N-Cadherin Is an In Vivo Substrate for Protein Tyrosine Phosphatase Sigma (PTP{sigma}) and Participates in PTP{sigma}-Mediated Inhibition of Axon Growth

Protein tyrosine phosphatase sigma (PTP ${sigma}$ ) belongs to the LARfamily of receptor tyrosine phosphatases and was previouslyshown to negatively regulate axon growth. The substrate forPTP ${sigma}$ and the effector(s) mediating this inhibitory effect wereunknown. Here we report the identification of N-cadherin asan in vivo substrate for PTP ${sigma}$ . Using brain lysates from PTP ${sigma}$ knockoutmice, in combination with substrate trapping, we identifieda hyper-tyrosine-phosphorylated protein of $~$ 120 kDa in the knockoutanimals (relative to sibling controls), which was identifiedby mass spectrometry and immunoblotting as N-cadherin. ß-Cateninalso precipitated in the complex and was also a substrate forPTP ${sigma}$ . Dorsal root ganglion (DRG) neurons, which highly expressendogenous N-cadherin and PTP ${sigma}$ , exhibited a faster growth ratein the knockout mice than in the sibling controls when grownon laminin or N-cadherin substrata. However, when N-cadherinfunction was disrupted by an inhibitory peptide or loweringcalcium concentrations, the differential growth rate betweenthe knockout and sibling control mice was greatly diminished.These results suggest that the elevated tyrosine phosphorylationof N-cadherin in the PTP ${sigma}$ ^–/– mice likely disruptedN-cadherin function, resulting in accelerated DRG nerve growth.We conclude that N-cadherin is a physiological substrate forPTP ${sigma}$ and that N-cadherin (and likely ß-catenin) participatesin PTP ${sigma}$ -mediated inhibition of axon growth.

INTRODUCTION

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INTRODUCTION

Protein tyrosine phosphatase sigma (PTP ${sigma}$ ) (also known as LAR-PTP2,PTP-P1, CRYP ${alpha}$ , PTP-NU3, PTP-NE3, and CPTP1 [42, 51, 58, 68, 71,81, 84]) belongs to the LAR family of receptor PTPs (class IIa)(3, 4, 13). In mammals, this family includes LAR, PTP ${sigma}$ , and PTP ${delta}$ ,whereas DLAR is the Drosophila melanogaster orthologue of allthree family members (13, 61, 62). In this class (IIa) of PTPs,the ectodomain is comprised of three immunoglobulin (Ig) domainsand usually four or eight FNIII repeats, resembling cell adhesionmolecules (CAMs) such as N-CAM and L1, a single pass transmembranedomain, and two tandem intracellular catalytic domains, thefirst of which (D1) is active and the second of which (D2) isinactive (13). Like other CAMs, LAR family PTPs have been demonstratedto play an important role in nervous system development (reviewedin references 13, 22, and 59).

In flies, DLAR and its relative PTP69D play a key role in axonguidance of motoneurons and axon pathfinding in the visual system(14, 17-19, 30, 34). Likewise, in the leech, HmLAR2 was shownto concentrate in the growth cone and direct growth of its processes(6, 26). Moreover, retinal ganglion outgrowth and growth coneintegrity in developing Xenopus laevis and avian embryos requirea functional PTP ${sigma}$ (CRYP ${alpha}$ ) (27, 31, 38, 47, 60). PTP ${delta}$ is also expressedin Xenopus retinal ganglion cells during axonogenesis (27),and it acts as an attractive cue for growth cone steering andfor driving axonal growth of forebrain neurons (63, 73).

In mammals, PTP ${sigma}$ is expressed primarily in the nervous systemand in select epithelia, and its expression is developmentallyregulated, generally declining with gestational age (29, 36,52, 71, 72). In view of this developmentally dependent expression,we (69) and others (21) previously generated PTP ${sigma}$ knockout mice.The PTP ${sigma}$ knockout animals display neuroendocrine and neuronaldefects, with severe reduction of growth hormone and prolactinproduction by the pituitary (8, 21), as well as central andperipheral nervous system (PNS) abnormalities (35, 36, 65).We previously showed that, in the central nervous system, knockoutof PTP ${sigma}$ causes hippocampal dysgenesis, reduction in thicknessof the corpus callosum and cerebral cortex, and spinal cordabnormalities (36). In the PNS, we demonstrated delayed myelinationand nerve conduction velocity in the developing sciatic nerveof newborn PTP ${sigma}$ knockout mice (69). Moreover, our analysis ofnerve regeneration of adult sciatic nerve following crush injury,nerve transection and immediate repair, or reciprocal allograftingbetween the knockout and sibling sciatic nerve revealed acceleratednerve regeneration and axon guidance errors in the PTP ${sigma}$ knockoutanimals (35). Enhanced axon outgrowth was also shown for theregenerating facial nerves and retinal ganglion cells of PTP ${sigma}$ knockout mice after injury (53, 65). These findings demonstratethat PTP ${sigma}$ is important for axon guidance in vivo in mammals andthat it is inhibitory to the rate of nerve growth. How PTP ${sigma}$ transmitsits inhibitory signal is unknown, as its substrate(s) and downstreamsignaling pathway(s) have not been identified.

By use of our PTP ${sigma}$ knockout mice in combination with substratetrapping and mass spectrometry, we report here the identificationof N-cadherin as an in vivo substrate for PTP ${sigma}$ and show thatN-cadherin (likely in collaboration with ß-catenin)participates in the downregulation of axon growth by PTP ${sigma}$ .

