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Fig. 5. Mutation of the second UIM of Eps15 abrogates EGF-induced Eps15 phosphorylation and re-distribution. (A) Wild-type and mutant Eps15 constructs used in this study. Eps15-UIM^- contains leucine to alanine substitutions at positions 883 and 885, indicated by an asterisk. UIM^-WT consists of amino acids 741-897 of murine Eps15. UIM^-mut in addition contains L883A/L885A substitutions. (B) CHO cells were co-transfected with EGFR-WT, c-Cbl and Eps15-WT or Eps15-UIM^-. FLAG-tagged Eps15 and EGFR were immunoprecipitated from stimulated or control cells. Immunoprecipitates (IP) and total cell lysates were separated on gel and immunoblotted (IB) using mAbs to PY, FLAG-tag or HA-tag. (C-N) For immunofluorescence, CHO cells expressing EGFR-WT, c-Cbl and Eps15-WT (C-H) or Eps15-UIM^- (I-N) were incubated with EGF-TxR (red) on ice for 1 hour. After stimulation at 37°C for 0 minutes (C,D,I,J), 1 minute (E,F,K,L) or 5 minutes (G,H,M,N), cells were stained with anti-FLAG mAb, followed by FITC-conjugated secondary antibody (green). The displayed confocal planes are from the basal half of the cells. XZ sections are shown below each panel.

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