CacyBP/SIP nuclear translocation regulates p27Kip1 stability in gastric cancer cells.
OBJECTIVE
To investigate the mechanism of calcyclin binding protein/Siah-1 interacting protein (CacyBP/SIP) nuclear translocation in promoting the proliferation of gastric cancer (GC) cells.


METHODS
The effect of CacyBP/SIP nuclear translocation on cell cycle was investigated by cell cycle analysis. Western blot analysis was used to assess the change in expression of cell cycle regulatory proteins and proteasome-mediated degradation of p27Kip1. Co-immunoprecipitation (co-IP) analysis was performed to examine the binding of CacyBP/SIP with Skp1. A CacyBP/SIP truncation mutant which lacked the Skp1 binding site was constructed and fused to a fluorescent protein. Subsequently, the effect on Skp1 binding with the fusion protein was examined by co-IP, while localization of fluorescent fusion protein observed by confocal laser microscopy, and change in p27Kip1 protein expression assessed by Western blot analysis.


RESULTS
CacyBP/SIP nuclear translocation induced by gastrin promoted progression of GC cells from G1 phase. However, while CacyBP/SIP nuclear translocation was inhibited using siRNA to suppress CacyBP/SIP expression, cell cycle was clearly inhibited. CacyBP/SIP nuclear translocation significantly decreased the level of cell cycle inhibitor p27Kip1, increased Cyclin E protein expression whereas the levels of Skp1, Skp2, and CDK2 were not affected. Upon inhibition of CacyBP/SIP nuclear translocation, there were no changes in protein levels of p27Kip1 and Cyclin E, while p27Kip1 decrease could be prevented by the proteasome inhibitor MG132. Moreover, CacyBP/SIP was found to bind to Skp1 by immunoprecipitation, an event that was abolished by mutant CacyBP/SIP, which also failed to stimulate p27Kip1 degradation, even though the mutant could still translocate into the nucleus.


CONCLUSIONS
CacyBP/SIP nuclear translocation contributes to the proliferation of GC cells, and CacyBP/SIP exerts this effect, at least in part, by stimulating ubiquitin-mediated degradation of p27Kip1.
Ying-Lin Niu, Department of Gastroenterology, Beijing Friendship Hospital Affiliated to Capital Medical University, Beijing 100050, China.
Ying-Lin
YL
Niu
Ying-Lin Niu, Department of Gastroenterology, Beijing Friendship Hospital Affiliated to Capital Medical University, Beijing 100050, China.
Ya-Jun
YJ
Li
Ying-Lin Niu, Department of Gastroenterology, Beijing Friendship Hospital Affiliated to Capital Medical University, Beijing 100050, China.
Jing-Bo
JB
Wang
Ying-Lin Niu, Department of Gastroenterology, Beijing Friendship Hospital Affiliated to Capital Medical University, Beijing 100050, China.
Yuan-Yuan
YY
Lu
Ying-Lin Niu, Department of Gastroenterology, Beijing Friendship Hospital Affiliated to Capital Medical University, Beijing 100050, China.
Zhen-Xiong
ZX
Liu
Ying-Lin Niu, Department of Gastroenterology, Beijing Friendship Hospital Affiliated to Capital Medical University, Beijing 100050, China.
Shan-Shan
SS
Feng
Ying-Lin Niu, Department of Gastroenterology, Beijing Friendship Hospital Affiliated to Capital Medical University, Beijing 100050, China.
Jian-Guo
JG
Hu
Ying-Lin Niu, Department of Gastroenterology, Beijing Friendship Hospital Affiliated to Capital Medical University, Beijing 100050, China.
Hui-Hong
HH
Zhai
D021581:Active Transport, Cell Nucleus; D000230:Adenocarcinoma / Q000235:genetics / Q000378:metabolism* / Q000473:pathology; D002135:Calcium-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D045744:Cell Line, Tumor; D049109:Cell Proliferation; D050760:Cyclin-Dependent Kinase Inhibitor p27 / Q000235:genetics / Q000378:metabolism*; D059585:G1 Phase Cell Cycle Checkpoints; D006801:Humans; D046988:Proteasome Endopeptidase Complex / Q000378:metabolism; D011485:Protein Binding; D055550:Protein Stability; D059748:Proteolysis; D034622:RNA Interference; D044843:SKP Cullin F-Box Protein Ligases / Q000378:metabolism; D015398:Signal Transduction; D013274:Stomach Neoplasms / Q000235:genetics / Q000378:metabolism* / Q000473:pathology; D014162:Transfection; D054875:Ubiquitination
C491778:CACYBP protein, human; C436291:CDKN1B protein, human; D002135:Calcium-Binding Proteins; D050760:Cyclin-Dependent Kinase Inhibitor p27; D044843:SKP Cullin F-Box Protein Ligases; D046988:Proteasome Endopeptidase Complex
Calcyclin binding protein/Siah-1 interacting protein; Cell cycle; Gastric cancer; P27kip1; Ubiquitin