FIGURE 5.

Amot130 induces AIP4-dependent ubiquitination of YAP. A, the levels of Myc-tagged AIP4, FLAG-tagged LATS1, and YFP-tagged Amot130 were detected by immunoblot (IB) from lysates and immunoprecipitations (anti-Myc) prepared from HEK 293T cells expressing these proteins in the indicated combinations. B, the levels of endogenous LATS1 were measured by immunoblot in MDA-MB-468 cells expressing mCherry-tagged AIP4 and YFP-tagged Amot130 alone or in combination. The ratio of pixel intensities of LATS1 over GAPDH is presented. C, the levels of Myc-tagged AIP4, YFP-tagged Amot130, and FLAG-tagged YAP2 were detected by immunoblot from lysates and immunoprecipitations (anti-FLAG) prepared from HEK 293T cells expressing these three proteins in the indicated combinations. D, the levels of FLAG-tagged YAP2 and Myc-tagged Amot130 wild-type, P-Y1F (Y109F), or P-Y2F (Y242F) mutants or control vector were detected by immunoblot from lysates and immunoprecipitations (anti-Myc) prepared from HEK 293T cells expressing these proteins in the indicated combinations. E, the levels of ubiquitinated FLAG-tagged YAP2 were detected by immunoblot following immunoprecipitation (anti-HA) from HEK 293T cells expressing HA-tagged Lys-0 ubiquitin (HA-K0 Ub) in combination with YFP-tagged Amot130 or control vector as well as Myc-tagged AIP4 and FLAG-tagged YAP2. F, the relative incorporation of HA-Lys-0 ubiquitin into immunoprecipitated FLAG-tagged YAP2 in HEK 293T cells co-expressing YFP-tagged Amot130, Myc-tagged AIP4, mutant AIP4 (C830A), or control vectors was detected by immunoblot. G, the levels of total and ubiquitinated FLAG-tagged YAP2 were detected by immunoblot following immunoprecipitation from HEK 293T cells expressing HA-tagged wild-type or Lys-0 ubiquitin in combination with YFP-tagged Amot130 or control vector as well as Myc-tagged AIP4 and FLAG-tagged YAP2. H, model of the binding and ubiquitination relationships of the Amot130·AIP4·YAP2 complex. A question mark denotes unknown sites of ubiquitination or binding.