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Regulation of Ankyrin‐repeat and SOCS‐box protein 9 (ASB9) in ovarian follicles and identification of binding partners
Author(s) -
Benoit Gabriel,
Lussier Jacques G,
Ndiaye Kalidou
Publication year - 2017
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.31.1_supplement.601.12
Subject(s) - ankyrin repeat , ankyrin , follicular phase , messenger rna , biology , western blot , theca , estrous cycle , andrology , microbiology and biotechnology , medicine , chemistry , endocrinology , gene , genetics
Ankyrin‐repeat and SOCS‐box protein 9 (ASB9) is a member of the large SOCS‐box containing proteins family and acts as the specific substrate recognition component of E3 ubiquitin ligases in the process of ubiquitination and proteasomal degradation. ASB9 is known to interact with creatine kinase B and negatively regulates cell growth. We previously identified ASB9 as a differentially expressed gene in granulosa cells (GC) of bovine ovulatory follicles. This study aimed to further investigate ASB9 mRNA and protein regulation and to identify its binding partners in GC of bovine ovulatory follicles. GC were obtained from small follicles (SF: 2–4 mm), dominant follicles at day 5 of the estrous cycle (DF), and ovulatory follicles, 24 hours following hCG injection (OF). RT‐PCR analyses showed a 104‐fold induction of ASB9 expression in GC of OF as compared to DF (P < 0.0001). Steady‐state mRNA levels of ASB9 in follicular walls (granulosa and theca cells) analyzed at 0, 6, 12, 18 and 24 h after hCG injection showed a significant induction of ASB9 expression at 12 and 18 h (P<0.001), reaching a maximum induction at 24 h post‐hCG (P<0.0001) as compared to 0 h. These results were confirmed in western blot analysis showing highest ASB9 protein amounts in OF. Yeast two‐hybrid screening of OF‐cDNAs library resulted in the identification of five potential ASB9 binding partners in GC, confirmed by in vitro co‐immunoprecipitation analyses. Overall, these results support a physiologically relevant role of ASB9 in the ovulation process, and provide, for the first time, insights into the regulation and mode of action of ASB9 in GC. Support or Funding Information This work was supported in part by internal funds from Université de Montréal to KN and by a Discovery Grant from Natural Sciences and Engineering Research Council of Canada (NSERC) to JGL.