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<article xmlns:xlink="http://www.w3.org/1999/xlink" xmlns:mml="http://www.w3.org/1998/Math/MathML" article-type="research-article"><?properties open_access?><front><journal-meta><journal-id journal-id-type="nlm-ta">J Cell Biol</journal-id><journal-id journal-id-type="iso-abbrev">J. Cell Biol</journal-id><journal-title-group><journal-title>The Journal of Cell Biology</journal-title></journal-title-group><issn pub-type="ppub">0021-9525</issn><issn pub-type="epub">1540-8140</issn><publisher><publisher-name>The Rockefeller University Press</publisher-name></publisher></journal-meta><article-meta><article-id pub-id-type="pmid">9744873</article-id><article-id pub-id-type="pmc">PMC1424221</article-id><article-categories><subj-group subj-group-type="heading"><subject>Regular Articles</subject></subj-group></article-categories><title-group><article-title>Regulation of Endosome Sorting by a Specific PP2A Isoform </article-title></title-group><contrib-group><contrib contrib-type="author"><name><surname>Molloy</surname><given-names>Sean S.</given-names></name><xref ref-type="aff" rid="N0x3769d00N0x358ef98">*</xref></contrib><contrib contrib-type="author"><name><surname>Thomas</surname><given-names>Laurel</given-names></name><xref ref-type="aff" rid="N0x3769d00N0x358ef98">*</xref></contrib><contrib contrib-type="author"><name><surname>Kamibayashi</surname><given-names>Craig</given-names></name><xref ref-type="aff" rid="N0x3769d00N0x358ef98">&#x02021;</xref></contrib><contrib contrib-type="author"><name><surname>Mumby</surname><given-names>Marc C.</given-names></name><xref ref-type="aff" rid="N0x3769d00N0x358ef98">&#x02021;</xref></contrib><contrib contrib-type="author"><name><surname>Thomas</surname><given-names>Gary</given-names></name><xref ref-type="aff" rid="N0x3769d00N0x358ef98">*</xref></contrib></contrib-group><aff id="N0x3769d00N0x358ef98">
<label>*</label>Vollum Institute, Oregon Health Sciences University, Portland, Oregon 97201; and <label>&#x02021;</label>Department of Pharmacology, The University of Texas Southwestern Medical Center, Dallas, Texas 75235</aff><pub-date pub-type="ppub"><day>21</day><month>9</month><year>1998</year></pub-date><volume>142</volume><issue>6</issue><fpage>1399</fpage><lpage>1411</lpage><history><date date-type="received"><day>19</day><month>6</month><year>1998</year></date><date date-type="rev-recd"><day>19</day><month>8</month><year>1998</year></date></history><permissions><copyright-year>1998</copyright-year><license license-type="openaccess"><license-p>This article is distributed under the terms of an Attribution&#x02013;Noncommercial&#x02013;Share Alike&#x02013;No Mirror Sites license for the first six months after the publication date (see <ext-link ext-link-type="uri" xlink:href="http://www.rupress.org/terms">http://www.rupress.org/terms</ext-link>). After six months it is available under a Creative Commons License (Attribution&#x02013;Noncommercial&#x02013;Share Alike 4.0 Unported license, as described at <ext-link ext-link-type="uri" xlink:href="http://creativecommons.org/licenses/by-nc-sa/4.0/">http://creativecommons.org/licenses/by-nc-sa/4.0/</ext-link>).</license-p></license></permissions><abstract><p>The regulated sorting of proteins within the <italic>trans</italic>-Golgi network (TGN)/endosomal system is a key determinant of their biological activity in vivo. For example, the endoprotease furin activates of a wide range of proproteins in multiple compartments within the TGN/endosomal system. Phosphorylation of its cytosolic domain by casein kinase II (CKII) promotes the localization of furin to the TGN and early endosomes whereas dephosphorylation is required for efficient transport between these compartments (Jones, B.G., L. Thomas, S.S. Molloy, C.D. Thulin, M.D. Fry, K.A. Walsh, and G. Thomas. 1995. <italic>EMBO [Eur. Mol. Biol. Organ.] J.</italic> 14:5869&#x02013;5883). Here we show that phosphorylated furin molecules internalized from the cell surface are retained in a local cycling loop between early endosomes and the plasma membrane. This cycling loop requires the phosphorylation state-dependent furin-sorting protein PACS-1, and mirrors the trafficking pathway described recently for the TGN localization of furin (Wan, L., S.S. Molloy, L. Thomas, G. Liu, Y. Xiang, S.L. Ryback, and G. Thomas. 1998. <italic>Cell</italic>. 94:205&#x02013;216). We also demonstrate a novel role for protein phosphatase 2A (PP2A) in regulating protein localization in the TGN/endosomal system. Using baculovirus recombinants expressing individual PP2A subunits, we show that the dephosphorylation of furin in vitro requires heterotrimeric phosphatase containing B family regulatory subunits. The importance of this PP2A isoform in directing the routing of furin from early endosomes to the TGN was established using SV-40 small t antigen as a diagnostic tool in vivo. The role of both CKII and PP2A in controlling multiple sorting steps in the TGN/endosomal system indicates that the distribution of itinerant membrane proteins may be acutely regulated via signal transduction pathways.</p></abstract><kwd-group><kwd>furin</kwd><kwd>endosome</kwd><kwd>PP2A</kwd><kwd>sorting</kwd><kwd>PACS-1</kwd></kwd-group></article-meta></front><body><p>T<sc>wo</sc> compelling and fundamental questions in cell biology are to identify the mechanisms by which proteins are routed to their correct intracellular compartments and how these sorting steps are regulated. The broad importance and complexity of the <italic>trans</italic>-Golgi network (TGN)/endosomal sorting system (for review see <xref rid="B60" ref-type="bibr">Robinson et al., 1996</xref>) has been highlighted by recent studies of such diverse processes as delivery of vacuolar proteins in yeast (<xref rid="B15" ref-type="bibr">Cowles et al., 1997<italic>a</italic>
</xref>,<italic>b</italic>; <xref rid="B57" ref-type="bibr">Piper et al., 1997</xref>; <xref rid="B80" ref-type="bibr">Voos and Stevens, 1998</xref>), transcytosis in polarized cells (<xref rid="B3" ref-type="bibr">Apodaca et al., 1996</xref>; <xref rid="B49" ref-type="bibr">Odorizzi et al., 1996</xref>; <xref rid="B88" ref-type="bibr">Zacchi et al., 1998</xref>), the mobilization of major histocompatability complex class 2 and Glut-4&#x02013;containing endosomes (for review see <xref rid="B35" ref-type="bibr">Keller and Simons, 1997</xref>), and synaptic vesicle biogenesis (<xref rid="B11" ref-type="bibr">Cameron et al., 1991</xref>; <xref rid="B83" ref-type="bibr">West et al., 1997</xref>; <xref rid="B54" ref-type="bibr">Partoens et al., 1998</xref>). Despite their central role to the physiology of cells, the regulation of these complex trafficking systems remain largely undetermined.</p><p>One factor that contributes to the dynamic capacity of the TGN/endosomal sorting system is regulation by phosphorylation. The link between protein trafficking and phosphorylation provides a means by which cells can rapidly and reversibly alter the distribution and function of a variety of transmembrane proteins. Kinase and phosphatase activities have been shown to control both general and cargo-specific trafficking. Phospholipid kinases and phosphatases (for reviews see <xref rid="B71" ref-type="bibr">Stack et al., 1995</xref>; <xref rid="B18" ref-type="bibr">De Camilli et al., 1996</xref>; <xref rid="B65" ref-type="bibr">Shepherd et al., 1996</xref>; <xref rid="B85" ref-type="bibr">Woscholski and Parker, 1997</xref>) modulate membrane dynamics throughout the TGN/endosomal system, including budding from the TGN (<xref rid="B66" ref-type="bibr">Simon et al., 1996</xref>; <xref rid="B13" ref-type="bibr">Chen et al., 1997</xref>; <xref rid="B33" ref-type="bibr">Jones et al., 1998</xref>) and recycling from endosomal compartments (<xref rid="B12" ref-type="bibr">Cardone and Mostov, 1995</xref>; <xref rid="B70" ref-type="bibr">Spiro et al., 1996</xref>; <xref rid="B14" ref-type="bibr">Chung et al., 1997</xref>; <xref rid="B38" ref-type="bibr">Luo and Chang, 1997</xref>; <xref rid="B40" ref-type="bibr">Malide and Cushman, 1997</xref>). Although protein kinase activities have long been recognized as important modulators of receptor transduction complexes at the cell surface and in signaling endosomes (<xref rid="B7" ref-type="bibr">Bevan et al., 1995</xref>; <xref rid="B82" ref-type="bibr">Wang et al., 1996</xref>; <xref rid="B27" ref-type="bibr">Grimes et al., 1997</xref>), there is growing evidence that protein kinases and phosphatases also control the sorting of itinerant membrane proteins (for review see <xref rid="B63" ref-type="bibr">Seaman et al., 1996</xref>). Regulation of protein traffic by phosphorylation can occur via both general e.g., modification of adaptin binding to clathrin (<xref rid="B84" ref-type="bibr">Wilde and Brodsky, 1996</xref>), and TGN export (<xref rid="B50" ref-type="bibr">Ohashi and Huttner, 1994</xref>; <xref rid="B5" ref-type="bibr">Austin and Shields, 1996</xref>) and specific, cargo-directed mechanisms. Examples of the latter include the transcytosis of the polymeric immunoglobulin receptor (<xref rid="B4" ref-type="bibr">Apodaca and Mostov, 1993</xref>; <xref rid="B52" ref-type="bibr">Okamoto et al., 1994</xref>), internalization of T cell receptors (CTLA-4 [<xref rid="B10" ref-type="bibr">Bradshaw et al., 1997</xref>] and CD4 [<xref rid="B55" ref-type="bibr">Pelchen-Matthews et al., 1993</xref>]), as well as the TGN localization of the endoprotease furin (<xref rid="B32" ref-type="bibr">Jones et al., 1995</xref>; <xref rid="B74" ref-type="bibr">Takahashi et al., 1995</xref>; <xref rid="B19" ref-type="bibr">Ditti&#x000e9; et al., 1997</xref>; <xref rid="B81" ref-type="bibr">Wan et al., 1998</xref>).