Characterization of the antiapoptotic Bcl-2 family member myeloid cell leukemia-1 (Mcl-1) and the stimulation of its message by gonadotropins in the rat ovary.
The majority of ovarian follicles undergo atresia mediated by apoptosis. Bcl-2-related proteins act as regulators of apoptosis via the formation of dimers with proteins inside and outside the Bcl-2 family. Previous studies have identified BAD as a proapoptotic Bcl-2 family member expressed in the ovary. It is known that BAD phosphorylation induced by survival factors leads to its preferential binding to 14-3-3 and suppression of the death-inducing function of BAD. To identify ovarian binding partners for hypophosphorylated BAD, we performed a yeast two-hybrid screening of a rat ovary complementary DNA library using as bait a mutant BAD incapable of binding to 14-3-3. Screening of yeast transformants yielded positive clones encoding the rat ortholog of Mcl-1 (myeloid cell leukemia-1), an antiapoptotic Bcl-2 protein. Amino acid sequence analysis revealed that rat and human Mcl-1 showed a complete conservation of the Bcl-2 homology domains BH1, BH2, and BH3. In the yeast two-hybrid system, Mcl-1 binds to the hypophosphorylated mutant of BAD and interacts preferentially with different proapoptotic (Bax, Bak, Bok, Bik, and BOD) compared with antiapoptotic Bcl-2 family members (Bcl-2, Bcl-xL, Bcl-w, Bfl-1, CED-9, and BHRF-1). Northern blot hybridization demonstrated expression of Mcl-1 transcripts of 2.3 and 3.7 kb in the ovary and diverse other rat tissues. In immature rats, PMSG treatment led to a transient increase in the 2.3-kb Mcl-1 transcript, peaking at 6 h after injection and returning to baseline levels after 24 h. Moreover, the same transcript was induced in the PMSG-primed preovulatory rat ovary 6 h after the administration of ovulatory doses of either hCG or FSH. In situ hybridization studies revealed that the gonadotropin stimulation of ovarian Mcl-1 message occurs in both granulosa and thecal cells. In conclusion, rat Mcl-1 was identified as an ovarian BAD-interacting protein and the message for the antiapoptotic Mcl-1 protein was induced after treatment with gonadotropins in granulosa and thecal cells of growing follicles.
Leo
C P
CP
Department of Gynecology and Obstetrics, Stanford University Medical Center, California 94305-5317, USA.
Hsu
S Y
SY
Chun
S Y
SY
Bae
H W
HW
Hsueh
A J
AJ
D000595:Amino Acid Sequence; D000818:Animals; D017209:Apoptosis; D015152:Blotting, Northern; D002645:Chickens; D006063:Chorionic Gonadotropin / Q000494:pharmacology; D019281:Dimerization; D005260:Female; D005640:Follicle Stimulating Hormone / Q000494:pharmacology; D015870:Gene Expression / Q000187:drug effects*; D006062:Gonadotropins / Q000494:pharmacology*; D006107:Granulosa Cells / Q000737:chemistry; D006801:Humans; D017403:In Situ Hybridization; D008969:Molecular Sequence Data; D064549:Myeloid Cell Leukemia Sequence 1 Protein; D009363:Neoplasm Proteins / Q000737:chemistry / Q000235:genetics* / Q000502:physiology*; D010053:Ovary / Q000378:metabolism*; D010060:Ovulation; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000502:physiology*; D012333:RNA, Messenger / Q000032:analysis / Q000096:biosynthesis; D051381:Rats; D017207:Rats, Sprague-Dawley; D017385:Sequence Homology
D006063:Chorionic Gonadotropin; D006062:Gonadotropins; D064549:Myeloid Cell Leukemia Sequence 1 Protein; D009363:Neoplasm Proteins; D019253:Proto-Oncogene Proteins c-bcl-2; D012333:RNA, Messenger; D005640:Follicle Stimulating Hormone