MATERIALS AND METHODS

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MATERIALS AND METHODS

Animals. PTP ${sigma}$ knockout mice were generated (on a C57BL/6/129 background)and bred as described previously (69) The PTP ${sigma}$ ^+/– miceare phenotypically indistinguishable from the wild-type mice.The PTP ${sigma}$ ^–/– mice were divided into three cohorts:60% died within 48 h after birth (cohort 1), $~$ 38% survived until2 to 3 weeks of age and most then died due to a wasting syndrome(cohort 2), and the remaining 2.5% survived to adulthood butwere roughly 25 to 50% smaller than the wild-type siblings byweight (cohort 3). Mice from the second cohort were used forall experiments.

Generation of PTP ${sigma}$ constructs. Rat PTP ${sigma}$ cDNA (GenBank accession number L11587) was obtainedfrom B. Goldstein. Flag-tagged full-length PTP ${sigma}$ , the first catalyticdomain (D1), or the tandem first and second catalytic domains(D1D2), were cloned into the pIRES mammalian expression vector.

Generation of GST-PTP ${sigma}$ fusion proteins. The sequences corresponding to the D1 domain (GenBank accessionnumber NP_035348, amino acids 1282 to 1607) and the D1D2 domain(amino acids 1282 to 1904) of PTP ${sigma}$ were subcloned into pGEX-2T(Amersham). The substrate trapping mutant with an Asp-to-Ala(D1472A) mutation was made by site-directed mutagenesis (Stratagene)using PCR and verified by sequencing. Glutathione S-transferase(GST) fusion proteins of PTP ${sigma}$ -D1 and PTP ${sigma}$ -D1D2 [GST- ${sigma}$ D1(WT), GST- ${sigma}$ D1(DA),GST- ${sigma}$ D1D2(WT), and GST- ${sigma}$ D1(DA)D2] (where WT is wild type and DAis an Asp-to-Ala substitution) were generated according to standardprocedure (e.g., see reference 70).

Generation of soluble N-cadherin-Fc chimeric protein. The protocol for generation of soluble N-cadherin-Fc chimericprotein was adapted from reference 67. Briefly, COS-7 cellswere plated on 150-mm tissue cultures dishes and allowed togrow until $~$ 70% confluent. They were then transfected with aconstruct expressing the extracellular domain of chicken N-cadherinfused to the Fc region of human IgG (Ncad-Fc) in the pIG1 vector(kind gift of P. Doherty), as described previously (67). Fortransfections, 150 µg of Ncad-Fc cDNA was mixed with 2.5ml of 5 mM chloroquine diphosphate and then 2.5 ml of 20 µg/mlDEAE dextran. This mix was diluted into 95 ml Dulbecco's modifiedEagle's medium (DMEM), and 10 ml was transferred into each ofthe dishes with COS-7 cells (for 4 h). The solution was aspirated,and the cells were osmotic shocked with 10 ml of 10% dimethylsulfoxide in phosphate-buffered saline (PBS) for 2 to 3 min.Cells were then washed with DMEM to remove the dimethyl sulfoxideand left overnight in DMEM plus 10% fetal bovine serum (FBS).One day after transfection, the medium was replaced with freshDMEM (plus 1% FBS) and the Ncad-Fc chimera was allowed to accumulatein the medium for 7 days. To purify the protein, the mediumwas collected, centrifuged to remove cell debris, and mixedfor 2 days with 0.25 g of protein A-Sepharose (Sigma). The beadswith bound Ncad-Fc were loaded onto a column and washed with30 ml PBS. Glycine-HCl, pH 2.7 (100 mM), was used to cleaveoff the Ncad-Fc. Fractions (0.5 ml) were collected into tubescontaining 50 µl 1 M Tris-HCl, pH 9.0, to neutralize thesolution, and absorbance at 280 nm was used to identify theNcad-Fc-containing fractions.

Preparation of mouse brain lysate. Whole brains were dissected from 2- to 3-week-old mice, quick-frozenin liquid nitrogen, and stored at –80°C. Brain tissueswere homogenized in ice-cold lysis buffer (150 mM NaCl, 50 mMHEPES, 1% Triton X-100, 10% glycerol, 1.5 mM MgCl₂, 1.0 mM EGTA,protease inhibitors [1 mM phenylmethylsulfonyl fluoride, 10µg/ml each of aprotinin, leupeptin, and pepstatin A])supplemented with 1 mM sodium orthovanadate and 5 mM iodoaceticacid (buffer A) to prevent tyrosine dephosphorylation of proteins.Any unreacted iodoacetic acid was inactivated with 10 mM dithiothreitol.Lysates were centrifuged at 14,000 rpm for 10 min at 4°Cto remove insoluble debris. Protein concentrations in the clearedlysates were determined using the Bradford assay (Bio-Rad).

Cell culture, transfection, and preparation of cell lysates. HEK293T cells were cultured in DMEM plus 10% FBS. Transienttransfection of N-cadherin and PTP ${sigma}$ into HEK293T cells was performedusing the calcium phosphate method. Prior to lysis, cells weretreated with 1 mM pervanadate (fresh solution of H₂O₂ and sodiumorthovanadate) for 10 min. After being washed with ice-coldPBS, cells were lysed in ice-cold buffer A and precleared bycentrifugation at 14,000 rpm for 10 min at 4°C.

Substrate trapping assays. Two milligrams of brain lysates from PTP ${sigma}$ ^–/– or siblingcontrol (PTP ${sigma}$ ^+/+ or PTP ${sigma}$ ^+/–) mice was incubated with $~$ 20µg each of GST, GST-PTP ${sigma}$ D1(WT), and GST-PTP ${sigma}$ D1(DA) fusionproteins for 1 to 2 h at 4°C. For the vanadate competitionassay, the GST and GST-PTP ${sigma}$ D1 fusion proteins were first incubatedin 10 mM sodium orthovanadate for 1 h prior to incubation withlysates. Precipitates (pull-downs) were washed twice each withlysis buffer and HNTG (20 mM HEPES, pH 7.5, 150 mM NaCl, 10%glycerol, 0.1% Triton X-100), and proteins were boiled in samplebuffer, separated by sodium dodecyl sulfate-polyacrylamide gelelectrophoresis (SDS-PAGE), and either immunoblotted with antiphosphotyrosine(anti-pTyr) antibody (PY99; Santa Cruz) or excised and trypsindigested for mass spectrometry. Mass spectrometry analysis wascarried out by MDS Proteomics, as described previously (23).