</p><p>Whereas a few of the itinerant membrane protein&#x02013;directed kinases involved in regulation of protein trafficking have been identified (e.g., CKII, <xref rid="B32" ref-type="bibr">Jones et al., 1995</xref>), both the phosphatases and the machinery responsible for the differential sorting of phosphoproteins remain largely uncharacterized. Furin is an excellent model for defining the cellular machinery involved in phosphorylation state-dependent protein sorting within the TGN/endosomal system. The endoprotease is routed through multiple proprotein processing compartments by virtue of defined trafficking signals within its cytosolic domain (cd)<sup>1</sup> (<xref rid="B32" ref-type="bibr">Jones et al., 1995</xref>; <xref rid="B62" ref-type="bibr">Sch&#x000e4;fer et al., 1995</xref>). Although the steady-state localization of furin is predominantly in the TGN (<xref rid="B9" ref-type="bibr">Bosshart et al., 1994</xref>; <xref rid="B45" ref-type="bibr">Molloy et al., 1994</xref>; <xref rid="B62" ref-type="bibr">Sch&#x000e4;fer et al., 1995</xref>; <xref rid="B64" ref-type="bibr">Shapiro et al., 1997</xref>), the protease cycles between this compartment and the cell surface via an endosomal pathway (<xref rid="B45" ref-type="bibr">Molloy et al., 1994</xref>; <xref rid="B32" ref-type="bibr">Jones et al., 1995</xref>; <xref rid="B36" ref-type="bibr">Liu et al., 1997</xref>).</p><p>Internalization of furin from the cell surface and export from the TGN are directed by canonical tyrosine and/or dileucine based clathrin-coated pit recruitment motifs interacting with the clathrin sorting machinery (<xref rid="B51" ref-type="bibr">Ohno et al., 1996</xref>; <xref rid="B62" ref-type="bibr">Sch&#x000e4;fer et al., 1995</xref>; <xref rid="B81" ref-type="bibr">Wan et al., 1998</xref>). Localization of furin to the TGN, however, requires a cluster of acidic residues (AC)<sup>1</sup> that constitute a CKII phosphorylation site (<xref rid="B9" ref-type="bibr">Bosshart et al., 1994</xref>; <xref rid="B32" ref-type="bibr">Jones et al., 1995</xref>; <xref rid="B62" ref-type="bibr">Sch&#x000e4;fer et al., 1995</xref>). Phosphorylation of this AC motif by CKII regulates the TGN localization of the protease by promoting its retrieval from immediate post-TGN compartments (<xref rid="B81" ref-type="bibr">Wan et al., 1998</xref>). The phosphofurin acidic cluster-sorting protein (PACS-1) directs this TGN retrieval step by linking the phosphorylated furin-cd to the clathrin sorting machinery (<xref rid="B81" ref-type="bibr">Wan et al., 1998</xref>).</p><p>Despite their importance for establishing processing compartments within endosomes and at the cell surface (<xref rid="B36" ref-type="bibr">Liu et al., 1997</xref>), the factors which control sorting of furin in peripheral compartments have not been well characterized. Phosphorylation of the furin-cd has, however, been implicated in the trafficking of the protease in the endosomal system (<xref rid="B32" ref-type="bibr">Jones et al., 1995</xref>). This observation indicates dual roles for both CKII and AC motifs in protein sorting. Furthermore, the ability of a phosphatase inhibitor (tautomycin) to alter the routing of internalized furin suggested that dephosphorylation is a critical determinant of furin sorting in early endosomes (<xref rid="B32" ref-type="bibr">Jones et al., 1995</xref>). The identity of the furin phosphatase, and the mechanism(s) by which these various factors act to regulate endosomal sorting, however, are not known.</p><p>The emerging complexity of the protein phosphatase (PP) 1 and 2A families suggests a myriad of roles for these enzymes. Indeed, isoforms of PP1 containing catalytic and regulatory or targeting subunits have been shown to control glycogen metabolism and myosin dephosphorylation (for review see <xref rid="B31" ref-type="bibr">Hubbard and Cohen, 1993</xref>). PP2A has been linked to the regulation of mitogen-signaling pathways, microtubule dynamics, and control of gene expression in the cell cycle (for reviews see <xref rid="B31" ref-type="bibr">Hubbard and Cohen, 1993</xref>; <xref rid="B42" ref-type="bibr">Mayer-Jaekel and Hemmings, 1994</xref>; <xref rid="B6" ref-type="bibr">Barford, 1996</xref>). The active form of PP2A in vivo is predominantly a heterotrimer which consists of a catalytic moiety (C subunit), as well as an A subunit which mediates the binding of variable regulatory subunits. Although neither phosphatase has been shown to direct protein trafficking, the demonstrated importance of subunit composition in determining their function in vivo suggests such a role is feasible. </p><p>A variety of PP2A regulatory subunit families genes, and splice variants have been reported (<xref rid="B43" ref-type="bibr">McCright et al., 1996</xref>; <xref rid="B90" ref-type="bibr">Zolnierowicz et al., 1996</xref>), yet specialized functions for these phosphatase isoforms remain to be established. Three unique PP2A regulatory subunit gene families have been identified to date in mammals based upon the characterization of tissue-specific isoforms and homology cloning. The B/PR55 family includes &#x003b1;, &#x003b2;, and &#x003b3; gene products, whereas the B&#x02032;/B56 family contains 5 genes (&#x003b1;, &#x003b2;, &#x003b3;, &#x003b4;, and &#x003b5;) some of which express multiple splice variants (&#x003b3;1&#x02013;5, &#x003b4;1&#x02013;3). The PR72/130 regulatory subunits are the product of differential splicing of a single gene. Although some of these regulatory subunits are associated with isoforms of PP2A highly expressed in particular tissues or cell types (<xref rid="B73" ref-type="bibr">Strack et al., 1998</xref>), their roles in vivo are largely undetermined.</p><p>Here we report the identity of a furin phosphatase and the importance of furin-cd phosphorylation state in regulating multiple steps in the trafficking of the endoprotease in vivo. We demonstrate that furin undergoes phosphorylation-dependent local cycling between early endosomes and the cell surface. This peripheral cycling loop mirrors that reported for the TGN localization of the protease (<xref rid="B81" ref-type="bibr">Wan et al., 1998</xref>), and requires the phosphorylation state-dependent sorting protein PACS-1. Analyses in vitro and in vivo show that the movement of furin between early endosomes and the TGN is regulated by specific PP2A isoforms containing B family regulatory subunits. These findings demonstrate a novel role for PP2A and reveal the importance of phosphorylation/dephosphorylation in the acute regulation of protein sorting within the TGN/endosomal system.</p><sec sec-type="materials|methods" id="MaterialsMethods"><title>Materials and Methods</title><sec><title>Antibodies and Reagents</title><p>Antibodies against PP2A regulatory subunits have been described previously (<xref rid="B34" ref-type="bibr">Kamibayashi et al., 1994</xref>; <xref rid="B76" ref-type="bibr">Tehrani et al., 1996</xref>). PP2A catalytic subunit antibody was obtained from B. Wadzinsky (Vanderbilt University, Nashville, TN). mAb M1 was from <named-content content-type="company" xlink:href="kodak">Eastman Kodak Co.</named-content> (Rochester, NY). PP1 and PP2A catalytic subunits were provided by A.A. DePaoli-Roach (Indiana University, Indianapolis, IN). All chemical reagents were obtained from <named-content content-type="company" xlink:href="sigma">Sigma Chemical Co.</named-content> (St. Louis, MO) unless otherwise noted.</p></sec><sec><title>Cell Culture and Immunofluorescence Analyses</title><p>BSC-40 and PACS-1 control (C1) and antisense (AS19) cells were cultured as previously described (<xref rid="B77" ref-type="bibr">Thorne et al., 1989</xref>; <xref rid="B81" ref-type="bibr">Wan et al., 1998</xref>). HeLa cells expressing TS-Dyn I under control of Tet-suppressing elements were obtained from S. Schmid (The Scripps Research Institute, La Jolla, CA), and maintained as described (<xref rid="B17" ref-type="bibr">Damke et al., 1995</xref>). For immunofluorescence analyses, cells were cultured directly on glass coverslips to a density of 50&#x02013;80% confluence before experimental manipulation as described in figure legends. Cells were fixed in 4% paraformaldehyde and processed for immunofluorescence as previously described (<xref rid="B45" ref-type="bibr">Molloy et al., 1994</xref>). mAb M1 was detected using either FITC- or TXR-conjugated goat anti&#x02013; mouse IgG2b-specific secondary antibodies (<named-content content-type="company" xlink:href="fisher">Fisher Scientific Co.</named-content>, Pittsburgh, PA).</p></sec><sec><title>Cloning and Expression Vectors</title><p>Epitope-tagged furin (fur/f), the CKII phosphorylation site point mutants (fur/f-DDD and fur/f-ADA), and the glutathione-S-transferase (GST)&#x02013; Fur<sub>cd</sub> fusion protein were generated previously (<xref rid="B45" ref-type="bibr">Molloy et al., 1994</xref>; <xref rid="B32" ref-type="bibr">Jones et al., 1995</xref>). Dynamin I WT and K44E cDNAs were each excised from pSVL (<xref rid="B28" ref-type="bibr">Herskovits et al., 1993</xref>) using EcoRI. The inserts were then blunted with Klenow and cloned into the vaccinia recombination vector pZVneo cut with StuI. SV-40 small t WT and the truncated small t mut 3 (<xref rid="B67" ref-type="bibr">Sontag et al., 1993</xref>) were excised from pCMV5 using EcoRI and BamHI, and then blunted with Klenow and inserted into pZVneo cut with StuI. Recombinant vaccinia expressing the various constructs were generated by standard methods (<xref rid="B79" ref-type="bibr">VanSlyke et al., 1995</xref>).