Study. Ankrd17 Function In Cell Cycle Regulation

cell cycle progression is principally governed by cyclin-dependent kinase that binds with cell cycle-specific regulatory subunits known as cyclins. A specific phase of the cell cycle is under the control of a specific class of cyclin/CDK complex. Previous studies have found that some proteins interacting with Cyclin/CDK2 complex play a critical role in cell cycle progression. Whereas, the mechanisms that Cyclin/CDK2 complex regulates cell cycle progression are not fully understood partly because few substrates of this complex have be identified.My lab had isolated a protein Ankrd17 that interacts with CDK2 by Tandem affinity purification (TAP). Some results have suggested that Ankrd17 is a substrate of CyclinE/CDK2 in human cells. This study was therefore designed to observe the effects of the protein Ankrd17 on cell cycle progression. First, to examine the expression of Ankrd17 mRNA in seven kinds of human different tissues and the mRNA lever of Ankrd17 throughout the cycle in tumour cell HCT116 and normal cell 2BS by RT-PCR. The results suggest Ankrd17 is constitutive expression in different kinds of human tissues and appears to be expressed in all phase of the cell cycle in 2BS and HCT116 cell. The second, to construct eukaryotic expressing vector of human Ankrd17 and then test its expression in human colorectal cancer cell line HCT116. To analyze the function of Ankrd17 when it interacts with CyclinE/CDK2 complex on cell cycle progression in HCT116 by selection of EGFP positive cells which were cotransfected with pBB14 and pDEST -flag-Ankrd17 vectors. The results suggest overexpression Ankrd17 increase the S-phase population, the protein may play a role at the G1/S boundry. At the same time, to constructe Prokaryotic expression vector of CyclinA1, expressed and purified the fusion protein GST-CyclinA1 for investigating further whether Ankrd17 is associated with CyclinA/CDK2 in vivo or whether it is also a substrate of this kinase complex. The third, to find effective recombinant retrovirus vectors expressing siRNA for Ankrd17 gene knockdown and then to establish Ankrd17-deficient stable cell line for analyzing the effect of Ankrd17 gene in cell cycle progression.