IPs and Western blot analysis. For immunoprecipitations (IPs) with anti-pTyr antibody, 20 µlof the antibody-conjugated agarose was mixed with 1 to 2 mglysates in lysis buffer for 1 h at 4°C. Beads were thenwashed twice each in lysis buffer and HNTG and eluted in samplebuffer. For IPs with monoclonal N-cadherin and ß-cateninantibodies (obtained from BD), 2 µg antibodies was incubatedwith 20 µl protein G-agarose and lysates as describedabove. Proteins were then separated by SDS-PAGE, transferredto a nitrocellulose membrane, and immunoblotted with anti-pTyr(1:2,000, blocked in 5% bovine serum albumin [BSA] in Tris-bufferedsaline), anti-N-cadherin (1:2,000), or anti-ß-catenin(1:1,500) antibodies, with the last two blocked in 5% nonfatdry milk in Tris-buffered saline. Blots were incubated withanti-mouse horseradish peroxidase-conjugated antibody (1:20,000;Calbiochem) and visualized by ECL-Plus (PerkinElmer). For IPswith boiled lysates, prior to the IP, 2.5 mg of brain lysateswas boiled in 6% SDS (for 10 min) to dissociate interactingproteins. Boiled lysates diluted in lysis buffer (<0.2% SDS,final) were then added to 20 µl of agarose-conjugatedanti-pTyr antibodies and mixed overnight (for $~$ 18 h) at 4°C.

In vitro dephosphorylation assay. N-cadherin and ß-catenin were immunoprecipitated fromlysates of HEK293T cells overexpressing N-cadherin, which werepretreated with 1 mM pervanadate to prevent Tyr dephosphorylationof N-cadherin and ß-catenin in the cells. Followingextensive washes of the immunoprecipitates, half of the immunoprecipitateswere incubated with $~$ 5 µg of active GST-PTP ${sigma}$ D1 in dephosphorylationbuffer [100 mM 2-(N-morpholino)ethanesulfonic acid (MES), pH5.5, 10 mM dithiothreitol, 150 mM NaCl, 2 mM EDTA] for 1 h atroom temperature. The reaction was stopped by adding samplebuffer. Proteins were then separated by SDS-PAGE, and the tyrosinephosphorylation states of the proteins were analyzed by immunoblottingwith anti-pTyr antibodies.

Primary cultures of DRG. Cervical, thoracic, and lumbar dorsal root ganglia (DRG) wereisolated from 3- to 4-day-old PTP ${sigma}$ ^–/– and siblingcontrol PTP ${sigma}$ ^+/+ or PTP ${sigma}$ ^+/– mice. Isolated DRG were incubatedin 0.25% trypsin (Gibco) in calcium- and magnesium-free Puck'ssaline G, pH 7.4 (140 mM NaCl, 5.4 mM KCl, 1 mM Na₂HPO₄ ·7H₂O, 1 mM KH₂PO₄, and 3.2 mM glucose), for 30 min at 37°C.Trypsin was then carefully aspirated, and the DRG were incubatedwith 0.2% collagenase (Worthington) in saline G (with 0.1 mMCa²⁺ and 0.6 mM Mg²⁺) for 10 min at 37°C. After collagenaseremoval, the DRG were resuspended in 1 ml neural medium (F-12:MEM[where MEM is minimal essential medium] [Gibco]) supplementedwith 10 mM HEPES, pH 7.3, 0.6% glucose, 25 ng/ml nerve growthfactor (Roche), 2x N2 solution (Gibco), 110 µg/ml pyruvicacid, and 1 mg/ml ovalbumin (Sigma). For the low-calcium experiments,the MEM was replaced with S-MEM (Gibco), which contains no calcium(final calcium concentration was 0.14 mM, compared to 1.05 mMin the regular neural medium). Next, the DRG explants were gentlydissociated by trituration with a glass Pasteur pipette (roughly50 to 100 passages). Cells were then seeded at a concentrationof $~$ 5,000 to 7,000 cells per well on six-well plates. For theN-cadherin blocking experiments, 0.1 mg/ml of inhibitory peptide(see below) was added to the neural medium during cell plating.Cells were plated on either N-cadherin-coated or poly-D-lysineplus laminin (PDLL)-coated dishes. Preparation of the N-cadherin-coatedplates was performed as described elsewhere (67). Briefly, 10µg/ml anti-human rabbit IgG, Fc specific (Jackson), inPBS was first used to coat the plates, followed by additionof 1% BSA to block nonspecific binding, PBS washes, and additionof 20 µg/ml Ncad-Fc (4°C, overnight). Ncad-Fc wasrinsed off with F-12:MEM prior to adding the cells. For PDLL-coatedplates, 10 µg/ml poly-D-lysine (Sigma) in water was addedto the plates overnight (room temperature). The next day, theplates were rinsed with water and allowed to dry before additionof 10 µg/ml laminin (Invitrogen) in PBS for 4 h. Priorto plating cells, laminin was removed and plates rinsed withF-12:MEM.