</p></sec><sec><title>Surface Labeling and Uptake Analysis</title><p>HeLa cells expressing the temperature-sensitive dominant-negative dynamin I were cultured in 35-mm plates for 3 d at permissive temperature in the absence of tetracycline. The cells were then infected with vaccinia virus expressing fur/f with either the serine to alanine (fur/f-ADA) or serine to aspartic acid (fur/f-DDD) substitutions within the cd CKII site (multiplicity of infection [m.o.i.] = 10) and allowed to express at nonpermissive temperature, 37&#x000b0;C. At 6 h postinfection the cells were placed on ice, rinsed with cold PBS and surface proteins were labeled for 1 h using 0.5 mg/ml EZ-link NHS-SS-biotin (<named-content content-type="company" xlink:href="pierce">Pierce Chemical Co.</named-content>, Rockford, IL) in PBS. After labeling, the cells were rinsed three times with PBS containing 50 mM glycine to quench unreacted biotin, and then refed with prewarmed medium (except for 0 time points) and placed at permissive temperature (31&#x000b0;C) for 10, 20, 30, or 40 min. At each time point, cells were transferred to ice, rinsed with cold PBS, and then either harvested directly in modified RIPA buffer (mRIPA, 50 mM Tris-HCl, pH 8, 150 mM NaCl, 1% NP-40 [<named-content content-type="company" xlink:href="calbio">Calbiochem-Novabiochem Corp.</named-content>, La Jolla, CA] and 1% sodium deoxycholate) for immunoprecipitation of total labeled furin using mAb M1 as previously described (<xref rid="B45" ref-type="bibr">Molloy et al., 1994</xref>), or were stripped of remaining surface biotin by washing three times for 10 min in Tris-buffered saline (TBS; 10 mM Tris-HCl, pH 8, 150 mM NaCl) with MesNa (50 mM). The surface-stripped samples were rinsed with PBS supplemented with Hepes (10 mM, pH 7.5) and harvested in mRIPA for immunoprecipitation of internalized labeled furin with mAb M1. The immunoprecipitates were resolved by SDS-PAGE on 8% gels, transferred to nitrocellulose membranes and probed with avidin HRP (1:2,000 dilution of 1 mg/ml in TBS + 0.05% Triton X-100) and developed by chemiluminescence (Renaissance; NEN Life Science Products, Boston, MA) to detect biotinylated furin. In double-strip analyses, cells processed as described above after the initial 20-min uptake period were then incubated for a second 20-min period at permissive temperature to allow for recycling of the internal pool to the cell surface. After the second chase period, cells were placed on ice and either harvested directly (control) or subjected to a second MesNa strip as described above before harvesting and analysis by Western blot.</p></sec><sec><title>PP2A Expression and Assays</title><p>Baculoviruses expressing PP2A catalytic, A, B&#x003b1;, and B&#x003b2; subunits have been described previously (<xref rid="B34" ref-type="bibr">Kamibayashi et al., 1994</xref>). The recombinant baculovirus encoding the B&#x02032;&#x003b1; subunit was generated as described (<xref rid="B76" ref-type="bibr">Tehrani et al., 1996</xref>). For expression experiments, Sf9 cells growing in log phase were seeded in 25-cm flasks (3 &#x000d7; 10<sup>6</sup> cells/flask) and infected with baculovirus recombinants (m.o.i. = 2). The cells were harvested at 64&#x02013;72 h postinfection by trituration and then pelleted and washed with PBS. The washed cell pellet was then resuspended in 0.5 ml of ice-cold harvest buffer (50 mM Tris, pH 7.0, 1 mM EDTA, and 2 mM DTT with pepstatin, leupeptin, PMSF, aprotinin, and E64) and the cells broken open by passage (10&#x000d7;) through a 25-gauge needle. The lysates were clarified by low speed centrifugation at 500 <italic>g</italic> and brought to 10% glycerol before performing phosphatase assays (see below).</p></sec><sec><title>Phosphatase Assays</title><p>Substrates for phosphatase assays were prepared by phosphorylating GST&#x02013;Fur<sub>cd</sub> fusion protein and phosphorylase b in vitro with CKII or phosphorylase kinase, respectively. The phosphorylation reactions (100 &#x003bc;l) contained 50 &#x003bc;g of substrate, and 100 &#x003bc;M of <sup>32</sup>P-labeled ATP (3,000 cpm/ pmol). After phosphorylation for 1 h at 30&#x000b0;C, the reactions were run over two G25 spin columns to remove free ATP. Dephosphorylation assays were conducted in a 50-&#x003bc;l reaction containing lysates, buffer (25 mM Tris, pH 7.0, 0.2 mM MnCl<sub>2</sub>, 1 mM DTT, and 0.2 mg/ml BSA) and &#x0223c;10 &#x003bc;M phosphorylated substrate (either phosphorylase a or GST&#x02013;Fur<sub>cd</sub>). Aliquots (5 &#x003bc;l in triplicate) of each reaction were removed at 15 and 30 min of incubation and applied to P81 filter paper (Whatman Inc., Clifton, NJ) squares that were washed three times with 75 mM phosphoric acid to remove free phosphate and then dried and subjected to scintillation counting. In some cases, the aliquots were mixed with SDS sample buffer, resolved by SDS-PAGE, and then analyzed using a PhosphoImager (model 445SI; Molecular Dynamics Inc., Sunnyvale, CA). Both measurement techniques gave identical results.</p></sec><sec><title>Microcystin Affinity Chromatography</title><p>Affinity chromatography for purification of endogenous furin-directed phosphatase from BSC-40 cell extract was performed essentially as described (<xref rid="B47" ref-type="bibr">Moorhead et al., 1994</xref>). In brief, microcystin (MC)-LR (LC Laboratories, Woburn, MA) was derivatized with aminoethanethiol to generate a primary amine group for coupling to NHS-activated sepharose (<named-content content-type="company" xlink:href="pharmacia">Pharmacia Biotech Inc.</named-content>, Piscataway, NJ). Approximately 0.25 mg of derivatized MC was linked to 1 ml of Sepharose resin. For the preparation of extracts, three 15-cm plates of confluent BSC-40 cells were rinsed three times with cold PBS and one time with harvest buffer (see above). The cells were then scraped from the plates and lysed by passage (10&#x000d7;) through a 25-gauge needle. The extract was then clarified by low-speed centrifugation (10 min at 500 <italic>g</italic>) and brought to 10% glycerol. The clarified extract was then cycled over the MC column (0.5 ml/min) for 1 h at 4&#x000b0;C to allow binding of the phosphatase. The column was then washed with 10 vol of harvest buffer and eluted with 3M NaSCN. Each eluted fraction (10 ml) was dialyzed over night against harvest buffer before assaying for phosphorylase a and GST&#x02013;Fur<sub>cd</sub>-directed phosphatase activities as described above.</p></sec></sec><sec id="Results"><title>Results</title><sec><title>Importance of Furin-Cd Dephosphorylation for Early Endosome to TGN Sorting</title><p>Phosphorylation of the furin-cd by CKII is necessary for maintaining the steady-state distribution of the endoprotease in the TGN and also for the localization of cell surface-internalized furin to early endosomes. By contrast, sorting of furin from early endosomes to the TGN requires dephosphorylation of the furin-cd (<xref rid="B32" ref-type="bibr">Jones et al., 1995</xref>). These findings were confirmed by antibody uptake studies (Fig. <xref ref-type="fig" rid="F1">1</xref>). Incubation of cells expressing epitope (FLAG)- tagged furin (fur/f) with mAb M1 demonstrated the retrieval of cell surface fur/f to the TGN (Fig. <xref ref-type="fig" rid="F1">1</xref>
<italic>A</italic>). However, treatment of cells with tautomycin, a potent inhibitor of PP1 and PP2A (<xref rid="B75" ref-type="bibr">Takai et al., 1995</xref>), resulted in the accumulation of internalized fur/f in peripheral punctate structures characteristic of early endosomes (Fig. <xref ref-type="fig" rid="F1">1</xref>
<italic>B</italic>). The effect of tautomycin is specific for a furin-directed phosphatase since (<italic>a</italic>) this drug has no effect on the internalization of a nonphosphorylatable furin construct, fur/ fS<sub>773,775</sub>A (ADA) (<xref rid="B32" ref-type="bibr">Jones et al., 1995</xref>), and (<italic>b</italic>) internalization of a phosphorylation mimic furin construct, fur/ fS<sub>773,775</sub>D (DDD), also resulted in accumulation in the peripheral punctate compartments (Fig. <xref ref-type="fig" rid="F1">1</xref>
