TY  - GEN
AV  - public
Y1  - 2007///
TI  - Functional characterization of hCdc14B phosphatase and its role during mitosis
UR  - https://archiv.ub.uni-heidelberg.de/volltextserver/7779/
A1  - Tumurbaatar, Indra
ID  - heidok7779
KW  - phosphatase 
KW  -  Cdc14 
KW  -  mitosis
N2  - Progression through the cell cycle requires coordinated activity of kinases and phosphatases. Cdk1/cyclin B is a major kinase that promotes entry into and progression through early mitosis. Exit from mitosis requires down-regulation of Cdk1/cyclin B as well as reversal of Cdk1/cyclin B-mediated phosphorylations. In budding yeast, the nucleolar phosphatase Cdc14 (yCdc14) triggers mitotic exit by dephosphorylating mitotic Cdk/cyclin target proteins, promoting degradation of mitotic regulators, and inactivating mitotic Cdk/cyclin complexes. In contrast to yeast, hCdc14B, a human homologue of yCdc14, is poorly characterized. The aim of this study was to investigate a potential regulatory function of hCdc14B in mitosis. To address this issue, a variety of experimental approaches were used, including the generation of transgenic human cell lines, gene silencing by RNAi, protein-interaction assays, determination of phosphatase and kinase activities, purification and size fractionation of cellular protein complexes as well as chromatin immunoprecipitation assay (ChIP) and fluorescence microscopy.  HeLa and U2OS cell lines were established that express Flag-, Flag-HA-, or TAP-tagged wildtype hCdc14B or a catalytically defective mutant, called hCdc14BPD. Analysis of U2OS cell lines that stably express either wildtype hCdc14B or the phosphatase-deficient mutant under the control of a tetracycline-inducible promoter showed that ectopic hCdc14B caused a delay in mitotic entry, while overexpression of the enzymatic inactive form prolonged mitosis and delayed exit from mitosis. These data were supported by siRNA-directed silencing of hCdc14B expression, demonstrating that ablation of hCdc14B leads to accumulation of bi- and multinucleated cells, delay in meta- to anaphase transition and finally to mitotic arrest and cell death.  To dissect the molecular mechanism underlying hCdc14B function, the phosphorylation state and activity of selected key regulators of mitosis were analyzed. These studies revealed that hCdc14B dephosphorylates the mitotic phosphatase Cdc25, which is required for activation of Cdk1/cyclin B at the G2/M transition and during early stages of mitosis. Overexpression of hCdc14B suppressed Cdc25 activity, leading to accumulation of Cdk1 phosphorylated at T14/Y15 and a substantial delay in Cdk1/cyclin B activation. In contrast, Cdk1/cyclin B activity remained elevated in cells depleted of hCdc14B. The results suggest that hCdc14B interrupts the positive feedback loop between Cdc25 and Cdk1/cyclin B and promotes progression through late mitosis.  ChIP revealed a preferred binding of hCdc14B to the intergenic spacer region of ribosomal RNA genes (rDNA). Binding to rDNA was abrogated during mitosis, indicating that hCdc14B associates with rDNA in a cell cycle-dependent fashion. In support of this, analysis of the subcellular distribution of hCdc14B throughout the cell cycle revealed that hCdc14B is bound to chromatin during interphase and released from chromatin from prometaphase until early G1-phase.  Size fractionation by gel filtration revealed that hCdc14B is contained in one major protein complex with a molecular weight of 400-600 kDa. During mitosis the majority of hCdc14B was monomeric with a small portion being contained in different protein complexes suggesting that during the cell cycle hCdc14B associates with different interaction partners.  To identify proteins that interact with and are dephosphorylated by hCdc14B, hCdc14B complexes were isolated from human cells that overexpress Flag-HA-tagged hCdc14B by sequential immunopurification, and co-purifying proteins were subjected to mass spectrometry. This analysis identified APC1, the largest subunit of the anaphase-promoting complex/cyclosome (APC/C), and ELP1, an RNA polymerase II specific elongation factor. The interaction between hCdc14B and APC/C was confirmed by co-immunoprecipitation experiments revealing that hCdc14B interacts with a distinct subform of the APC/C complex, APC/CCdh1 that forms during late mitosis and regulates proteosome-dependent degradation of mitotic regulators at the exit from mitosis and during G1-phase. Studies carried out in the present work provide first evidence that hCdc14B is an essential regulator of late mitotic events and reveal the molecular mechanisms underlying hCdc14B function. 
ER  -