N-cadherin blocking peptides. The linear peptide N-acetyl-INPISGQ-NH₂, corresponding to aminoacids 212 to 218 in the N-cadherin sequence (GenBank accessionno. BAA23549), has previously been shown to effectively inhibitN-cadherin function (76). This peptide was generated at theAdvanced Protein Technology Centre of the Hospital for SickChildren (Toronto, Ontario, Canada).

Quantification of neurite outgrowth. Growth of DRG cells was monitored with an inverted microscope(Leica). Neurons were randomly chosen, and images were capturedusing a charge-coupled-device camera (QImaging) with OpenLabsoftware (Improvision). To assess general neurite growth, thenumbers of cells with and without neurites extending from theircell body were counted. A cell was counted positive if it hadneurites greater than twice its body length.

Quantification of SC-axon alignment. Schwann cells (SCs) were scored to align with axons when atleast two of their processes overlapped with neurites. The resultsare expressed as percentages of SCs aligned with axons.

LacZ staining. DRG cultured cells were washed twice with PBS, fixed with 0.5%glutaraldehyde (Sigma) for 15 min (room temperature), washedwith PBS (3x), and stained with X-Gal solution (5 mM ferricyanide,5 mM ferrocyanide, 2 mM MgCl₂, 1.0 mg/ml X-Gal [5-bromo-4-chloro-3-indolyl-ß-D-galactopyranoside]in PBS) for 8 h (37°C) in the dark. The reaction was stoppedby rinsing the cells with PBS.

RESULTS

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RESULTS

Hyper-tyrosine-phosphorylated proteins in brain lysates of PTP ${sigma}$ knockout mice. To identify potential in vivo substrates for PTP ${sigma}$ , we searchedfor proteins that are hyperphosphorylated on tyrosine residuesin the PTP ${sigma}$ knockout mice; these proteins are likely substratesof PTP ${sigma}$ because they cannot be dephosphorylated in its absence.Figure 1 demonstrates the presence of several such hyper-Tyr-phosphorylatedproteins/bands in brain lysates (which highly express PTP ${sigma}$ ) obtainedfrom the PTP ${sigma}$ knockout mice relative to sibling controls (PTP ${sigma}$ ^+/+or PTP ${sigma}$ ^+/–). PTP ${sigma}$ ^+/– mice do not show any adversephenotype and are identical to the PTP ${sigma}$ ^+/+ animals in all aspectsstudied (8, 35, 69). Substrate trapping (24) with a catalyticallyinactive first phosphatase domain of PTP ${sigma}$ [PTP ${sigma}$ -D1(DA)] was subsequentlycarried out to capture these hyper-Tyr-phosphorylated proteinsfrom brain lysates of the PTP ${sigma}$ knockout animals. The band at $~$ 120 kDa was cut out, trypsin digested, and analyzed by tandemmass spectrometry by MDS Proteomics, as detailed elsewhere (23).The sequence identified was that of N-cadherin.

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FIG. 1. Brain lysates from PTP ${sigma}$ ^–/– mice contain several hyper-tyrosine-phosphorylated proteins. Equal amounts of protein from brain lysates of PTP ${sigma}$ knockout (–/–) mice and their sibling (Sib) controls (PTP ${sigma}$ ^+/+ and PTP ${sigma}$ ^+/–) were either separated by SDS-PAGE and immunoblotted with anti-pTyr antibodies or immunoprecipitated with anti-pTyr antibodies prior to immunoblotting with the same antibodies. The arrow indicates the protein band ( $~$ 120 kDa) that was cut out for further analysis by mass spectrometry and identified as N-cadherin.

N-cadherin is a substrate of PTP ${sigma}$ . To validate the mass spectrometry identification, we testedsubstrate trapping of N-cadherin with PTP ${sigma}$ -D1(DA). PTP ${sigma}$ -D1(DA)or PTP ${sigma}$ -D1 (WT control) was incubated with brain lysates fromthe PTP ${sigma}$ ^–/– mice or sibling controls, and the precipitatedproteins were immunoblotted with anti-N-cadherin antibodies.As seen in Fig. 2A and B, PTP ${sigma}$ -D1(DA) was able to trap N-cadherinfrom brain lysates of the PTP ${sigma}$ knockout animals (Fig. 2B) butnot from lysates of the sibling controls (Fig. 2A). Moreover,the control PTP ${sigma}$ -D1(WT) fusion protein was able to trap onlya small amount of N-cadherin relative to the amount trappedby the catalytically inactive PTP ${sigma}$ -D1(DA) fusion protein (Fig.2B, right panel). As expected, substrate (N-cadherin) trappingwas impaired in the presence of vanadate (Fig. 2D). To verifythat the N-cadherin trapped is Tyr phosphorylated, Tyr-phosphorylatedproteins from brain lysates of the PTP ${sigma}$ knockout mice or theirsibling controls were immunoprecipitated with anti-pTyr antibodiesand the precipitate immunoblotted with N-cadherin antibodies.Figure 2C demonstrates that Tyr-phosphorylated N-cadherin wasindeed precipitated from the PTP ${sigma}$ knockout animals but not fromthe sibling controls. In support of the substrate trapping experimentswith brain lysates, we were also able to trap N-cadherin expressedin HEK293T cells with PTP ${sigma}$ -D1(DA), as well as with PTP ${sigma}$ -D1(DA)D2(i.e., when both intracellular catalytic domains are used fortrapping) (Fig. 2E). Together, these results suggest that tyrosine-phosphorylatedN-cadherin binds to and is an in vivo substrate for PTP ${sigma}$ .