<italic>C</italic>). Double-labeling experiments showed that internalized fur/f-DDD and transferrin colocalized within these peripheral vesicles (Fig. <xref ref-type="fig" rid="F1">1</xref>, <italic>D</italic> and <italic>E</italic>). Together these results demonstrate that after internalization, phosphorylated furin localizes to early endosomes and sorting of the endoprotease from this compartment to the TGN requires the activity of a tautomycin-sensitive furin phosphatase. </p></sec><sec><title>Surface Recycling of Furin</title><p>The dynamic nature of the early endosomal sorting system suggests that the phosphorylation state-dependent accumulation of furin in early endosomes is not likely due to static retention in these structures. Therefore, we tested the possibility that this accumulation reflected a directed cycling of phosphorylated furin between the cell surface and early endosomes. To facilitate quantitative analyses of internalization and recycling, we first investigated the mechanism of furin endocytosis as a means by which to increase the surface pool of the protease. The presence of functional tyrosine- and dileucine-based internalization signals within the furin-cd suggested that endocytosis occurs via a clathrin-dependent pathway (<xref rid="B62" ref-type="bibr">Sch&#x000e4;fer et al., 1995</xref>; <xref rid="B51" ref-type="bibr">Ohno et al., 1996</xref>). Therefore, we expressed a dominant-negative form of the GTPase dynamin I, K44E, to effectively block clathrin-dependent endocytosis (<xref rid="B28" ref-type="bibr">Herskovits et al., 1993</xref>; <xref rid="B78" ref-type="bibr">van der Bliek et al., 1993</xref>) and examined its effect on furin trafficking. Immunofluorescence analysis of furin internalization from the cell surface (Fig. <xref ref-type="fig" rid="F2">2</xref>) showed that although overexpression of native dynamin I had no effect on uptake (compare Fig. <xref ref-type="fig" rid="F2">2</xref>
<italic>A</italic> with Fig. <xref ref-type="fig" rid="F1">1</xref>
<italic>A</italic>), the K44E dominant-negative mutant blocked uptake and resulted in accumulation of the enzyme at the cell surface (Fig. <xref ref-type="fig" rid="F2">2</xref>
<italic>B</italic>). This effect on furin internalization mimicked the block in TfR endocytosis as observed with TRITC-transferrin uptake (Fig. <xref ref-type="fig" rid="F2">2</xref>, <italic>C</italic> [Dyn I] and <italic>D</italic> [K44E]). </p><p>Based on this result, quantitative analyses of furin trafficking were performed in stably transfected HeLa cells expressing a temperature-sensitive and reversible dominant-negative form of dynamin I (TS-Dyn I). Incubation of cells expressing fur/f with TS-Dyn I at nonpermissive temperature resulted in a dramatic increase in the amount of furin detected by surface biotinylation when compared with control cells at permissive temperature (data not shown). Therefore, we used this system to compare the trafficking of fur/f-DDD, to fur/f-ADA (Fig. <xref ref-type="fig" rid="F3">3</xref>). After surface labeling, cells expressing either fur/f-DDD or fur/f-ADA were chased for the increasing times to allow endocytosis/recycling. At each time point duplicate samples were either processed to detect internalized (<italic>I</italic>) or total (<italic>T</italic>) surface labeled furin (Fig. <xref ref-type="fig" rid="F3">3</xref>
<italic>A</italic>). The ratio of protected/total furin was plotted quantitatively as a percentage of surface furin internalized versus time at the permissive temperature (Fig. <xref ref-type="fig" rid="F3">3</xref>
<italic>B</italic>). At time points up to 20 min the internalization of fur/f-DDD and fur/f-ADA were similar. However, at longer incubation times the percentage of fur/ f-DDD in internal compartments decreased while the percentage of internalized fur/f-ADA continued to increase. Additional double-strip experiments showed that a significant portion (&#x0223c;50%) of the fur/f-DDD found in internal compartments at the 20-min time point was reexpressed at the cell surface during a subsequent 20-min chase (Fig. <xref ref-type="fig" rid="F3">3</xref>
<italic>B</italic>, <italic>inset</italic>). Reexpression of substantial amounts of fur/f-DDD at the cell surface within 20&#x02013;30 min is consistent with the recycling time of transferrin receptor in HeLa cells (<xref rid="B8" ref-type="bibr">Bleil and Bretscher, 1982</xref>), and suggests that the phosphorylated form of furin undergoes a similar local cycling between early endosomes and the cell surface. </p></sec><sec><title>PACS-1 Is Required for Early Endosome Localization of Phosphorylated Furin</title><p>The cell surface/early endosome recycling of furin requires multiple sorting motifs within the enzyme's cd. Internalization of furin from the cell surface is directed by the canonical tyrosine and di-leucine-based endocytosis signals (<xref rid="B62" ref-type="bibr">Sch&#x000e4;fer et al., 1995</xref>). Recycling from the early endosome to the cell surface, however, requires the CKII-catalyzed phosphorylation of the furin-cd AC (Fig. <xref ref-type="fig" rid="F3">3</xref>). Whereas the hydrophobic endocytosis signals bind directly to the clathrin adaptor AP-2 (<xref rid="B51" ref-type="bibr">Ohno et al., 1996</xref>), association of the phosphorylated furin-cd AC with the clathrin sorting machinery requires a connector protein, PACS-1 (<xref rid="B81" ref-type="bibr">Wan et al., 1998</xref>). A requirement for PACS-1 for the early endosomal localization of phosphorylated furin was demonstrated by monitoring furin internalization in PACS-1&#x02013;deficient cells (Fig. <xref ref-type="fig" rid="F4">4</xref>). Parallel plates of control or PACS-1&#x02013;deficient cells expressing fur/f were incubated with mAb M1 for 1 h in the presence of tautomycin. In agreement with Fig. <xref ref-type="fig" rid="F1">1</xref>, tautomycin treatment caused the accumulation of internalized fur/f in transferrin-containing early endosomes (Fig. <xref ref-type="fig" rid="F4">4</xref>
<italic>A</italic>). By contrast, in the absence of PACS-1, internalized fur/f failed to localize to early endosomes and instead showed a faint and dispersed staining pattern throughout the cytosol (Fig. <xref ref-type="fig" rid="F4">4</xref>
<italic>C</italic>). Early endosomal localization of internalized transferrin, however, was unaffected by PACS-1 depletion (Fig. <xref ref-type="fig" rid="F4">4</xref>, compare <italic>B</italic> with <italic>D</italic>). The inability of internalized fur/f to localize to early endosomes in the PACS-1&#x02013;deficient cells was not the result of defective furin endocytosis since after short times of antibody uptake both the control and PACS-1&#x02013;deficient cells showed similar punctate staining patterns (Fig. <xref ref-type="fig" rid="F4">4</xref>, <italic>E</italic> and <italic>G</italic>). Rather, the faint staining pattern in panel B reflects a shared requirement for PACS-1 in the localization of phosphorylated furin both to early endosomes and to the TGN (<xref rid="B81" ref-type="bibr">Wan et al., 1998</xref>). Indeed, mAb M1 uptake in the absence of tautomycin showed that in control cells fur/f is retrieved to the TGN and to a peripheral endosome/lysosome population (Fig. <xref ref-type="fig" rid="F4">4</xref>
<italic>F</italic>). In PACS-1 antisense cells, however, internalized fur/f fails to localize to either the TGN or to the peripheral endosome/lysosome population (Fig. <xref ref-type="fig" rid="F4">4</xref>
<italic>H</italic>). Together, these observations show that the sorting, but not the internalization, of furin requires PACS-1. </p></sec><sec><title>Identification of Furin Phosphatase</title><p>After internalization into early endosomes, phosphorylated furin may either (<italic>a</italic>) undergo a PACS-1&#x02013;dependent recycling to the cell surface (Figs. <xref ref-type="fig" rid="F3">3</xref> and <xref ref-type="fig" rid="F4">4</xref>) or (<italic>b</italic>) be directed to the TGN by a tautomycin-sensitive furin phosphatase (refer to Fig. <xref ref-type="fig" rid="F1">1</xref>) (<xref rid="B32" ref-type="bibr">Jones et al., 1995</xref>). The sensitivity to tautomycin suggested the furin phosphatase is either PP1 or PP2A. Therefore, the ability of both PP1 and PP2A catalytic (C) subunits to dephosphorylate the phosphorylated furin-cd in vitro was determined (Fig. <xref ref-type="fig" rid="F5">5</xref>). Surprisingly, neither phosphatase was able to dephosphorylate the furin-cd, whereas both enzymes efficiently dephosphorylated a control substrate, phosphorylase a. Similar results were obtained for PP2B (data not shown). </p><p>Whereas neither the PP1 nor the PP2A catalytic subunits were active against the phosphorylated furin-cd, both cell extracts and bovine brain cytosol contained a tautomycin- and okadaic acid-sensitive furin phosphatase activity (Fig. <xref ref-type="fig" rid="F6">6</xref>
<italic>A</italic>). Because of the high total protein concentrations required to detect furin phosphatase activity, we were unable to identify the phosphatase by differential sensitivity to these inhibitors. Therefore, an affinity chromatography procedure was used to isolate the furin phosphatase. Cell extract containing furin phosphatase activity was applied to a microcystin (a potent PP1/PP2A inhibitor) affinity column (<xref rid="B47" ref-type="bibr">Moorhead et al., 1994</xref>, <xref rid="B46" ref-type="bibr">1995</xref>). Although microcystin normally covalently binds to both PP1 and PP2A (<xref rid="B39" ref-type="bibr">MacKintosh et al., 1995</xref>), derivatization for coupling to the column matrix blocks the reactive group and changes the reagent to a tight-binding reversible inhibitor. Bound material was then eluted with NaSCN, dialyzed and assayed for phosphatase activity using either the phosphorylated furin-cd or phosphorylase a as substrate (Fig. <xref ref-type="fig" rid="F6">6</xref>
