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Human Phosphatase CDC14A Regulates Actin Organization Through Dephosphorylation of Epithelial Protein Lost in Neoplasm

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Human Phosphatase CDC14A Regulates Actin Organization Through Dephosphorylation of Epithelial Protein Lost in Neoplasm Human phosphatase CDC14A regulates actin organization through dephosphorylation of epithelial protein lost in neoplasm Nan-Peng Chena,b, Borhan Uddina,b, Robert Hardta, Wen Dinga, Marko Panica,b, Ilaria Lucibelloa, Patricia Kammererc, Thomas Rupperta, and Elmar Schiebela,1 aZentrum für Molekulare Biologie der Universität Heidelberg (ZMBH), Deutsches Krebsforschungszentrum (DKFZ)–ZMBH Allianz, Universität Heidelberg, 69120 Heidelberg, Germany; bHartmut Hoffmann-Berling International Graduate School of Molecular and Cellular Biology (HBIGS), Universität Heidelberg, 69120 Heidelberg, Germany; and cMax Planck Institute of Biochemistry, 82152 Martinsried, Germany Edited by Thomas D. Pollard, Yale University, New Haven, CT, and approved April 11, 2017 (received for review November 24, 2016) CDC14 is an essential dual-specificity phosphatase that counteracts Because there have been no systematic attempts to identify CDK1 activity during anaphase to promote mitotic exit in Saccharo- phospho-sites that are regulated by hCDC14A, we know relatively myces cerevisiae. Surprisingly, human CDC14A is not essential for little about the identity of either the proteins that are dephos- cell cycle progression. Instead, it regulates cell migration and cell phorylated by hCDC14A or, indeed, the kinases that counteract adhesion. Little is known about the substrates of hCDC14A and hCDC14A in human cells. The only known substrate of hCDC14A the counteracting kinases. Here, we combine phospho-proteome that acts within the actin cytoskeleton is the protein Kibra (20, 21). profiling and proximity-dependent biotin identification to identify However, considering the broad distribution of hCDC14A hCDC14A substrates. Among these targets were actin regulators, throughout the actin network, it is highly likely that hCDC14A including the tumor suppressor eplin. hCDC14A counteracts EGF- dephosphorylates additional actin-associated proteins. induced rearrangements of actin cytoskeleton by dephosphorylating To identify hCDC14A substrates and decipher the molecular eplin at two known extracellular signal-regulated kinase sites, serine mechanisms by which hCDC14A controls cell migration, we com- PD 362 and 604. hCDC14A and eplin knockout cell lines exhibited bined phospho-proteome analysis with the biotin identification down-regulation of E-cadherin and a reduction in α/β-catenin at (BioID) proximity assay (22). These approaches identified the cell–cell adhesions. Reduction in the levels of hCDC14A and eplin potent tumor suppressor eplin as a key actin-associated hCDC14A mRNA is frequently associated with colorectal carcinoma and is cor- substrate. We show that hCDC14A controls actin-bundling activity related with poor prognosis. We therefore propose that eplin de- of eplin to locally modulate actin rearrangements by counteracting phosphorylation by hCDC14A reduces actin dynamics to restrict EGF-induced ERK-mediated phosphorylation of eplin. Eplin also tumor malignancy. links F-actin to the E-cadherin–α/β catenin complex (23). Consis- tently, we found a reduction in the enrichment of α/β catenin at CDC14 | eplin | actin dynamics cell–cell junctions and decreased E-cadherin levels when either hCDC14A phosphatase activity was ablated or eplin was removed. he highly dynamic nature of the actin network of nonmuscle This striking correlation indicates that hyperphosphorylated eplin Tcells contributes to its functions in cell migration, cell synaptic lacks the ability to interconnect the E-cadherin–α/β catenin com- transmission, endocytosis, and the immune response (1–3). It is plex with F-actin. As down-regulation of hCDC14A and eplin is a therefore not surprising that deregulation of actin is associated with common feature of colorectal cancer and is associated with poor a range of human diseases, including leukemia, kidney disorders, prognosis, our data strongly suggest that loss of hCDC14A–eplin and cancer development (4, 5). Long lists of actin-associated pro- teins from Rho GTPases, kinases, phosphatases, and actin-binding Significance proteins regulate actin filament dynamics (1, 3, 4, 6–8). Actin- bundling proteins such as α-actinin, fascin, and filamin cross-link Human hCDC14A phosphatase has been shown to regulate cell actin filaments into bundles and play important roles in regulating migration and adhesion by modulating actin dynamics. Here we membrane protrusions and cell mobility (9–13). Cofilin severs identify the potent tumor suppressor eplin as an hCDC14A sub- existing actin filaments to generate more free ends and thus plays a strate. Through dephosphorylation of serine