Rap1GAP is ubiquitinated and degraded in a β-TrCP dependent manner.

<p>(A) 293T cells were co-transfected with Flag-Rap1GAP and the indicated Myc-tagged E3 ligases constructs (β-TrCP1, SKP2, Cdc20, Cdh1 and Fbw5). At 24 h after transfection, cells were harvested and lysed. Cell lysates were subjected to immunoprecipitation with anti-Myc antibody. The immunoprecipitates were immunoblotted with anti-Flag and anti-Myc antibodies, respectively. (B) 293T cells were co-transfected with HA-Rap1GAP and Flag- β-TrCP1/2 constructs. Cell lysates were prepared and subjected to immunoprecipitation with anti-HA or anti-Flag antibodies, respectively. (C) Endogenous Rap1GAP interacts with endogenous β-TrCP in HeLa cells. At the 4 h before harvesting, cells were treated with the proteasome inhibitor, MG132. HeLa cell lysates were incubated with protein A/G sepharose conjugated with either control IgG or Rap1GAP antibody. The immunoprecipitates were immunoblotted with Rap1GAP or β-TrCP antibodies, respectively. (D) Alignment of amino acids corresponding to the DSGxxS sequence with Rap1GAP1 orthologs and other β-TrCP substrates. (E) 293T cells were co-transfected with Flag-β-TrCP1/2 and wild type HA-Rap1GAP or SA mutant (S525A, S529A and S525/529A) constructs. At 24 h after transfection, cells were harvested and lysed. Cell lysates were subjected to immunoprecipitation with anti-HA antibody. The immunoprecipitates were immunoblotted with anti-Flag and anti-HA antibodies, respectively. (F) Flag-Rap1GAP (WT or S525/529A), HA-ubiquitin, and β-TrCP1 (WT or ΔF Box mutant) constructs were co-transfected into 293T cells. Rap1GAP proteins were immunoprecipitated by anti-Flag antibody. The polyubiquitinated forms of Rap1GAP were immunoblotted with anti-HA antibody. (G) Control siRNA or β-TrCP-specific siRNA were transfected in HeLa cells that were synchronized by double thymidine block followed by release into nocodazole-containing media for the indicated times. Cell lysates were subjected immunoblotting with the indicated antibodies. (H) Flag-Rap1GAP wild type or S525/529A mutant was transfected into HeLa cells that were synchronized by double thymidine block followed by release into nocodazole-containing media for the indicated times. Cell lysates were subjected to immunoblotting with the indicated antibodies.