<italic>B</italic>). The microcystin affinity column efficiently bound both the phosphorylase a and furin-cd phosphatase activities from the load sample. However, only the phosphorylase a activity could be recovered from the eluate. The elution conditions were not responsible for the loss of furin-directed activity since treatment of extract with NaSCN followed by dialysis did not inactivate the furin phosphatase. Furthermore, neither the pooling of all eluted fractions from the column before dialysis nor the addition of the column flow-through to the assay was able to restore furin-directed phosphatase activity (data not shown). These results suggested that the depletion of furin phosphatase activity was not simply the result of separating the catalytic moiety from a required cofactor during purification. </p><p>The enhanced total phosphorylase a&#x02013;directed activity recovered in the eluate fractions is consistent with previous reports (<xref rid="B47" ref-type="bibr">Moorhead et al., 1994</xref>, <xref rid="B46" ref-type="bibr">1995</xref>), and reflects the purification of PP1 away from endogenous inhibitors. Interestingly, despite the ability of microcystin to bind and inhibit both PP1 and PP2A, only PP1 isoforms from muscle myofibrils and glycogen particles have been successfully isolated using this affinity technique (<xref rid="B47" ref-type="bibr">Moorhead et al., 1994</xref>, <xref rid="B46" ref-type="bibr">1995</xref>). It is possible, therefore, that exposure of bound PP2A to the high concentrations of chaotropic agent required to dissociate it from the resin leads to an irreversible inhibition of the enzyme. These results, together with those in Fig. <xref ref-type="fig" rid="F5">5</xref>, indicate that the furin phosphatase is indeed a PP1/PP2A-like enzyme (tautomycin-sensitive, binds to the microcystin column). Furthermore, the inability to recover the furin phosphatase activity from the affinity column implicates PP2A.</p></sec><sec><title>Role of PP2A Regulatory Subunits in Determining Specificity for Furin</title><p>Although the PP2A C subunit is active towards numerous substrates in vitro, the predominant form of the enzyme in vivo is a trimeric complex with additional A and B subunits (for reviews see <xref rid="B6" ref-type="bibr">Barford, 1996</xref>; <xref rid="B31" ref-type="bibr">Hubbard and Cohen, 1993</xref>; <xref rid="B42" ref-type="bibr">Mayer-Jaekel and Hemmings, 1994</xref>). The A subunit promotes association of the C subunit with one of a variety of regulatory B subunits. Recent cloning studies reveal several unique families of regulatory subunits with multiple members derived from both separate genes and alternative splicing. Although the role of complex formation was originally envisioned as restricting the otherwise broad substrate specificity of the isolated C subunit, recent studies show that the B subunits can act as positive regulators to enhance catalytic activity toward particular substrates both in vitro and in vivo (<xref rid="B69" ref-type="bibr">Sontag et al., 1996</xref>). Moreover, the distinct subcellular localization of the different B subunits could act to target isoforms of PP2A to particular locations within the cell (<xref rid="B68" ref-type="bibr">Sontag et al., 1995</xref>; <xref rid="B43" ref-type="bibr">McCright et al., 1996</xref>; <xref rid="B53" ref-type="bibr">Okamoto et al., 1996</xref>). Together, these findings suggest a potentially high degree of isoform-specific PP2A characteristics.</p><p>To test the possibility that such isoform-specific composition could modulate the activity of PP2A toward furin, we adopted a strategy of phosphatase isoform reconstitution by baculovirus expression (<xref rid="B34" ref-type="bibr">Kamibayashi et al., 1994</xref>). Sf9 cells were coinfected with baculovirus recombinants expressing combinations of the C, A, and one of the various B subunits (Fig. <xref ref-type="fig" rid="F7">7</xref>
<italic>A</italic>). Furin phosphatase activity was strictly dependent on B subunit composition. Whereas expression of C subunit alone or coexpression of C and A subunits failed to generate increased furin phosphatase activity, coexpression of the C and A subunits with B family regulatory subunits (B&#x003b1; and B&#x003b2;) resulted in a selective increase in furin phosphatase activity (two- to fourfold). Importantly, the furin phosphatase activity was B family&#x02013; specific since coexpression of C and A subunits with a member of the B&#x02032; family, B&#x02032;&#x003b1;, failed to stimulate furin phosphatase activity. Indeed, B&#x02032;&#x003b1; selectively inhibited the basal furin-directed activity. This effect is most likely due to displacement of endogenous Sf9 cell B family regulatory subunits from holoenzyme complexes by the overexpressed B&#x02032;&#x003b1;, which has a higher affinity for the AC complex in vitro (<xref rid="B76" ref-type="bibr">Tehrani et al., 1996</xref>). As expected, both the phosphorylase a and furin phosphatase activities were inhibited completely by submicromolar concentrations of okadaic acid and tautomycin. Furthermore, Western blot analyses of the Sf9 extracts using subunit-specific antisera show that (<italic>a</italic>) the appropriate regulatory subunits were indeed expressed by the recombinant baculoviruses and (<italic>b</italic>) similar levels of catalytic subunit were expressed in each combinatorial infection (Fig. <xref ref-type="fig" rid="F7">7</xref>
<italic>B</italic>). This latter observation indicates that the dramatic changes in furin dephosphorylation are not the result of differential stabilization of the catalytic subunit, but rather reflect the inherent ability of the regulatory subunits to facilitate the recognition of furin by PP2A holoenzyme. </p></sec><sec><title>PP2A Regulates Furin Trafficking In Vivo</title><p>The in vitro data described above clearly implicate PP2A isoforms containing B family subunits in the dephosphorylation of furin. The characterization of SV-40 virus-transforming factors has also provided a mechanism through which to probe the importance of these particular PP2A isoforms in vivo. The small t antigen of SV-40 contributes to the transforming capacity of the large T antigen, at least in part, by disrupting the mitogen-activated protein (MAP) kinase signaling pathway. Recent studies have shown that this effect of small t derives from the ability of the SV-40 protein to displace B family regulatory subunits from PP2A, resulting in a change in PP2A substrate specificity and hyperphosphorylation of several members of the MAP kinase cascade (<xref rid="B67" ref-type="bibr">Sontag et al., 1993</xref>). We used this characteristic of small t antigen to examine directly the importance of PP2A for the correct routing of furin in vivo.</p><p>To test this possibility, replicate plates of BSC-40 cells were coinfected with vaccinia virus recombinants expressing fur/f and either small t or an inactive small t mutant (mut3) lacking the PP2A binding site (<xref rid="B67" ref-type="bibr">Sontag et al., 1993</xref>). The effect of small t expression on the recycling of furin from the cell surface was then assessed in immunofluorescence studies as described above (Fig. <xref ref-type="fig" rid="F8">8</xref>). As seen previously, antibody uptake showed that recycling furin is primarily localized to the TGN/late endosome in control cells (Fig. <xref ref-type="fig" rid="F8">8</xref>
<italic>A</italic>). Coexpression of small t, however, resulted in an accumulation of the internalized furin in peripheral endosome-like structures (Fig. <xref ref-type="fig" rid="F8">8</xref>
<italic>C</italic>) as seen in cells treated with tautomycin (Fig <xref ref-type="fig" rid="F8">8</xref>
<italic>B</italic>). This effect required the PP2A-binding function of small t since the inactive mut3 construct had no discernible effect on furin trafficking (Fig. <xref ref-type="fig" rid="F8">8</xref>
<italic>D</italic>). These results complement the in vitro data described in Fig. <xref ref-type="fig" rid="F7">7</xref> and show that PP2A isoforms containing B family regulatory subunits control the dephosphorylation dependent transfer of furin from early endosomes to the TGN. </p></sec></sec><sec id="Discussion"><title>Discussion</title><p>The mechanisms by which the endosomal system achieves and controls its complex sorting functions are largely unknown. Studies of furin recycling, however, demonstrate several factors that affect sorting within the early endosomal system. Here we report the identification of cellular machinery that directs the phosphorylation state-dependent trafficking of furin through endosomal compartments. Cell surface furin is endocytosed in a clathrin-/dynamin I&#x02013;dependent step by direct interaction of the tyrosine- and dileucine-like internalization motifs with the AP-2 adaptor (refer to Fig. <xref ref-type="fig" rid="F2">2</xref>) (<xref rid="B62" ref-type="bibr">Sch&#x000e4;fer et al., 1995</xref>; <xref rid="B51" ref-type="bibr">Ohno et al., 1996</xref>). After localization to transferrin containing early endosomes (refer to Fig. <xref ref-type="fig" rid="F1">1</xref>), furin undergoes a phosphorylation state-dependent sorting step that is controlled by the activities of CKII, and PP2A isoforms containing B family regulatory subunits (<xref rid="B32" ref-type="bibr">Jones et al., 1995</xref>) (refer to Fig. <xref ref-type="fig" rid="F7">7</xref>). Phosphorylated furin molecules bind the connector protein PACS-1 and are placed in a local cycling loop between the early endosome and the cell surface (refer to Figs. <xref ref-type="fig" rid="F3">3</xref> and <xref ref-type="fig" rid="F4">4</xref>). By contrast, furin molecules dephosphorylated by PP2A are sorted to the TGN (refer to Figs. <xref ref-type="fig" rid="F5">5</xref>&#x02013;<xref ref-type="fig" rid="F8">8</xref>).