residues 362 and critical role in actin dynamics (9, 10, 14, 15). The protein epithelial 604 of eplin, hCDC14A promotes F-actin bundling and counteracts protein lost in neoplasm (eplin) bundles F-actin filaments and can EGF-induced membrane ruffling. Cells lacking hCDC14A activity or CELL BIOLOGY control G-actin nucleation by regulating the activity of the Arp2/3 eplin expression showed reduced E-cadherin levels and β-catenin actin complex (16). Interestingly, the activity of eplin is regulated retention at cell–cell junctions and formed more invasive through phosphorylation by extracellular signal-regulated kinase colonies than wild-type cells. Expression of a phospho-inhibitory, (ERK) at serine residues 362 and 604 (17). However, the phos- but not a phospho-mimetic, eplin mutant rescued these pheno- phatase that reverses these important phosphorylations is unknown. types. Database analysis indicated that hCDC14A and eplin are In Saccharomyces cerevisiae, the dual-specificity phosphatase frequently inactivated in human colorectal cancer and this is as- CDC14 counteracts cyclin-dependent kinase (Cdk1) activity to sociated with poor prognosis. Therefore, the hCDC14A and eplin drive mitotic exit (18, 19). Therefore, CDC14 is essential for the axis plays an important role in limiting tumor malignancy. viability of S. cerevisiae. In contrast, its ortholog, CDC14A,isnot Author contributions: N.-P.C. and E.S. designed research; N.-P.C., B.U., R.H., W.D., M.P., essential for the viability of either human or chicken DT40 cells. I.L., and P.K. performed research; T.R. contributed new reagents/analytic tools; N.-P.C., Surprisingly, cells in which the hCDC14A coding sequence had B.U., R.H., and W.D. analyzed data; and N.-P.C. and E.S. wrote the paper. been disrupted did not show any obvious defects in cell cycle The authors declare no conflict of interest. progression (18, 19). However, we recently reported that This article is a PNAS Direct Submission. hCDC14A colocalizes with F-actin at the leading edge of cells 1To whom correspondence should be addressed. Email: [email protected]. and stress fibers where it regulates actin dynamics. This regula- This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. tion is important for cell migration and cell adhesion (20). 1073/pnas.1619356114/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1619356114 PNAS | May 16, 2017 | vol. 114 | no. 20 | 5201–5206 Downloaded by guest on September 30, 2021 regulation may be a key step in driving the malignancy of invasive Hypo-phosphorylated peptides were analyzed with the Perseus colorectal cancers. program to generate a consensus dephosphorylation motif for hCDC14A (Fig. 1C). This analysis is consistent with structural Results data (26) indicating that hCDC14A shares a common preference Phospho-Proteome Analysis Revealed Multiple Actin Regulators as with cyclin-dependent kinases in being proline directed with a hCDC14A Substrates. We applied affinity purification of phospho- preference for lysine or arginine three residues after the phos- peptides followed by large-scale phospho-proteomics to identify phorylation site (25). The presence of proline residues at positions + hCDC14A substrates. HeLaTetON-hCDC14A-YFP and TetON-YFP -1, -2, and 2 indicates that hCDC14A may also counteract other cells [stable integration into the flippase recognition target kinases such as ERK1/2 and glycogen synthase kinase 3 (GSK-3β) (FRT) site] were grown in heavy (Arg13C15NLys13C15N) and light that emulate cyclin-dependent kinases in being proline-directed (Arg12C14NLys12C14N) medium, respectively (Fig. 1A). After kinases yet have a preference for additional proline residues sur- eight passages, addition of doxycycline (Dox) to the medium in- rounding the phosphorylation site (www.phosphosite.org/). Con- duced hCDC14A-YFP and YFP expression. Twenty-four hours sistent with hCDC14A’s localization and function along F-actin, later, proteins were extracted, the extracts were mixed at a ratio of gene ontology enrichment revealed that the majority of hCDC14A 1:1, and, after phospho-peptide enrichment, peptides were ana- substrates were either involved in the organization of the actin lyzed by LC-MS/MS. Over 14,000 phospho-sites were identified in cytoskeleton or associated with adherens junctions and focal ad- each of three independent replicate experiments. Surprisingly, hesion sites (Fig. 1D). Bioinformatic analysis using STRING and only 68 phospho-sites (0.5%) belonging to 51 proteins (threshold = BioGrid revealed a molecular network of multiple actin regulators 0.5) were hypo-phosphorylated upon hCDC14A-YFP expression (Fig. 1E) that contained a high number of actin-bundling proteins compared with the YFP control (Fig. 1B and Table S1). Of the such as eplin, drebrin, filamin, and supervillin. 68 hypo-phosphorylated peptides, 65 (95.6%) contained phospho- To validate the data arising
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