|
POSTER NO: 235 Characterization of Human Calcyclin(S100A6)-binding protein (CacyBP)
Jing Wu, Weixia Liu, Yan Zhou, Xiaozhong Peng, Jiangang Yuan, Boqin Qiang The cDNA of human CacyBP was cloned from human liver gt11 cDNA library by one EST isolated from DDRT-PCR. It encodes a cytoplasmic protein of 228 amino acids which are 93% identical and 97% similar to mouse Calcyclin-binding protein. Software analysis suggests that this protein contains one nuclear localization sequence and many phosphorylation sites. Northern blot shows that the gene has a 2.1kb and 1.35kb isoforms in multiple adult tissues. The mRNA amount of this gene in heart, brain, and skeletal muscle is significantly higher than that of lung, liver, kidney, placenta and pancreas. The results of immunohistochemistry shows that the subcellular distribution of CacyBP changes from cytoplasm to nucleus and perinuclear cytoplasm after induction of differentiation in both glioma cell BT325 and neuroblastoma cell SH-SY5Y. Western blot showed that the protein expression level didn't change before and after differentiation. After glioma cell BT325 was synchronized by double thymidine block, we released the cells by reculture in normal medium for different periods to obtain cells in different phases of cell-cycle. Northern blot shows that the mRNA level of this gene is different in different phases of cell-cycle. As suggested by possible function associated with Calcyclin, we performed the pull-down experiment. The results show that CacyBP can form complex with Calcyclin in vitro. As Calcyclin, a Ca2+-binding protein, plays multiple roles in various cellular activities, we assume that CacyBP may take part in many physiological activities associated with Calcyclin especially in the progress of differentiation and cell-cycle regulation. |