FIGURE 1.

Plk1 phosphorylates MyoGEF on Thr-574. A, schematic diagram of MyoGEF. The numbers indicate the amino acids. T574, threonine 574. DH, Dbl homology domain. PH, pleckstrin homology domain. B, Plk1 phosphorylates MyoGEF in vitro. Four GST-tagged MyoGEF polypeptides were incubated with or without purified Plk1 in the presence of [γ-³²P]ATP. The proteins were resolved on SDS-PAGE gel and visualized by autoradiography (upper panel) or Coomassie Blue staining (lower panel). C, Plk1 phosphorylates MyoGEF in vivo. HeLa cells were transfected with plasmids encoding wild type full-length MyoGEF (Myc-WT) or mutated full-length MyoGEF (Myc-T574A, Myc-T585A, or Myc-T620A). Anti-Myc-conjugated agarose was used to precipitate Myc-tagged proteins from transfected cell lysates, followed by immunoblot analysis with anti-phosphothreonine antibody. D, threonine-phosphorylation of MyoGEF in transfected cells is sensitive to Plk1 depletion. HeLa cells were transfected with a plasmid encoding Myc-MyoGEF and control siRNA (siCont; lanes 1 and 2) or Plk1 siRNA (siPlk1; lanes 3 and 4). The unsynchronized transfected cells were subjected to immunoprecipitation with normal control IgG or anti-Myc antibody. WB, Western blot; IP, immunoprecipitation.