Molecular Elucidation Of HCdc14A In Cell Cycle Coordination

Cell cycle progression is a highly ordered and tightly regulated process. Deregulation of the cell cycle is one of the most frequent alterations during tumor development. Cdc14 is a key regulator of cell cycle. In budding yeast Saccharomyces cerevisiae, full activation of Cdc14 under the help of MEN (Mitotic Exit Network) and FEAR (Cdc Fourteen Early Release) network is requested to mitosis exit and reentry into next G1 phase. There are two homologues in human, named hCdcl4A and hCdcl4B which share 50% homogeneity. HCdcl4B seems to bundle and stabilize microtubules while hCdc14A appears to play a role in the regulation of the centrosome cycle, mitosis, and cytokinesis, thereby influencing chromosome partitioning and genomic stability in human cells. But the exact regulatory network of hCdc14A is still elusive. We propose to search for candidate interaction partners of hCdc14A using yeast two-hybrid screen system. We also plan to study further on one or more such related proteins in order to elucidate the molecular regulation of hCdc14A in cell cycle. Part IScreening of hCdc14A candidate interaction partners using yeast two- hybrid systemObjective To screen the hCdcl4A candidate interacting partners using yeast two-hybrid system. Methods The MATCHMAKER GAL4 Two-Hybrid Systems from BD Biosciences was used to identify interacting proteins for hCdc14A. Briefly, the coding region of hCdc14A was fused to the GAL4 DNA binding domain to be used as bait, and a human testis cDNA library was fused to the GAL4 transactivation domain to be screened for interaction partners. The yeast strain AH109 was transformed with the bait plasmid, then with the cDNA library using lithium acetate method. Cells were first spread on plates lacking Leu, Trp and His, then the positive colonies were streaked on selective plates lacking Leu, Trp, His and Ade, and assayed for expression ofβ-galactosidase by an x-α-gal overlay test. After that, yeast plasmid DNA was extracted from individual colony and transformed into E. coli. DNA recovered from E. coli was co-transformed into yeast AH109 with bait plasmid again for another screening assay on selective plates lacking Leu, Trp, His and Ade with x-α-gal overlay. Finally, the positive reactive plasmids were sequenced and characterized using BLAST database (http://www.ncbi.nlm.nih.gov/BLAST/.'). Results Auto-activation assay identified that bait protein couldn't activate the expression of reporter genes, indicating that bait plasmid could be used in yeast two-hybrid screening. Initial screening of 1 X 10. colonies of human testis cDNA library identified more than 20 clones that showed specific activation of His, Ade andβ-galactosidase reporter genes. Conclusions We found 23 candidate partners of hCdc14A including cell cycle related proteins, ubiquitin relating proteins, skeleton proteins and unidentified proteins, which was a foundation stone of further study on regulatory network of hCdcl4A. Part IIPhosphotase activity of hCdc14A is regulated by infra-molecular associationObjective To elucidate the biological features of full length and deletions of hCdc14A, and test the phosphatase activity of functional domain in vitro. Methods GFP-tagged different deletion expression plasmids were constructed and transformed into Hela cells. Their localizations in cell cycle were viewed under fluorescence microscopy. Flow cytometry assay tested their effects on cell cycle progress when overexpression. The physical interactions were verified by Pulldown assay and coimmunoprecipitation assay. MFP protein phosphatase assay kit was used to detect protein phosphatase activity. Results The full length, N terminal and C terminal of hCdc14A all located on centrosomes and mitotic spindle poles in mitotic phase. Overexpression of N-terminal catalytic domain of hCdcl4A prevented cells entering into mitosis with a prolonged arrest in G2/M phase, which was also validated by flow cytometry. Pulldown assay and coimmunoprecipitaion assay verified the interactions between N terminal and C terminal. MFP protein phosphatase assay indicated that C terminal inhibited the phosphatase activity of N terminal of hCdcl4A. Conclusions The C terminal of hCdcl4A could inhibit the phosphatase activity of N terminal, indicating that hCdcl4A formed an intra-molecular association which inhibited its phosphatase activity. Part IIIPlk1 phosphorylated hCdc14A and facilitated its phosphatase activityObjective To explore the functional association of Plk1 and hCdcl4A. Methods FLAG-tagged Plk1 and GFP-tagged hCdc14A expression plasmids were constructed and cotransformed into Hela cells to detect their cellular localizations. Pulldown assay and coimmunoprecipitation assay were used to test the physical interactions. The effects of Plk1, wild type, nonphospho-mimicking or phospho-mimicking mutants of hCdc14A on cell cycle were investigated under fluorescence microscopy when overexpression. Cell cycle progression was viewed when transformed with different mutants using real time fluorescence microscopy. In vitro phosphorylation assay was utilized to detect whether Plk1 could phosphorylate hCdc14A. MFP protein phosphatase assay kit was used to detect protein phosphatase activity. Results Plk1 and hCdc14A co-localized on mitotic spindle poles in cell cycle. Their physical interaction was verified by Pulldown assay and coimmunoprecipitation assay. Overexpression of phosphor-mimicking mutant of hCdcl4A made lagging chromosome, as the same with cotransformed with Plk1 and wild type hCdcl4A. The real time fluorescence microscopy movie showed that phosphor-mimicking mutant of hCdc14A interphase aberrantly longer. Plk1 could phosphorylate hCdc14A in vitro. The phosphatase activity of the phosphor-mimicking mutant was more significant than wild type. Conclusions Plk1 could phosphorylate hCdc14A and facilitated its phosphatase activity. The phosphorylation modification of hCdc14Aby Plk1 might release its intra-molecular inhibition of phosphatase activity.