STUDIES ON PHOSPHORYLATION MODIFICATION OF SPINDLIN IN GIBEL CARP OOCYTES

Spindlin, first isolated in mouse, was a substrate in the MOS/MAP kinase pathway in oocytes. In previousstudy, we isolated a homolog in gibel carp and nominated it as Carassius auratus gibelio Spindlin (CagSpin). CagSpinwas an oocyte-specific expression protein and presented dynamic distribution in nucleio in different stage of oocytesdevelopment. CagSpin associated with β-tubulin and chromosomes played important roles during oocyte maturation andoocyte-to-embyro transition. In this study, in order to explore the modification state of CagSpin in matured oocytes, wefirst analyzed the primary structure of CagSpin. The data showed that there were many possible phosphorylation sites inCagSpin. We performed dephosphorylation assay and Western blot detection to confirm the predicted posttranslationalmodification. 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 shiftclearly demonstrated that CagSpin was phosphorylated during oocyte maturation. Furthermore, Capillary Electrophoresis(CE) was utilized to detect the modified amino acid of CagSpin. The hydrolyzed amino acids were comparativelyanalyzed with the standard amino acids: L-Glu and L-Asp, and one specific peak was observed after the peaks of standardamino acids L-Glu and L-Asp. To further identify the phosphoamino acid residue, the standard phosphoamino acidThr 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 weprovided direct evidence that CagSpin was phosphorylated in gibel carp oocytes, and suggested that the phosphorylatedCagSpin might play important roles during oogenesis, oocyte maturation and oocyte-to-embryo transition.