Murine pituitary tumor-transforming gene functions as a securin protein in insulin-secreting cells.
Human pituitary tumor-transforming gene 1 (PTTG1) encodes a securin protein critically important in regulating chromosome separation. Murine PTTG (mPTTG) is 66% homologous to human PTTG1 and PTTG-null (PTTG-/-) mice exhibit pancreatic beta-cell hypoplasia and abnormal nuclear morphology with resultant diabetes. As we show that ductal beta-cell neogenesis is intact in PTTG-/- mice, we explored mechanism for defective beta-cell replication. We tested whether mPTTG exhibits securin properties in mouse insulin-secreting insulinoma MIN6 cells, using a live-cell system to monitor mitosis in cells transfected with an enhanced green fluorescent protein (EGFP)-tagged mPTTG conjugate (mPTTG-EGFP). To fulfill the criteria for securin properties, the protein should undergo degradation immediately before the metaphase-to-anaphase transition when expression levels are low, and should inhibit metaphase-to-anaphase transition when expression levels are high. EGFP itself did not undergo degradation throughout mitosis and high levels of EGFP per se did not affect normal mitosis progression (n=25). However, mPTTG-EGFP was degraded 2 min before the metaphase-to-anaphase transition when expression levels were low (n=19), and high mPTTG-EGFP levels blocked metaphase-to-anaphase transition in 13 cells. mPTTG-EGFP inhibited MIN6 cell proliferation and caused apoptosis. Immunocoprecipitation demonstrated binding of mPTTG-EGFP and separase. These results show that mPTTG exhibits properties consistent with a murine securin in insulin-secreting mouse cells and mPTTG overexpression inhibits cell proliferation, suggesting that defective beta-cell proliferation observed in PTTG-/- mice is likely due to abnormal cell-cycle progression.
Yu
Run
R
UCLA School of Medicine, Cedars-Sinai Research Institute, Cedars-Sinai Medical Center, 8700 Beverly Blvd, Room D3066, Los Angeles, California 90048, USA. run.yu@cshs.org
Cruz-Soto
Martha
M
Li Calzi
Sergio
S
Hui
Hongxiang
H
Melmed
Shlomo
S
D000705:Anaphase; D000818:Animals; D017209:Apoptosis; D015153:Blotting, Western / Q000379:methods; D018797:Cell Cycle Proteins / Q000378:metabolism; D002460:Cell Line; D010450:Endopeptidases / Q000378:metabolism; D005455:Fluorescent Antibody Technique; D015870:Gene Expression; D049452:Green Fluorescent Proteins / Q000235:genetics; D006801:Humans; D047468:Immunoprecipitation; D050417:Insulin-Secreting Cells / Q000166:cytology / Q000378:metabolism*; D007340:Insulinoma; D008677:Metaphase; D051379:Mice; D018345:Mice, Knockout; D009363:Neoplasm Proteins / Q000235:genetics* / Q000378:metabolism; D064249:Securin; D064247:Separase; D014162:Transfection / Q000379:methods
D018797:Cell Cycle Proteins; D009363:Neoplasm Proteins; D064249:Securin; C485184:enhanced green fluorescent protein; C578002:pituitary tumor-transforming protein 1, human; D049452:Green Fluorescent Proteins; D010450:Endopeptidases; C577987:ESPL1 protein, human; D064247:Separase