Fig 6.

PTTG1 containing an H¹³⁴ mutation activates ESP1. (A) PTTG1^H134R promotes autocleavage of ESP1. HeLa cells were cotransfected with plasmids expressing ESP1 and FLAG-tagged PTTG1, PTTG2, or PTTG1^H134R. The FLAG-tagged constructs were turned on or off with doxycycline as indicated. Lysates were prepared, and the expression of ESP1 was detected with an antibody against the V5 tag. Uniform loading was confirmed by immunoblotting for actin. (B) Both PTTG1^H134R and PTTG1^H134R+R153G promote cleavage of ESP1. HeLa cells were cotransfected with plasmids expressing ESP1 and the indicated FLAG-tagged PTTG1 proteins. The FLAG-tagged constructs were turned on or off with doxycycline as indicated. Lysates were prepared, and the expression of ESP1 was detected with an antibody against the V5 tag. Uniform loading was confirmed by immunoblotting for actin. (C) PTTG1^H134R induces ESP1 cleavage in both G₂ phase and mitosis. HeLa cells were cotransfected with plasmids expressing ESP1 and FLAG-tagged PTTG1 or PTTG1^H134R. The FLAG-tagged constructs were turned on or off with doxycycline as indicated. The cells were incubated with RO3306 or NOC for 16 h to enrich the cells in G₂ phase and mitosis, respectively. Lysates were prepared and analyzed by immunoblotting. Mitosis was confirmed by immunoblotting for phospho-histone H3^S10. (D) PTTG1^H134R stimulates ESP1 cleavage in both S phase and mitosis. HeLa cells were cotransfected with plasmids expressing ESP1 and FLAG-tagged PTTG1 or PTTG1^H134R. The FLAG-tagged constructs were turned on or off with doxycycline as indicated. The cells were incubated with thymidine or NOC for 16 h to trap cells in S phase and mitosis, respectively. Lysates were prepared and analyzed by immunoblotting. (E) H134 is required to inhibit ESP1 cleavage. Cells were cotransfected with plasmids expressing ESP1 and FLAG-tagged PTTG1, PTTG1^H134R, or PTTG1^H134A. The PTTG1 constructs were controlled with doxycycline as indicated. Lysates were prepared, and the expression of ESP1 was detected with an antibody against the V5 tag. Uniform loading was confirmed by immunoblotting for actin. (F) PTTG1^H134R facilitates ESP1 cleavage in vitro. HeLa cells depleted of PTTG1 with siRNA were transfected with plasmids expressing V5-tagged ESP1. The cells were treated with NOC for 16 h before mitotic cells were collected by mechanical shake off. Lysates were prepared and subjected to immunoprecipitation with V5 antibodies. The ESP1 immunoprecipitates were then incubated with bacterially expressed GST-PTTG1 or GST-PTTG1^H134R at 37°C for 1 h. Autocleavage of ESP1 was detected by immunoblotting. The positions of ESP1, cleaved ESP1, and IgG from the immunoprecipitation are indicated.