The Numb-binding domain of TBC1D15 targets p53 for proteolysis.

<p>(A) Following transfection with the indicated expression vectors, HEK-293A cells were exposed to vehicle or Nutlin-3 (10 μM) for 16 hr as indicated, then lysed and analyzed by immunoblotting. (B) Depletion of endogenous <i>TBC1D15</i> increases steady-state levels of p53. HEK-293A cells were stably transduced with lentivirus vectors encoding a non-targeting, scrambled shRNA control (sh-<i>Scr</i>) or two different shRNAs targeting <i>TBC1D15</i>, or transfected with Flag-p53 or myc-TBC1D15, followed by preparation of lysates, SDS-PAGE and immunoblotting using the indicated antibodies. (C) Depletion of TBC1D15 sensitizes cells to apoptosis. HEK-293A cells harboring a non-targeting shRNA or sh1-<i>TBC1D15,</i> or expressing either myc-TBC1D15 or Flag-p53, were treated with DMSO vehicle or with etoposide (34 μM, 16 hr) followed by propidium iodide staining and FACS analysis. The percentage of sub-G1 DNA content is indicated in each histogram. (D) The amino-terminal domain of TBC1D15 targets p53 for proteolysis. Myc-p53 was expressed in HEK-293A cells with empty vector or with the indicated Flag-TBC1D15 constructs. Lysates were immunoprecipitated using either anti-Numb or anti-myc agarose. GFP was co-transfected as an internal control for transfection efficiency. (E) Functional dissection of the TBC1D15 amino-terminal domain in p53 destabilization. Myc-p53 was transfected with GFP or with the indicated GFP-TBC1D15 fusions, followed by lysis, Numb immunoprecipitation and immunoblotting. Lysates represent 10% of the input volume used in the immunoprecipitation.