotyrosine antibody 4G10. (B) The USH-deficient mutant (HIP1Δ753-799) and the HPM Y-to-F mutants (HIP1 4xYF) were not phosphorylated by EGFR or EGFRvIII. (C) Y152 of HIP1 inhibits EGFR phosphorylation. Phosphorylation-resistant mutants (lanes 5 and 7) were phosphorylation sensitive when the Y152F point mutation was included (lanes 6 and 8). The levels of EGFR were increased in the presence of wild-type HIP1 but not in the presence of phosphorylation-resistant mutants (bottom panel). (D) HEK293T cells were transfected with Myc-tagged HIP1 or the HIP1/HPM(4xYF) mutant and assayed for HIP1 levels by Western blotting with anti-Myc antibody over a 96-hour period. (E) Low GFP levels that were associated with cotransfected HIP1/HPM(4xYF) (lanes 7 to 9) were rescued by cotransfection of HIP1/HPM(4xYF)-ires-GFP with wild-type HIP1 (lanes 10 to 12). Wild-type HIP1 also increased GFP levels (lanes 4 to 6) compared to those in cells transfected with wild-type HIP1-ires-GFP (lanes 1 to 3). (F) Cos-7 cells were transfected with Myc-tagged wild-type HIP1, HIP1/HPM(4xYF), and HIP1/ΔANTH DNA constructs. Cells were stained with mouse monoclonal anti-Myc antibody (cytoplasmic staining) to allow for analysis of transfected cells and with DAPI to show nuclear morphology. Cells were scored at 24 h posttransfection and deemed apoptotic if nuclear condensation or fragmentation was observed (arrowheads). These images are representative of the overall results, where the HIP1-transfected cells were found more frequently with smooth, nonblebbed nuclei and contained large regions of HIP1-expressing cytoplasm. In comparison, the mutant cells displayed more condensed cytoplasm, and the nuclei were less frequently intact. (G) Apoptotic cells in the three different transfections described for panel F were quantitated. The experiment was performed on three separate occasions, and data were averaged. Error bars denote standard deviations. At least 100 cells from each sample in each experiment were scored for apoptosis by two blinded investigators (A.A.W. and A.C.), according to nuclear morphology. The percentages of apoptotic cells were compared to the baseline cell death frequency (15%) of surrounding HIP1-negative cells in each experiment.