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FIG. 2. N-cadherin is an in vivo and an in vitro substrate of PTP ${sigma}$ . (A and B) Immobilized PTP ${sigma}$ substrate trapping mutant GST- ${sigma}$ D1(DA) and the corresponding wild type, GST- ${sigma}$ D1(WT), as well as GST alone, were incubated with brain lysates from (A) sibling (Sib) controls or (B) PTP ${sigma}$ knockout (–/–) mice. Precipitated proteins were then separated by SDS-PAGE and immunoblotted with anti-pTyr or anti-N-cadherin (anti-N-cad) antibodies, as indicated. (C) Tyrosine-phosphorylated N-cad is detected in brain lysates of PTP ${sigma}$ knockout mice but not sibling controls. Brain lysates from PTP ${sigma}$ knockout mice or sibling controls were immunoprecipitated with anti-pTyr antibodies and immunoblotted with anti-N-cad or anti-pTyr antibodies. (D) Vanadate (VO₄) reduces trapping of N-cad. Brain lysates from PTP ${sigma}$ ^–/– mice were incubated with the indicated trapping constructs in the presence or absence of the PTP inhibitor vanadate (10 mM), and precipitated proteins were separated by SDS-PAGE and immunoblotted with anti-N-cad antibodies. (E) N-cad is trapped with catalytically inactive first or both PTPase domains of PTP ${sigma}$ . HEK293T cells transfected with N-cad were lysed, lysates were incubated with the GST fusion protein, active (WT) or inactive (DA mutant), of the first (D1) or both (D1D2) catalytic domains of PTP ${sigma}$ , and precipitated proteins were immunoblotted for N-cad. Since the second domain (D2) is naturally inactive, only the first domain (D1) was inactivated using the DA mutation [GST- ${sigma}$ D1(DA) and GST ${sigma}$ D1(DA)D2 represent the inactive first or both catalytic domains, respectively]. For panels A, B, D, and E, the lower parts of the panels depict Ponceau S staining of the blots, shown to verify that equal amounts of substrate trapping proteins were used for precipitating N-cad. (F) Ectopic expression of PTP ${sigma}$ in cells leads to dephosphorylation of endogenous N-cad. Flag-tagged PTP ${sigma}$ (full length, D1, or D1D2; all WT) were transfected into HEK293T cells, the cells were lysed, lysates were incubated with anti-pTyr antibodies, and precipitated proteins were immunoblotted with anti-N-cad antibodies to detect the extent of N-cad dephosphorylation. The lower panel shows the levels of expression of the indicated Flag-tagged proteins. tfxn, transfection. (G) PTP ${sigma}$ can directly dephosphorylate N-cad. N-cad overexpressed in HEK293T cells was immunopurified with N-cad antibodies and incubated (or not) with purified, GST-tagged PTP ${sigma}$ D1(WT). Proteins were then immunoblotted with anti-pTyr antibodies to analyze the extent of N-cad dephosphorylation or with anti-N-cad antibodies to demonstrate equal amounts of N-cad precipitated in the experiment.

To further verify that PTP ${sigma}$ can directly dephosphorylate N-cadherin,we transfected PTP ${sigma}$ into HEK293T cells (full length, the firstcatalytic domain [D1, which is active], or both domains [D1D2])and tested for the ability to dephosphorylate endogenous N-cadherin.As seen in Fig. 2F, ectopic expression of PTP ${sigma}$ (or its intracellularcatalytic domains) in these cells led to a decrease in Tyr phosphorylationof N-cadherin. Furthermore, the addition of purified PTP ${sigma}$ -D1domain to immunopurified and Tyr-phosphorylated N-cadherin ledto dephosphorylation of N-cadherin in vitro (Fig. 2G), suggestingthat N-cadherin is a direct substrate of PTP ${sigma}$ . Collectively,these data suggest that PTP ${sigma}$ can dephosphorylate N-cadherin invitro and in vivo.

ß-Catenin is also a substrate of PTP ${sigma}$ . The intracellular region of N-cadherin (and other cadherins)is known to bind to ß-catenin, itself associated with ${alpha}$ -catenin, which binds actin (1, 15). This allows a connectionbetween N-cadherin and the actin cytoskeleton, promoting regulationof cell adhesion and motility by the cadherin-catenin complex.We first verified that N-cadherin and ß-catenin caninteract with each other by use of coimmunoprecipitation assays,as shown in Fig. 3A. Since the antibodies used in this experimentcannot distinguish between phosphorylated and nonphosphorylatedspecies of N-cadherin and ß-catenin, it is likelythat these proteins bind each other in the nonphosphorylatedstate, as previously suggested (7, 10, 45, 50).

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FIG. 3. ß-Catenin binds N-cadherin (N-cad) in vivo and is also a substrate for PTP ${sigma}$ . (A) Coimmunoprecipitation of N-cad with ß-catenin (ß-cat). Brain lysates from sibling (Sib) control or PTP ${sigma}$ ^–/– (–/–) mice were immunoprecipitated with anti-N-cad antibodies. The immunoprecipitates were separated by SDS-PAGE and immunoblotted with either anti-N-cad or anti-ß-cat antibodies. (B) Substrate trapping of ß-cat. PTP ${sigma}$ ^–/– brain lysates were incubated with immobilized GST or the PTP ${sigma}$ substrate trapping mutant GST- ${sigma}$ D1(DA). The associated proteins were then analyzed by probing with antibodies against N-cad or ß-cat. Ponceau S staining to visualize the amount of GST or GST- ${sigma}$ D1(DA) substrate trapping proteins used is shown in the lower panel. (C and D) Hyper-tyrosine phosphorylation of ß-cat in knockout mice. Brain lysates from Sib or PTP ${sigma}$ ^–/– mice were immunoprecipitated with anti-pTyr antibodies and immunoblotted for ß-cat. For panel D, lysates were boiled in 6% SDS prior to the IP. (E) Vanadate (VO₄) impairs substrate trapping of ß-catenin. Brain lysates from PTP ${sigma}$ ^–/– mice were incubated with the indicated trapping constructs in the presence or absence of the PTP inhibitor vanadate (10 mM), and precipitated proteins were separated by SDS-PAGE and immunoblotted with anti-ß-cat antibodies. Ponceau S staining to verify equal amounts of trapping proteins is shown in Fig. 2D. (F) Ectopic expression of PTP ${sigma}$ in cells leads to dephosphorylation of endogenous ß-cat. Flag-tagged PTP ${sigma}$ constructs (full length, D1, or D1D2; all WT) were transfected into HEK293T cells, the cells were lysed, lysates were incubated with anti-pTyr antibodies, and precipitated proteins were immunoblotted with anti-ß-cat antibodies to detect ß-cat dephosphorylation. Levels of expression of the indicated Flag-tagged proteins are depicted in Fig. 2F. tfxn, transfection. (G) PTP ${sigma}$ can directly dephosphorylate ß-cat. ß-Catenin from HEK293T cells overexpressing N-cad was immunopurified with ß-cat antibodies and incubated (or not) with purified, GST-tagged PTP ${sigma}$ D1(WT). Proteins were then immunoblotted with anti-pTyr antibodies to analyze ß-cat dephosphorylation or with anti-ß-cat antibodies to demonstrate similar amounts of precipitated ß-cat in the experiment.

