WWP1 promotes LATS1 degradation by polyubiquitination and the proteasome pathway.

<p>A) Ubiquitination of LATS1 by WWP1 <i>in vivo</i>. Ubiquitin-HA and different combination of WWP1-myc, WWP1-ligase-dead mutant (WWP1-C890A-myc) and LATS1-FLAG were transfected into HEK293T cells. Ubiquitinated LATS1 was detected by immunoprecipitation of LATS1 with anti-FLAG antibody, followed by detection of ubiquitin using anti-HA antibody. B) Ubiquitination of LATS1 by WWP1 <i>in vitro</i>. Immunoprecipitated LATS1-myc or LATS1-Y376A/Y559A-myc on beads was used as a substrate in an ubiquitination assay with a ligase buffer containing E1, E2, Ubiquitin-FLAG, ATP, and WWP1-GST or WWP1-C890A-GST. After the reaction, beads containing LATS1-myc were washed extensively with modified RIPA buffer, followed by western blot analysis using anti-FLAG antibody. Cell lysate expressing FLAG-tagged LATS1 was used as positive control (lane 1). C) Inhibition of WWP1-mediated LATS1 degradation by proteasome inhibitor MG132. COS7 cells transfected with either LATS1-FLAG alone or together with WWP1-myc were treated with either DMSO (control) or proteasome inhibitor (MG132), followed by examination of LATS1 protein levels by western blot.