Figure 2.

SH3P2 interacts with Myo1E via its N-terminal proline-rich region and C-terminal acidic amino acid cluster. (A) Glutathione-Sepharose beads coupled with GST or GST-tagged wild-type or indicated mutant forms of human SH3P2 were incubated with MKN1 cell lysates, after which bead-bound proteins as well as the whole-cell lysates (10% of the input for the pull-down assays) were subjected to SDS-PAGE followed by staining with Coomassie brilliant blue (CBB) or immunoblot analysis (IB) with antibodies to Myo1E. Arrowheads indicate the Myo1E band. (B) MKN1 cells were transfected for 24 h with vectors encoding EGFP-tagged wild-type or mutant forms of human SH3P2, lysed, and subjected to immunoprecipitation (IP) with antibodies to GFP. The resulting precipitates as well as the whole-cell lysates (10% of the input for immunoprecipitation) were subjected to immunoblot analysis with antibodies to Myo1E and to GFP. Amino acid sequences of the PR region and the C-terminal acidic amino acid cluster are shown. The RSK phosphorylation site (Ser²⁰²), acidic amino acids, basic amino acids, and proline are indicated in yellow, red, blue, and green, respectively. All data are representative of at least three separate experiments.