To test whether ß-catenin itself is a substrate forPTP ${sigma}$ , the interaction trap experiment was repeated, where PTP ${sigma}$ -D1(DA)was incubated with brain lysate from the PTP ${sigma}$ knockout mice,leading to trapping of N-cadherin (Fig. 3B, top panel). Theblot was then stripped and reprobed for ß-catenin.As seen in Fig. 3B, middle panel, ß-catenin was alsotrapped with the PTP ${sigma}$ -D1(DA) protein, suggesting that ß-cateninitself may be a substrate for PTP ${sigma}$ . To test if ß-cateninis indeed a subject for PTP ${sigma}$ -mediated dephosphorylation, we analyzedits level of Tyr phosphorylation in the lysates of our knockoutanimals. Figure 3C shows that although only a small fractionof ß-catenin is tyrosine phosphorylated in the totalcellular pool, there was more Tyr-phosphorylated ß-cateninin the knockout mice than in the sibling control. This was notsimply an effect of more ß-catenin coprecipitatingwith the increased amount of Tyr-phosphorylated N-cadherin inthese animals (Fig. 3B), because boiling the lysate in 6% SDSprior to the immunoprecipitation with anti-pTyr antibodies (todissociate the N-cadherin:ß-catenin complex) stillrevealed elevated Tyr phosphorylation of ß-catenin(Fig. 3D). Similarly to results with N-cadherin, trapping ofß-catenin with PTP ${sigma}$ -D1 was largely inhibited in thepresence of vanadate (Fig. 3E). Together, these results suggestthat ß-catenin itself is also a substrate for PTP ${sigma}$ .In support, ectopic expression of PTP ${sigma}$ in HEK293T cells led todephosphorylation of endogenous ß-catenin (Fig. 3F);moreover, immunopurified ß-catenin was directly dephosphorylatedby PTP ${sigma}$ -D1 in vitro (Fig. 3G). Thus, in addition to N-cadherin,ß-catenin appears to be a target for PTP ${sigma}$ -mediateddephosphorylation.

Axon growth rate of DRG neurons is accelerated in PTP ${sigma}$ knockout mice. Our previous work has demonstrated an accelerated rate of axongrowth in the sciatic nerve of the PTP ${sigma}$ knockout mice followingnerve injury (35). Since DRG have been demonstrated to expresshigh levels of N-cadherin in both neurons and Schwann cells(57, 74), we focused our studies on DRG. These cells also expressPTP ${sigma}$ (Fig. 4A) (35), as assessed by LacZ staining reporting anendogenous pattern of expression of this phosphatase, sincethe knockout cassette includes the ß-galactosidasegene downstream of the endogenous promoter (69). To quantifyaxon growth rate, we isolated DRG from the knockout animalsand their sibling controls, dissociated their cells, and grewthem as primary cultures on PDLL-coated plates. As seen in Fig.4B, axon growth was faster in the DRG cells from the PTP ${sigma}$ knockoutmice than in those from the sibling controls, and this differencewas especially noticeable after 24 h of growth in culture, suggestinga lag period in the manifestation of the difference in growthrate. The different growth rates between the knockout animalsand their sibling controls were apparent in terms of both thenumber of neurons extending neurites (Fig. 4B) and the totallength of neurites per cell (not shown).

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FIG. 4. DRG neurons from PTP ${sigma}$ ^–/– mice exhibit a faster rate of neurite outgrowth. (A) LacZ staining of PTP ${sigma}$ ^–/– DRG culture, showing endogenous expression (blue) of PTP ${sigma}$ in the cell bodies of neurons (N) and, to a lesser extent, of Schwann cells (S). There was no LacZ staining seen in WT (+/+) DRG (not shown). Bar, 40 µm. (B and C) DRG cultures were prepared from sibling (Sib) controls and PTP ${sigma}$ ^–/– mice, and cells were plated on either (B) PDLL or (C) N-cadherin (N-cad). Neurite growth was measured at the indicated times. Data are expressed as the percentages of cells counted that have neurite extensions. A cell is counted positive if it has neurites greater than twice its body length. Data are summaries of seven independent experiments, and a total of at least 750 cells were counted for each condition. Error bars represent 95% confidence intervals. **, P < 0.0001 (chi-square test).