</p><p>Several findings indicate that phosphorylation/dephosphorylation of the furin-cd by CKII and PP2A directly affect furin trafficking in the TGN/endosomal system (<xref rid="B32" ref-type="bibr">Jones et al., 1995</xref>; <xref rid="B19" ref-type="bibr">Ditti&#x000e9; et al., 1997</xref>; <xref rid="B81" ref-type="bibr">Wan et al., 1998</xref>). For example, the accumulation of furin in early endosomes observed upon treatment of cells with tautomycin (refer to Fig. <xref ref-type="fig" rid="F1">1</xref>) (<xref rid="B32" ref-type="bibr">Jones et al., 1995</xref>) is replicated by the fur/f-DDD point mutant designed to mimic phosphorylation at the CKII site. Furthermore, this effect of tautomycim requires an intact, phosphorylatable CKII site in the furin-cd (<xref rid="B32" ref-type="bibr">Jones et al., 1995</xref>). Similarly, the effect of depletion of the phosphorylation state-dependent furin-binding protein PACS-1 upon trafficking is selective for furin and proteins containing related AC motifs (<xref rid="B81" ref-type="bibr">Wan et al., 1998</xref>). Although these results suggest direct effects on furin sorting, it seems likely that CKII and PP2A also have additional global roles in controlling membrane traffic within the TGN/endosomal system.</p><sec><title>Regulation of Isoform-specific Phosphatase Function</title><p>The regulation of furin trafficking by the combined activities of CKII and PP2A indicates a link between signaling pathways and control of protein localization within the TGN/endosomal system. Together with their known function in cell cycle progression, our studies support a broad role for CKII and PP2A in regulating diverse cellular processes. Although modulation of CKII activity in vivo has not been demonstrated (<xref rid="B2" ref-type="bibr">Allende and Allende, 1995</xref>), PP2A can be regulated in several ways. The importance of holoenzyme composition in generating a furin-directed phosphatase (refer to Fig. <xref ref-type="fig" rid="F7">7</xref>) illustrates the high level of substrate specificity determined by subunit composition. Similarly, the B family is required for the PP2A-catalyzed dephosphorylation of tau both in vitro and in vivo. Although a selective role for B&#x02032; family subunits has not been demonstrated in mammalian cells, deletion of the B&#x02032; homologue in yeast, Rts1, results in a temperature-sensitive growth defect that can be rescued by rabbit B&#x02032; family subunits (<xref rid="B89" ref-type="bibr">Zhao et al., 1997</xref>). Together, these studies show that the PP2A regulatory subunits can act as positive effectors for select substrates.</p><p>Regulatory subunit composition may also modulate PP2A by targeting the phosphatase to select compartments. For example, B&#x02032; subunits differentially partition between the nucleus and cytoplasm (<xref rid="B43" ref-type="bibr">McCright et al., 1996</xref>), whereas B&#x003b1; subunits target a population of PP2A to microtubules where they are positioned for efficient dephosphorylation of tau (<xref rid="B68" ref-type="bibr">Sontag et al., 1995</xref>). The microtubule-associated PP2A also undergoes cell cycle-dependent modulation of its activity, indicating an additional level of regulation. The observation that both the catalytic and regulatory subunits of PP2A are phosphoproteins (<xref rid="B42" ref-type="bibr">Mayer-Jaekel and Hemmings, 1994</xref>; <xref rid="B43" ref-type="bibr">McCright et al., 1996</xref>) introduces the possibility that this second tier of regulation represents rapid, reversible changes in PP2A function linked to second messenger signaling pathways.</p></sec><sec><title>Role of PP2A in Protein Sorting</title><p>The effect of SV-40 small t expression on furin sorting (Fig. <xref ref-type="fig" rid="F8">8</xref>) provides an unequivocal demonstration of PP2A's role in directing trafficking in the endocytic pathway. The selective displacement of B family regulatory subunits by small t offers a sensitive and direct diagnostic tool for delineating the role of PP2A isoforms in vivo. Our data do not, however, exclude a potential contribution by PP1 to the regulation of furin sorting. At limiting concentrations in vitro, PP1 is at least an order of magnitude more sensitive to tautomycin whereas PP2A is more sensitive to okadaic acid (<xref rid="B75" ref-type="bibr">Takai et al., 1995</xref>). Initial studies of the phosphorylation state-dependent sorting of furin, however, showed that, at low concentrations (100 nM), tautomycin affected sorting while similar concentrations of okadaic acid had no effect. Okadaic acid at high concentrations (e.g., 1 &#x003bc;M) not only affected furin trafficking, but also caused a dramatic dispersal of the paranuclear staining, consistent with disruption of the microtubule network and fragmentation of the TGN and Golgi (<xref rid="B37" ref-type="bibr">Lucocq, 1992</xref>; <xref rid="B59" ref-type="bibr">Reaven et al., 1993</xref>; <xref rid="B30" ref-type="bibr">Horn and Banting, 1994</xref>), precluding the use of this inhibitor in evaluating protein sorting. This preferential inhibition of furin trafficking by tautomycin could reflect cell type differences in the permeability of the inhibitors and/or their relative ability to penetrate the cellular compartments associated with PP2A-dependent sorting. Although the empirical determination of effective concentrations by examination of residual PP1 and PP2A activities in treated cells can facilitate the application of inhibitors as diagnostic tools (<xref rid="B21" ref-type="bibr">Favre et al., 1997</xref>), the possibility of resistant or sensitive pools of the enzymes remains a complicating factor.</p></sec><sec><title>TGN and Endosomal Protein Sorting Share Common Machinery</title><p>The requirement of PACS-1 for the sorting of phosphorylated furin in early endosomes points to several commonalties between the early endosome/cell surface furin cycling loop and the phosphorylation- and PACS-1&#x02013;dependent localization of the endoprotease to the TGN (<xref rid="B81" ref-type="bibr">Wan et al., 1998</xref>). In the model shown in Fig. <xref ref-type="fig" rid="F9">9</xref>, the cell surface/early endosome local cycling loop represents a mirror image of a TGN/endosome cycling pathway. As for endocytosis of cell surface furin to early endosomes (<xref rid="B62" ref-type="bibr">Sch&#x000e4;fer et al., 1995</xref>), efficient export of TGN-localized furin to a post-TGN endosomal compartment requires the canonical tyrosine- and/or dileucine-based internalization signals (<xref rid="B81" ref-type="bibr">Wan et al., 1998</xref>). These hydrophobic sorting signals bind directly to the clathrin adaptor AP-2 at the cell surface, whereas budding from the TGN uses AP-1 (<xref rid="B1" ref-type="bibr">Alconada et al., 1996</xref>; <xref rid="B29" ref-type="bibr">Honing et al., 1996</xref>; <xref rid="B51" ref-type="bibr">Ohno et al., 1996</xref>; <xref rid="B81" ref-type="bibr">Wan et al., 1998</xref>). Although furin in the peripheral recycling pathway colocalizes with TfR, demonstrating its presence in early endosomes, the identity of the post-TGN recycling compartment is not established. The recovery of phosphorylated furin from immature secretory granules in neuroendocrine cells (<xref rid="B19" ref-type="bibr">Ditti&#x000e9; et al., 1997</xref>), however, provides an analogy for the TGN recycling loop. PACS-1 directs the retrieval step of both cycling loops by linking phosphorylated furin to the clathrin sorting machinery. Binding assays in vitro show PACS-1 connects the phosphorylated furin-cd to AP-1, which is consistent with a role for this adaptor in the TGN localization of membrane proteins. This finding, however, does not exclude the possibility that other adaptors (e.g., AP-3) may also mediate retrieval. The composition of the adaptor species used in the PACS-1&#x02013;directed recycling between early endosomes and the plasma membrane has yet to be established. Interestingly, the prevalence on endosomes of clathrin-coated buds which contain neither &#x003b1; or &#x003b3; adaptin (<xref rid="B72" ref-type="bibr">Stoorvogel et al., 1996</xref>) indicates that unique adaptor species may mediate the retrieval step within the peripheral cycling loop. </p><p>As reflected in Fig. <xref ref-type="fig" rid="F9">9</xref>, depletion of PACS-1 leads to sorting defects at both the level of endosomal trafficking and TGN localization. This model, however, does not address why nonphosphorylatable furin, fur/f-ADA (which does not bind PACS-1), displays some trafficking characteristics unique from fur/f in PACS-1 antisense cells (compare Fig. <xref ref-type="fig" rid="F4">4</xref> and <xref rid="B81" ref-type="bibr">Wan et al., 1998</xref> with <xref rid="B74" ref-type="bibr">Takahashi et al., 1995</xref> and <xref rid="B32" ref-type="bibr">Jones et al., 1995</xref>). Most notable is the finding that fur/f-ADA mislocalizes from either the TGN or early endosomes to larger punctate membrane structures whereas fur/f in PACS-1 antisense cells shows very fine, dispersed vesicular staining. There are several possible explanations for these results, including (<italic>a</italic>) the presence of additional phosphorylated-furin binding proteins, (<italic>b</italic>) multiple roles for PACS-1 in organizing endosomal sorting compartments, and (<italic>c</italic>) the presence of binding proteins which interact selectively with the nonphosphorylated furin cd but not with fur/f-ADA. This latter possibility implies that nonphosphorylated AC motifs constitute positive sorting signals for trafficking within the TGN/endosomal system.