Fig 5.

H¹³⁴ of PTTG1 is critical for securin function. (A) Chimeras between PTTG1 and PTTG2 reveal the importance of H¹³⁴ or R¹⁵³ for securin function. A schematic diagram of domain-swapping constructs between PTTG1 (gray) and PTTG2 (black) is shown to scale. A summary of the ESP1 binding is shown. Together with the data from the C-terminally deleted mutants, the region between 123 and 161 is likely to be critical for ESP1 binding. The positions and amino acids of the only 2 residues that are different between PTTG1 and PTTG2 within this region are indicated. (B) PTTG1-PTTG2 chimeras define a region critical for ESP1 binding. ESP1- and FLAG-tagged PTTG1-PTTG2 chimeras were expressed in HeLa cells as indicated (cf. panel A). Binding to ESP1 was detected by immunoprecipitation with FLAG antibodies, followed by elution and immunoblotting. (C) H¹³⁴ of PTTG1 is crucial for ESP1 binding. H¹³⁴ and R¹⁵³ of PTTG1 (gray) were mutated to the corresponding sequence in PTTG2 (black) either individually or together. ESP1- and the indicated FLAG-tagged PTTG1 constructs were expressed in HeLa cells. Binding to ESP1 was assayed by immunoprecipitation with FLAG antibodies, followed by elution and immunoblotting. The expression of PTTG1^R153G was lower in this experiment, which may explain the lower level of coimmunoprecipitated ESP1. There is no evidence from other experiments that PTTG1^R153G binds ESP1 more weakly than wild-type PTTG1.