水生生物学报2011，Vol.35Issue(4)：616-621,6.DOI:10.3724/SP.J.1035.2011.00616
Spindlin在银鲫卵母细胞中磷酸化修饰分析
STUDIES ON PHOSPHORYLATION MODIFICATION OF SPINDLIN IN GIBEL CARP OOCYTES
摘要
Abstract
Spindlin, first isolated in mouse, was a substrate in the MOS/MAP kinase pathway in oocytes. In previous study, we isolated a homolog in gibel carp and nominated it as Carassius auratus gibelio Spindlin (CagSpin). CagSpin was an oocyte-specific expression protein and presented dynamic distribution in nucleio in different stage of oocytes development. CagSpin associated with p-tubulin and chromosomes played important roles during oocyte maturation and oocyte-to-embyro transition. In this study, in order to explore the modification state of CagSpin in matured oocytes, we first analyzed the primary structure of CagSpin. The data showed that there were many possible phosphorylation sites in CagSpin. We performed dephosphorylation assay and Western blot detection to confirm the predicted posttranslational modification. Compared with the specific 28 kD protein band in normal matured egg extracts, a smaller protein band, about 27 kD, was detected in CIP (calf intestine alkaline phosphatase) treated matured egg extracts. The mobility shift clearly demonstrated that CagSpin was phosphorylated during oocyte maturation. Furthermore, Capillary Electrophore-sis (CE) was utilized to detect the modified amino acid of CagSpin. The hydrolyzed amino acids were comparatively analyzed with the standard amino acids: L-Glu and L-Asp, and one specific peak was observed after the peaks of standard amino acids L-Glu and L-Asp. To further identify the phosphoamino acid residue, the standard phosphoamino acid Thr was added into the hydrolyzed products. The same extra peaks implicates CagSpin is phosphorylated on Thr residue. The data suggest that CagSpin might be involved in the MOS/MAP kinase pathway during oocytes maturation. Here we provided direct evidence that CagSpin was phosphorylated in gibel carp oocytes, and suggested that the phosphorylated CagSpin might play important roles during oogenesis, oocyte maturation and oocyte-to-embryo transition.关键词
Spindlin；磷酸化；去磷酸化；卵子发生；卵母细胞成熟Key words
Spindlin/ Phosphorylation/ Dephosphorylation/ Oogenesis/ Oocyte maturation分类
生物科学引用本文复制引用
孙敏,王小磊,桂建芳..Spindlin在银鲫卵母细胞中磷酸化修饰分析[J].水生生物学报,2011,35(4):616-621,6.基金项目
973项目(2010CB126301)；国家自然科学基金(30630050)资助 （2010CB126301）