N-cadherin differentially affects growth of DRG neurons from PTP ${sigma}$ ^–/– mice versus sibling controls. Since our biochemical studies described above suggest that N-cadherinis a substrate for PTP ${sigma}$ and since N-cadherin can support neuritegrowth in vitro (9, 12, 67), we investigated the effect of N-cadherinon growth of DRG neurons harvested from the knockout mice andtheir sibling controls by culturing the neurons on purifiedN-cadherin. Culturing these neurons on control substrata suchas BSA or Fc antibody did not support their growth (data notshown). The accelerated growth exhibited by the PTP ${sigma}$ knockoutneurons compared to that of the sibling control neurons whengrown on PDLL (Fig. 4B) was partially attenuated (delayed) onthe N-cadherin substratum (Fig. 4C). These results are consistentwith the idea that N-cadherin can support neurite growth ofperipheral sensory neurons. Moreover, they suggest that PTP ${sigma}$ may collaborate with N-cadherin to exert its inhibitory effecton neurite growth, since this inhibition was slower/less effectiveupon loss of PTP ${sigma}$ expression in the knockout animals.

To further investigate this hypothesis, we utilized the N-cadherininhibitory peptide INPISGQ (76). This peptide is derived fromthe ectodomain of N-cadherin, binds to a sequence near the HAVmotif necessary for N-cadherin homophilic interactions, andis unique to N-cadherin among the cadherin family (56, 76).Interference with the HAV motif by this and other peptides providesa potent antagonistic effect to N-cadherin function in cells,including DRG neurons and Schwann cells (74, 75). When the N-cadherinfunction was disrupted by the inhibitory peptide, sibling controlDRG neurons cultured on PDLL exhibited a marked increase inneurite growth (Fig. 5A and B). In contrast, this effect wasmuch smaller (and not statistically significant) in the PTP ${sigma}$ knockout neurons (Fig. 5C and D). These results suggest thatthe already accelerated growth of the PTP ${sigma}$ knockout neurons cannotbe further enhanced by inhibiting N-cadherin function, suggestingthat PTP ${sigma}$ and N-cadherin may be part of the same signaling pathwaythat normally inhibits (or slows down) neurite growth.

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FIG. 5. N-cadherin blocking peptide causes increased neurite outgrowth on PDLL of siblings but not PTP ${sigma}$ ^–/– neurons. DRG neurons from (A and B) sibling (Sib) controls or (C and D) PTP ${sigma}$ ^–/– mice were cultured on PDLL and treated with (gray bars) or without (black or white bars) 0.1 mg/ml of the N-cadherin (N-cad) inhibitory peptide INPISGQ. In panels A and C, data represent the percentages of cells counted that have neurite extensions at the indicated times, and these results are normalized to no-treatment controls in panels B and D, respectively. Data are summaries of four or five independent experiments, with a total of at least 500 cells counted for each condition. Error bars represent 95% confidence intervals. *, P < 0.001; **, P < 0.0001 (chi-square test). (E and F) As for panels A and C, only DRG were cultured on N-cad substratum. Data are summaries of four independent experiments, and a total of at least 500 cells were counted for each condition. Error bars represent 95% confidence intervals.

When the neurons were cultured on N-cadherin, the inhibitorypeptide had no significant effect on the growth of either groupof neurons (Fig. 5E and F), most likely because the amount ofpeptide used could not compete with the N-cadherin used as asubstratum (larger amounts of N-cadherin inhibitory peptidewere toxic to cells and hence could not be used).

Difference in growth rate observed between DRG neurons from the knockout and sibling mice is abolished in low calcium. The role of N-cadherin as a cell-cell adhesion molecule is calciumdependent. Calcium is required to stabilize the extracellulardomains of N-cadherin to allow homodimerization (64). Loweringthe extracellular calcium concentration is one way to perturbN-cadherin function, and this was shown to be an effective meansto disrupt Schwann cell contact with axons, a process that isdependent on N-cadherin (74). We studied the role of N-cadherinin DRG neurite outgrowth by reducing the calcium concentrationfrom 1.05 mM in the normal neuronal medium to 0.14 mM, a concentrationthat would render the calcium-dependent N-cadherin nonfunctional.As expected, compared to cells cultured on N-cadherin in normalneuronal medium, the DRG neurons cultured in the low-calciummedium adhered loosely onto the surface, they did not extendneurites, and the Schwann cell processes were much shorter (seeFig S1, bottom panels, in the supplemental material). When thecells were cultured on PDLL, the effect was reversed. The numberof cells extending neurites in the low-calcium medium was increasedmore than 1.5-fold at each time point for the sibling controlcells and increased only slightly for the PTP ${sigma}$ knockout cells(Fig. 6A to D). Moreover, the difference that we observed inthe growth rates between the sibling controls and PTP ${sigma}$ knockoutneurons in normal calcium medium was greatly reduced under low-calciumconditions (compare Fig. 4B to Fig. 6E). This indicates thatthe PTP ${sigma}$ knockout neurons are not sensitive to lowering of extracellularcalcium level, likely because the N-cadherin function/pathwayin the PTP ${sigma}$ knockout neurons is already impaired. These resultsare consistent with those presented in Fig. 5A to D and suggestthat disruption of N-cadherin function by lowering calcium levelsor adding an N-cadherin inhibiting peptide lead to the sameoutcome, an acceleration of neurite growth, which is dependenton PTP ${sigma}$ , since it is lost in the PTP ${sigma}$ knockout DRG.

