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Human Phosphatase CDC14A Is Recruited to the Cell Leading Edge to Regulate Cell Migration and Adhesion

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Human Phosphatase CDC14A Is Recruited to the Cell Leading Edge to Regulate Cell Migration and Adhesion Human phosphatase CDC14A is recruited to the cell leading edge to regulate cell migration and adhesion Nan-Peng Chena,b, Borhan Uddina,b, Renate Voitc, and Elmar Schiebela,1 aZentrum für Molekulare Biologie der Universität Heidelberg (ZMBH), Deutsches Krebsforschungszentrum (DKFZ)-Zentrum für Molekulare Biologie (ZMBH) Allianz, 69120 Heidelberg, Germany; bHartmut Hoffmann-Berling International Graduate School of Molecular and Cellular Biology, Universität Heidelberg, 69120 Heidelberg, Germany; and cMolecular Biology of the Cell II, Deutsches Krebsforschungszentrum (DKFZ), 69120 Heidelberg, Germany Edited by James E. Cleaver, University of California, San Francisco, CA, and approved December 17, 2015 (received for review August 6, 2015) Cell adhesion and migration are highly dynamic biological pro- With mitotic exit, the proteins that were phosphorylated by cesses that play important roles in organ development and CDK1 are dephosphorylated, so that cells can return to the cancer metastasis. Their tight regulation by small GTPases and nonmitotic, interphase status (13). In Saccharomyces cerevisiae, protein phosphorylation make interrogation of these key pro- cell-division cycle 14 (Cdc14) is the major phosphatase that cesses of great importance. We now show that the conserved dual- counteracts Cdk1 activity after the metaphase–anaphase transi- specificity phosphatase human cell-division cycle 14A (hCDC14A) tion (14). The function of Cdc14 in budding yeast has been well associates with the actin cytoskeleton of human cells. To un- studied and two regulatory networks have been identified that derstand hCDC14A function at this location, we manipulated native promote the release of Cdc14 from the RENT complex in the PD loci to ablate hCDC14A phosphatase activity (hCDC14A )inun- nucleolus: “Cdc Fourteen Early Anaphase Release” (FEAR) and transformed hTERT-RPE1 and colorectal cancer (HCT116) cell lines “Mitotic Exit Network” (MEN) (15–17). and expressed the phosphatase in HeLa FRT T-Rex cells. Ectopic Despite the conservation of the N-terminal catalytic phos- expression of hCDC14A induced stress fiber formation, whereas PD PD phatase domain and the ability of human CDC14B to comple- stress fibers were diminished in hCDC14A cells. hCDC14A cells ment the essential functions of budding yeast CDC14 (18), displayed faster cell migration and less adhesion than wild-type Cdc14 phosphatases play divergent roles in different organisms. controls. hCDC14A colocalized with the hCDC14A substrate kidney- Schizosaccharomyces pombe Cdc14/Flp1 primarily participates and brain-expressed protein (KIBRA) at the cell leading edge and CELL BIOLOGY in the regulation of the phosphatase Cdc25 and cytokinesis overexpression of KIBRA wasabletoreversethephenotypesof hCDC14APD cells. Finally, we show that ablation of hCDC14A ac- (19). Vertebrate CDC14s have been linked to diverse functions tivity increased the aggressive nature of cells in an in vitro tumor ranging from centrosome maturation and separation, DNA formation assay. Consistently, hCDC14A is down-regulated in many damage checkpoint control, DNA repair, and cytokinesis control – tumor tissues and reduced hCDC14A expression is correlated with (20 24). These studies have unraveled novel functions of mam- poorer survival of patients with cancer, to suggest that hCDC14A malian CDC14 phosphatases; however, they reveal striking may directly contribute to the metastatic potential of tumors. inadequacies in our understanding of this important phos- Thus, we have uncovered an unanticipated role for hCDC14A in phatase family. cell migration and adhesion that is clearly distinct from the mi- Here, we have addressed the function of hCDC14A (human totic and cytokinesis functions of Cdc14/Flp1 in budding and cell-division cycle 14A) using human genetically engineered fission yeast. hCDC14A phosphatase dead cell lines (PD). Mobility and spread- ing were both enhanced by ablation of hCDC14A, whereas cell–cell CDC14 | cell migration | cell adhesion | phosphatase adhesion was reduced. Moreover, ectopic hCDC14A expression ell migration and adhesion play key roles in embryonic de- Significance Cvelopment, tissue remodeling and cancer metastasis (1). Many oncoproteins, such as Yes-associated protein 1 (YAP), Cell migration and adhesion play critical roles in animal de- STAT3, and K-RAS, regulate cancer metastasis by enhancing velopment and tumor metastasis and are regulated by protein cell migration and invasion (2–4). The dynamic behavior of the phosphorylation. Here we identified the human phosphatase actin cytoskeleton drives migration and invasion and is