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

作　　者：孙敏[1,2] 王小磊[1,2] 桂建芳[1] 

机构地区：[1]中国科学院水生生物研究所,淡水生态与生物技术国家重点实验室,武汉430072 [2]中国科学院研究生院,北京100049

出　　处：《水生生物学报》2011年第4期616-621,共6页Acta Hydrobiologica Sinica

基　　金：973项目(2010CB126301);国家自然科学基金(30630050)资助

摘　　要：Spindlin最早在小鼠中被发现和命名,是MOS/MAP激酶通路的底物。实验室之前在银鲫卵母细胞中克隆并分离鉴定出具有同小鼠相似序列特征和表达特性的Spindlin,命名为CagSpin(Carassius auratus gibelio Spindlin)。CagSpin是一个母源表达的蛋白,存在于卵母细胞生长、减数成熟以及早期胚胎发育过程中。研究结合去磷酸化和Western blot分析,检测到CagSpin在卵母细胞中发生磷酸化。进一步通过毛细管电泳(Capillary Electrophoresis,CE)对蛋白水解后的氨基酸残基进行分析,确定在银鲫卵母细胞中CagSpin蛋白的苏氨酸残基(threonine,Thr)被磷酸化,这一结果为CagSpin在卵母细胞中的磷酸化修饰提供了证据,表明磷酸化的CagSpin在银鲫卵子发生、卵母细胞成熟和卵—胚转换中可能起了重要作用。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 β-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 stan-dard 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 

分 类 号：Q344.13[生物学—遗传学]