PTEN is a negative regulator of mitotic checkpoint complex during the cell cycle

Abstract

Nuclear PTEN plays an important role during mitosis. To understand the molecular basis by which PTEN mediates mitotic progression, we examined whether PTEN regulated the formation of mitotic checkpoint complex (MCC). We observed that arsenic trioxide, a mitotic inducer, stimulated nuclear translocation of PTEN in a time-dependent manner. PTEN physically interacted with Cdc20 and Mad2, two important components of MCC. Arsenic treatment diminished the physical association of PTEN with BubR1 and Bub3 but not with Cdc20 and Mad2. Our further studies revealed that downregulation of PTEN via RNAi enhanced formation of MCC during the cell cycle. Moreover, PTEN silencing induced chromosomal instability. Given the crucial role of PTEN in suppressing tumor development, our study strongly suggests that PTEN also functions to maintain chromosomal stability, partly through suppressing unscheduled formation of MCC.

Keywords: Chromosomes; Mitosis; Mitotic checkpoint complex; Nuclear localization; PTEN.

Fig. 1

Nuclear translocation of PTEN after arsenic treatment. HeLa cells were treated with arsenic trioxide [As(III)] for various concentrations as indicated, after which cells were collected for obtaining cytoplasmic and nuclear fractions. Equal amounts of cytoplasmic and nuclear proteins were blotted for PTEN, tubulin, PARP, and phospho-histone H3

Fig. 2

PTEN negatively regulates formation of mitotic checkpoint complex. a HEK293 cells were transfected with Flag-Cdc20 expression plasmid or empty vector (ev) for 24 h and then treated with arsenic (1 μM) for 18 h. Equal amounts cell lysates were immunoprecipitated with the anti-Flag antibody. Flag immunoprecipitates, along with lysate inputs, were blotted for Flag, Mad2, PTEN, BubR1 and Bub3. b HEK293 cells were co-transfected with Flag-Cdc20 and/or PTEN siRNAs, or control siRNAs (siRNAs to luciferase), for 24 h and treated with arsenic for 18 h. Equal amounts of cell lysates were then immunoprecipitated with the anti-Flag antibody. Flag immunoprecipitates, along with lysate inputs, were blotted for Flag, Mad2, PTEN, BubR1 and Bub3

Fig. 3

PTEN suppresses MCC formation during the cell cycle. HeLa cells co-transfected with siRNAs to PTEN (siPTEN), or to luciferase (siLuc) as control, and Flag-Mad2 expression plasmid were synchronized at G1/S junction via double-thymidine treatment. At various times after releasing into the cell cycle, cells were collected and lysed. Equal amounts of cell lysates were immunoprecipitated with the anti-Flag antibody. Flag immunoprecipitates, along with lysate inputs, were blotted for Flag, PTEN, Cdc20, BubR1 and Bub3. TT release double thymidine release

Fig. 4

PTEN silencing induces chromosomal instability. a HeLa cells seeded in chamber slides were transfected with siRNAs to PTEN or luciferase for 48 h, after which cells were fixed and stained with antibodies to tubulin and CREST. DAPI was also used to label DNA. Stained cells were examined under a fluorescent microscope and representative images are shown. b, c Percentages of normal or abnormal mitotic cells as shown in a were summarized and presented