</p></sec><sec><title>Phosphorylation-dependent Sorting Regulates Furin Processing Compartments</title><p>While PACS-1 directs the retrieval of phosphorylated furin in both the TGN and peripheral cycling loops, the transport of furin between these loops requires dephosphorylation by PP2A. This dephosphorylation-dependent trafficking step provides a mechanism by which cells may control the distribution of furin between the biosynthetic and plasma membrane/endosomal processing compartments. Furin at the cell surface is tethered by interaction with the actin binding protein ABP-280 (<xref rid="B36" ref-type="bibr">Liu et al., 1997</xref>). This interaction modulates the rate of furin internalization and may also act to generate processing sites at the cell surface (e.g., bacterial toxin activation) (<xref rid="B26" ref-type="bibr">Gordon et al., 1995</xref>). The cycling of furin between the cell surface and endosomes may reflect a requirement for the formation of peripheral processing compartments in which the enzyme can be concentrated with its substrates. In addition, the internal milieu of the early endosomes (e.g., acidic pH) facilitates the processing of some substrates such as pseudomonas and diphtheria toxins (<xref rid="B26" ref-type="bibr">Gordon et al., 1995</xref>). Thus, due to the highly dynamic and transitory nature of the endosomal system, continuous cycling of phosphorylated furin between the plasma membrane and early endosomes could be the best mechanism for maintaining functional concentrations of the protease within the peripheral processing compartments. Similarly, the TGN cycling loop might function to optimize processing of substrates in the biosynthetic pathway. This concept is consistent with the observation that, in some cells, endogenous furin can be predominantly localized to a post-TGN processing compartment (<xref rid="B61" ref-type="bibr">Sariola et al., 1995</xref>).</p></sec><sec><title>A Broad Role for PACS-1/PP2A in Protein Sorting</title><p>The PACS-1&#x02013;directed trafficking of furin suggests that the localization of additional proteins containing acidic cluster motifs may be regulated via the phosphorylation state-dependent sorting machinery. Indeed, a number of itinerant secretory pathway membrane proteins including the cation-independent (<xref rid="B44" ref-type="bibr">Meresse et al., 1990</xref>) and cation-dependent (<xref rid="B41" ref-type="bibr">Mauxion et al., 1996</xref>) mannose 6 phosphate receptors, carboxypeptidase D (<xref rid="B86" ref-type="bibr">Xin et al., 1997</xref>), and sortilin (<xref rid="B56" ref-type="bibr">Petersen et al., 1997</xref>) have CKII phosphorylatable ACs within their cytosolic domains. In the case of the MPRs this phosphorylation has been linked to the efficient function of the receptor, however, it is not clear how this modification effects trafficking. Interestingly, many herpes virus envelope glycoproteins (e.g., VZV-gE, HSV-1-gE, and HCMV-gB) have phosphorylatable AC sorting motifs on their cds (<xref rid="B20" ref-type="bibr">Edson et al., 1987</xref>; <xref rid="B48" ref-type="bibr">Norais et al., 1996</xref>; <xref rid="B87" ref-type="bibr">Yao et al., 1993</xref>). Recent data indicate that the phosphorylation of these sites can influence trafficking of the envelope proteins (<xref rid="B1" ref-type="bibr">Alconada et al., 1996</xref>; <xref rid="B24" ref-type="bibr">Fish et al., 1998</xref>) and that PACS-1 binds VZV-gE cytosolic domain (<xref rid="B81" ref-type="bibr">Wan et al., 1998</xref>), raising the possibility that the CKII-mediated sorting system is used in viral biogenesis and/or spread.</p><p>Our studies of the regulation of furin sorting between the early endosome and plasma membrane also reveal several parallels with the cellular machinery that controls the phosphorylation-dependent resensitization of G protein&#x02013; coupled receptors. The trafficking of both furin and &#x003b2;AR depend upon (<italic>a</italic>) the phosphorylation state of the cytosolic domain (<xref rid="B23" ref-type="bibr">Ferguson et al., 1995</xref>, <xref rid="B22" ref-type="bibr">1996</xref>), (<italic>b</italic>) the presence of a phosphorylation state-dependent connector protein (either PACS-1 or &#x003b2; arrestin) (<xref rid="B25" ref-type="bibr">Goodman et al., 1996</xref>) that provides a link to the clathrin-sorting machinery, and (c) dephosphorylation by specific endosome-associated PP2A isoforms (<xref rid="B58" ref-type="bibr">Pitcher et al., 1995</xref>).</p><p>In summary, our results reveal the integrated roles of identified components of the endosomal trafficking machinery that direct the phosphorylation state-dependent sorting of furin. The importance of kinase (CKII) and isoform-specific phosphatase (PP2A) activities in regulating protein routing also indicates that these sorting events are likely under the control of second messenger systems. Future studies, therefore, will focus on how this dynamic endosomal sorting system responds to intracellular signaling pathways in order to control the distribution and activity of membrane proteins in vivo.</p></sec></sec></body><back><ack><p>We thank B.G. Jones (University of Sheffield, Sheffield, UK) for contributions to the early part of this study. We also wish thank S.L. Schmid for the TS-Dyn I cell line, A.A. DePaoli-Roach for purified PP1 and PP2A, Wadzinsky for PP2A catalytic subunit antibody, R. Valle (Worcester Foundation, Shrewsbury, MA) for dynamin I and K44E plasmids, and P. Cohen (University of Dundee, Dundee, UK) for helpful discussions. </p><p>This work was supported by National Institutes of Health grants to G. Thomas (DK-44629 and DK-37274).</p></ack><fn-group><fn><p>Address all correspondence to G. Thomas, Vollum Institute, Oregon Health Sciences University, Portland, OR 97201. Tel.: (503) 494-6955. Fax: (503) 494-4534. E-mail: <email>thomasg@ohsu.edu</email>
</p></fn><fn><p>C. Kamibayashi's present address is Simmons Cancer Center, University of Texas Southwestern Medical Center, Dallas, TX 75235.</p></fn></fn-group><glossary><title>Abbreviations used in this paper</title><def-list><def-item><term>AC</term><def><p>acidic cluster</p></def></def-item><def-item><term>cd</term><def><p>cytosolic domain</p></def></def-item><def-item><term>GST</term><def><p>glutathione-S-transferase</p></def></def-item><def-item><term>MC</term><def><p>microcystin</p></def></def-item><def-item><term>m.o.i.</term><def><p>multiplicity of infection</p></def></def-item><def-item><term>PACS-1</term><def><p>phosphofurin acidic cluster-sorting protein</p></def></def-item><def-item><term>PP2A</term><def><p>protein phosphatase 2A</p></def></def-item></def-list></glossary><ref-list><title>References</title><ref id="B1"><element-citation publication-type="journal">
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<lpage>8262</lpage>
<pub-id pub-id-type="pmid">9079645</pub-id></element-citation></ref><ref id="B90"><element-citation publication-type="journal">
<person-group person-group-type="author"><name><surname>Zolnierowicz</surname><given-names>S</given-names></name><name><surname>Van Hoof</surname><given-names>C</given-names></name><name><surname>Andjelkovic</surname><given-names>N</given-names></name><name><surname>Cron</surname><given-names>P</given-names></name><name><surname>Stevens</surname><given-names>I</given-names></name><name><surname>Merlevede</surname><given-names>W</given-names></name><name><surname>Goris</surname><given-names>J</given-names></name><name><surname>Hemmings</surname><given-names>BA</given-names></name></person-group>
<article-title>The variable subunit associated with protein phosphatase 2A0 defines a novel multimember family of regulatory subunits</article-title>
<source>Biochem J</source>
<year>1996</year>
<volume>317</volume>
<fpage>187</fpage>
<lpage>194</lpage>