regulated hCDC14A (human cell-division cycle 14A) as F-actin binding by a combined impact of Rho GTPases, membrane phospho- protein at the leading edge that regulates both processes. Loss lipids, and protein phosphorylation (5). The switch of phos- of hCDC14A activity promotes cell mobility and reduces cell phorylation at the cell leading edge is crucial for rapid turnover adhesion. Importantly, cells devoid of hCDC14A activity were of actin filaments. For example, focal adhesion kinase (FAK) can more invasive in a colony-forming assay. Database analysis be activated by integrins and various growth factors. Once acti- indicates that hCDC14A expression is frequently down-regu- vated, FAK regulates actin polymerization, membrane pro- lated in cancer tissues and is associated with poor patient trusion and cell migration by promoting the phosphorylation of prognosis. Thus, the promigratory impact of hCDC14A inactiva- the actin cytoskeleton remodelers p130cas, GRB2/7, and WASP tion upon the F-actin cytoskeleton supports tumor proliferation (5, 6). The tyrosine phosphatase SHP2 increases cell mobility and metastasis. This hCDC14A function is clearly distinct from through activation of the SRC kinase family to promote tumor the mitotic functions of the budding and fission yeast orthologs metastasis (7). Conversely, the lipid phosphatase PTEN inhibits Cdc14/Flp1. tumor invasion by suppressing the activation of RAC GTPases Author contributions: N.-P.C. and E.S. designed research; N.-P.C. and B.U. performed re- (8). There is also extensive evidence for control of cancer cell search; R.V. contributed new reagents/analytic tools; N.-P.C. and B.U. analyzed data; and migration and invasion through the phosphatase PP2A upon N.-P.C. and E.S. wrote the paper. Wnt/beta-catenin signaling, metal matrix proteases, and ERK The authors declare no conflict of interest. kinase (9–11). This article is a PNAS Direct Submission. At the G2/M transition, cyclin-dependent kinase 1 (CDK1) is 1To whom correspondence should be addressed. Email: [email protected]. activated to trigger mitotic entry and its kinase activity remains This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. high until metaphase to maintain the cell in a mitotic state (12). 1073/pnas.1515605113/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1515605113 PNAS Early Edition | 1of6 Downloaded by guest on September 26, 2021 inhibited migration and the actin cytoskeleton was remodeled when hCDC14A activity was impaired. Consistent with these actin-modulating functions, a pool of hCDC14A associated with F-actin filaments at the leading edge where it colocalized with the Hippo pathway component kidney- and brain-expressed pro- tein (KIBRA). KIBRA overproduction rescued the migration and adhesion defects in the hCDC14APD cells. Our study therefore reveals a previously unidentified function of hCDC14A. As hCDC14A expression is down-regulated in a variety of cancers, including colorectal, and this down-regulation is associated with poor prognosis, our results suggest that hCDC14A regu- lates tumor metastasis and is therefore of considerable clinical relevance. Results A Pool of hCDC14A Localizes to the Cell Leading Edge and F-Actin Fibers. Whether hCDC14 phosphatases impact upon actin-related functions is yet to be addressed. To this end, we monitored the distribution of a hCDC14A-YFP fusion protein stably integrated in the HeLa cell genome as a single copy via the Flp-in T-Rex system. The levels of ectopic hCDC14A expression achieved by induction were much lower than those arising from transient transfection (Fig. S1A) and did not generate obvious toxicity or ad- verse impact upon cell cycle progression based on the propidium iodide DNA staining (Fig. S1 B–D). Phosphohistone 3 serine 10 staining revealed that the ectopic expression of hCDC14A only slightly increased the mitotic index (Fig. S1 E and F). This was further confirmed by live cell imaging, which revealed only a mod- Fig. 1. hCDC14A colocalizes with F-actin at the cell leading edge. est increase in the duration of mitosis upon ectopic hCDC14A (A) hCDC14A-YFP colocalizes with F-actin at the leading edge of HeLa FRT T-Rex expression (Fig. S1 G and H). hCDC14A-YFP cells (Upper). (Scale bar: 10 μm.) (Lower) Regional magnifica- As in previous reports (22, 25), hCDC14A was found in the tion of the boxed area in the Upper panel. (Scale bar: 5 μm.) The white line cytoplasm, at centrosomes of interphase cells and at the midbody indicates the scanned segment in B.(B) Line scan of hCDC14A-YFP and during cytokinesis (Fig. S2A). In the Flp-in T-Rex cell line, we F-actin from A.(C) hCDC14A-YFP relocalizes with F-actin remnants after failed to detect the reported association of hCDC14A with mi- blebbistatin treatment. (Lower) Regional magnification of the area high- crotubules (18). However, strong overexpression of hCDC14A- lighted
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