<pub-id pub-id-type="pmid">8694763</pub-id></element-citation></ref></ref-list></back><floats-group><fig id="F1" position="float"><label>Figure 1</label><caption><p>Phosphorylation-dependent routing of furin in early endosomes (<italic>top</italic>). Epitope-tagged furin showing the FLAG insertion (<italic>cross-hatched</italic>), the catalytic (<italic>shaded</italic>) and transmembrane domains (<italic>stippled</italic>), as well as the sequence of the cd with CKII phosphorylation site substitutions. (<italic>Bottom</italic>) BSC-40 cells were infected with vaccinia recombinants (m.o.i. = 10) expressing either fur/f with the native cytosolic domain (<italic>A</italic> and <italic>B</italic>) or with serine to aspartic acid substitutions within the CKII site (fur/f-DDD, <italic>C&#x02013;E</italic>). At 6 h postinfection, the cells were incubated with mAb M1 (15 &#x003bc;g/ml) and TRITC-transferrin (40 ng/ml) (<italic>E</italic>) for 1 h before fixation and processing for immunofluorescence. The cells in <italic>B</italic> were treated with 100 nM tautomycin during the uptake period. Internalized mAb M1 was visualized using FITC goat anti&#x02013;mouse IgG (<italic>A&#x02013;D</italic>).</p></caption><graphic xlink:href="JCB9806103.f1"/></fig><fig id="F2" position="float"><label>Figure 2</label><caption><p>Internalization of furin is dependent upon dynamin function. BSC-40 cells were infected with vaccinia recombinants (m.o.i. = 5 each) expressing fur/f and either native dynamin I (<italic>A</italic> and <italic>C</italic>) or the dominant-negative dynamin construct K44E (<italic>B</italic> and <italic>D</italic>) in the presence of 10 &#x003bc;M hydroxyurea. At 16 h postinfection, cells were incubated with either mAb M1 (<italic>A</italic> and <italic>B</italic>) or TRITC-transferrin (<italic>C</italic> and <italic>D</italic>) for 1 h in culture before fixation and analysis by immunofluorescence. The extended culture time postinfection was required to allow sufficient expression levels for optimum K44E block.</p></caption><graphic xlink:href="JCB9806103.f2"/></fig><fig id="F3" position="float"><label>Figure 3</label><caption><p>Recycling of phosphorylated furin from endosomes to the cell surface. (<italic>A</italic>) HeLa TS-Dyn I cells were infected with vaccinia recombinants (m.o.i. = 10) expressing fur/f mutants mimicking either nonphosphorylated (fur/f <italic>ADA</italic>) or constitutively phosphorylated furin (fur/f <italic>DDD</italic>). After accumulation at the cell surface during incubation at nonpermissive temperature (37&#x000b0;C) for 6 h, surface proteins were biotinylated (refer to Materials and Methods) and then allowed to internalize for the indicated times at permissive temperature (31&#x000b0;C). The total (<italic>T</italic>) and internalized (<italic>I</italic>) pools of labeled furin were then determined by immunoprecipitation and Western analysis. (<italic>B</italic>) The percent of furin internalized at each time point was quantified by densitometric analysis (data averaged from four independent experiments, error bars = SEM). (<italic>Inset</italic>) Double-strip analyses were conducted in which the pool of internalized furin following an initial 20-min chase period was assessed for reexpression at the cell surface. After a second 20-min chase, total biotinylated furin was compared with surface localized furin using a second MesNa strip (&#x02212; and +, respectively).</p></caption><graphic xlink:href="JCB9806103.f3"/></fig><fig id="F4" position="float"><label>Figure 4</label><caption><p>Endocytic sorting of furin requires the phosphorylated cd binding protein PACS-1. Fur/f was expressed in control cells (<italic>A</italic>, <italic>B</italic>, <italic>E</italic>, and <italic>F</italic>) and PACS-1&#x02013;deficient antisense cells (<italic>C</italic>, <italic>D</italic>, <italic>G</italic>, and <italic>H</italic>) using vaccinia recombinants (m.o.i. = 10). At 4 h postinfection, the cells were exposed to mAb M1 as a marker for endocytosed furin and TRITC-labeled transferrin (<italic>B</italic> and <italic>D</italic>) for either 1 h (<italic>A&#x02013;D</italic>, <italic>F</italic>, and <italic>H</italic>) or 10 min. (<italic>E</italic> and <italic>G</italic>) before fixation and processing for immunofluorescence microscopy. The cells in <italic>A&#x02013;D</italic> were exposed to 100 nM tautomycin during the 1-h incubation period with mAb M1 and transferrin. Internalized mAb M1 was visualized using FITC-conjugated goat anti&#x02013;mouse IgG2b (<italic>A</italic>, <italic>C</italic>, and <italic>E&#x02013;H</italic>).</p></caption><graphic xlink:href="JCB9806103.f4"/></fig><fig id="F5" position="float"><label>Figure 5</label><caption><p>Dephosphorylation of furin by purified phosphatase. PP1 and PP2A catalytic subunits were incubated with either <sup>32</sup>P-labeled GST-Fur<sub>cd</sub> fusion protein (<italic>GST&#x02013;furin<sub>cd</sub></italic>) phosphorylated with CKII, or a <sup>32</sup>P-labeled control substrate (phosphorylase a, <italic>Phos. a</italic>) for the indicated times (in min). Aliquots from the dephosphorylation reactions were then separated by SDS-PAGE and analyzed by autoradiography. Parallel experiments analyzed by quantitative filter paper assays also indicated no measurable dephosphorylation of the furin-cd by either enzyme (data not shown).</p></caption><graphic xlink:href="JCB9806103.f5"/></fig><fig id="F6" position="float"><label>Figure 6</label><caption><p>Endogenous furin-directed phosphatase activity. (<italic>A</italic>) Extracts from bovine brain (shown) and cultured cells were incubated with <sup>32</sup>P-labeled GST&#x02013;Fur<sub>cd</sub> in vitro in the absence (<italic>Control</italic>) or presence of 100 nM tautomycin for the indicated times (in min). Aliquots of the reactions were separated by SDS-PAGE and analyzed by autoradiography. (<italic>B</italic>) Affinity chromatography of endogenous furin-directed phosphatase activity in BSC-40 cells. Cell extract was applied to a microcystin affinity column and the bound phosphatase eluted with 3M NaSCN. Aliquots of the original extract (<italic>Load</italic>), the unbound protein (<italic>FT</italic>), and eluted material (<italic>E1&#x02013;3</italic>) were assayed for total PP1/PP2A activity using <sup>32</sup>P-labeled phosphorylase a (<italic>open bars</italic>) and furin-directed phosphatase activity using <sup>32</sup>P-labeled GST&#x02013;Fur<sub>cd</sub> (<italic>hatched bars</italic>). The total activities were calculated based on fraction volume and expressed as pmol/min. Assays were performed in triplicate with relative error (one standard deviation) of less than 5%. Similar results were obtained in several independent experiments.</p></caption><graphic xlink:href="JCB9806103.f6"/></fig><fig id="F7" position="float"><label>Figure 7</label><caption><p>Isoform-specific dephosphorylation of furin by PP2A. (<italic>A</italic>) Baculovirus recombinants (m.o.i. = 2) were used to express PP2A subunits, either alone or in combination, in Sf9 cells. At 64&#x02013;72 h postinfection, cells were harvested and lysates were assayed for phosphatase activity with both <sup>32</sup>P-labeled phosphorylase a (<italic>open bars</italic>) and phosphorylated <sup>32</sup>P-labeled GST&#x02013;Fur<sub>cd</sub> (<italic>hatched bars</italic>). Noninfected Sf9 cells (data not shown) expressed a detectable level of endogenous furin-directed phosphatase activity which was not increased by expression of the catalytic subunit either alone or in combination with the A subunit (<italic>C</italic>, and <italic>A</italic>+<italic>C</italic>, respectively). The C and A subunits were also expressed with either the &#x003b1; or &#x003b2; isoforms of the B family of regulatory subunits (<italic>A</italic>+<italic>C</italic>+<italic>B&#x003b1;</italic> and <italic>A</italic>+<italic>C</italic>+<italic>B&#x003b2;</italic>, respectively), as well as the &#x003b1; subunit from the unrelated B&#x02032; family (<italic>A</italic>+<italic>C</italic>+<italic>B&#x02032;&#x003b1;</italic>). As a control, lysates from <italic>A</italic>+<italic>C</italic>+<italic>B&#x003b2;&#x02013;</italic>expressing cells were exposed to 100 nM tautomycin and okadaic acid (+<italic>Inhib.</italic>) during the phosphatase assays. Assays were performed in triplicate with relative error &#x0003c;5%. The data shown are representative of several independent experiments. (<italic>B</italic>) Western analysis of PP2A subunit expression. Lysates of Sf9 cells infected with baculovirus recombinants encoding the indicated PP2A subunits were resolved by SDS-PAGE and screened by Western blot using antisera specific for the catalytic subunit or the B&#x003b1;, B&#x003b2;, or B&#x02032;&#x003b1; regulatory subunits.</p></caption><graphic xlink:href="JCB9806103.f7"/></fig><fig id="F8" position="float"><label>Figure 8</label><caption><p>Disruption of endogenous PP2A affects furin trafficking in vivo. BSC-40 cells were infected with vaccinia recombinants (m.o.i. = 5 each) expressing fur/f alone (<italic>A</italic> and <italic>B</italic>) or in combination with viruses expressing either SV-40 small t (<italic>C</italic>) or truncated and inactive small t mut3. At 6 h (<italic>D</italic>) postinfection, the cells were incubated with mAb M1 for 1 h in culture before fixation. The cells in <italic>B</italic> were incubated in the presence of 100 nM tautomycin during mAb M1 uptake The cells were then processed for immunofluorescence to localize internalized furin.</p></caption><graphic xlink:href="JCB9806103.f8"/></fig><fig id="F9" position="float"><label>Figure 9</label><caption><p>Model of furin trafficking in the endosomal system. Furin molecules are internalized via dynamin-sensitive, clathrin-dependent endocytosis. Within early endosomes shared by TfR, furin phosphorylated by CKII is directed toward a cell surface recycling pathway by virtue of its selective interaction with PACS-1, which links phosphorylated furin to components of the clathrin sorting machinery (<xref rid="B81" ref-type="bibr">Wan et al., 1998</xref>). This endosome to plasma membrane recycling pathway mirrors the local cycling loop which localizes furin to the TGN (<xref rid="B81" ref-type="bibr">Wan et al., 1998</xref>). Furin molecules dephosphorylated by PP2A isoforms containing B regulatory subunits are sorted from the plasma membrane/early endosome cycling loop to a TGN retrieval pathway. Black shading of either the tyrosine or AC motifs denotes their &#x0201c;active&#x0201d; state whereas gray shading reflects a &#x0201c;silencing&#x0201d; of these signals.</p></caption><graphic xlink:href="JCB9806103.f9"/></fig></floats-group></article>