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FIG. 6. Low-calcium medium leads to increased neurite outgrowth on PDLL of siblings but not PTP ${sigma}$ ^–/– neurons. Sibling (Sib) control (A and B) and PTP ${sigma}$ ^–/– (C and D) DRG neurons grown on PDLL were cultured in normal neuronal medium (1.05 mM Ca²⁺) (black or white bars) or low-calcium medium (0.14 mM Ca²⁺) (gray bars). In panels B and D, data represent percentages of cells with neurite extensions normalized to those in panels A and C, respectively. (E). Neurite growth on PDLL in low-calcium medium (0.14 mM Ca²⁺) abolishes the difference in growth rate observed between the sibling control and PTP ${sigma}$ ^–/– neurons. Data are summaries of six independent experiments, and a total of at least 800 cells were counted for each condition. Error bars represent 95% confidence intervals. *, P < 0.001; **, P < 0.0001 (chi-square test).

Knockout of PTP ${sigma}$ does not disrupt axon-Schwann cell alignment. In the PNS, growth of neurons is supported by SCs. SC-SC andSC-axon interactions are essential during early nerve developmentand in the organization of the peripheral nervous tissues. Theseinteractions have previously been shown to involve N-cadherin(32, 74). Alignment of SCs along neurites in cultured chickenDRG was disrupted upon treatment with N-cadherin blocking antibodiesand by lowering the calcium concentration in the medium (32).We thus investigated whether tyrosine phosphorylation of N-cadherinand PTP ${sigma}$ has a role in this alignment process. DRG neurons wereisolated from both sibling control and PTP ${sigma}$ knockout mice andcultured on PDLL or purified N-cadherin. After 2 days, alignmentof SCs with axons was assessed. As expected, there was a significantreduction in SC-axon alignment when cells were cultured in low-calciummedium. In a low-calcium environment, of the SCs that made contactwith axons, only about 20% aligned their processes along theaxons, whereas 55 to 60% were aligned in the cultures with normalmedium (Fig. 7A). No difference in alignment of neurons withSCs was observed between the sibling control and the PTP ${sigma}$ knockoutneurons when grown on PDLL or N-cadherin substrates in normalmedium (Fig. 7A and B). Neurite growth on N-cadherin of bothknockout and sibling controls was inhibited in low calcium (seeFig S1, bottom panels, in the supplemental material), as expected.

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FIG. 7. Alignment of Schwann cells to axons is not affected in the PTP ${sigma}$ ^–/– DRG. DRG cells from sibling (Sib) control and PTP ${sigma}$ ^–/– mice were plated on either (A) PDLL or (B) N-cadherin (N-cad). Alignment of SCs to axons was assessed after the cells were allowed to grow for (A) 40 h or (B) 46 h. An SC is counted positive for alignment when at least two of its processes are aligned with axons. Data represent means ± standard deviations of percentages of SCs aligned with axons from six independent experiments. A total of at least 700 cells were counted for each condition.

Collectively, these results suggest that PTP ${sigma}$ and N-cadherincollaborate to regulate the rate of growth of DRG neurons butnot their alignment with Schwann cells.

DISCUSSION

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DISCUSSION

Our work here identified N-cadherin as an in vivo (and in vitro)substrate for PTP ${sigma}$ and suggests that N-cadherin (likely via ß-catenin)participates in the inhibitory function of PTP ${sigma}$ in axon growth.We propose that dephosphorylation of N-cadherin (and possiblyß-catenin) by PTP ${sigma}$ leads to increased adhesion andattenuated axon growth, an effect reversed in the PTP ${sigma}$ knockoutaxons (Fig. 8). This conclusion is supported by our observedloss of the difference in axon growth between the PTP ${sigma}$ ^–/–mice and their sibling controls upon inhibition of N-cadherinfunction with an inhibitory peptide or by lowering Ca²⁺ levels.This correlation between our biochemical and axon growth datasuggests that PTP ${sigma}$ and N-cadherin are part of the same signalingpathway that regulates rates of axon growth. Interestingly,we found that only the rate of axon growth, not the alignmentof axons with Schwann cells (previously shown to be regulatedby N-cadherin [74]), was affected in the PTP ${sigma}$ knockout mice,suggesting that PTP ${sigma}$ regulates some but not all functions ofN-cadherin.

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FIG. 8. Model for PTP ${sigma}$ -regulated axon growth via the N-cadherin pathway. When PTP ${sigma}$ is present on the plasma membrane, it can dephosphorylate N-cadherin and recruit ß-catenin (ß-cat) to the complex. ß-Catenin itself also becomes dephosphorylated, and this allows the association of the N-cadherin complex with the actin cytoskeleton, resulting in increased adhesion and reduced rate of axon growth. Conversely, when PTP ${sigma}$ is absent (e.g., in the knockout mice), N-cadherin and ß-catenin remain phosphorylated on tyrosine residues. This prevents interaction between the two proteins, resulting in a loss of N-cadherin adhesive function and faster rate of axon growth. ${alpha}$ -cat, ${alpha}$ -catenin.

It is well established that the adhesive function of cadherindepends on its stable connection to the actin cytoskeleton viacatenin interactions (1, 2) and that tyrosine phosphorylationof ß-catenin leads to its dissociation from cadherinand loss of cadherin adhesiveness (7, 49, 50). Tyrosine phosphorylationof the cadherins themselves has not been well characterized,especially in regard to neurite growth. In this report, we havedemonstrated that tyrosine (de)phosphorylation of N-cadherinitself could regulate neurite growth. The cadherin-catenin complexhas been reported to play a role in the regulation of neuritegrowth by other receptor PTPs. LAR (39), PTPß (37),PTPµ (11, 12), and PTP ${kappa}$ (25) can dephosphorylate ß-catenin.Although until now there have not been any reports on cadherinsbeing direct substrates for PTPs, PTPµ and PTP1B havebeen suggested to associate with N-cadherin and this associationis important for the regulation of N-cadherin-dependent axongrowth (12, 43). Moreover, LAR was recently shown to indirectlyassociate with the cadherin-catenin complex via liprin- ${alpha}$ andGRIP (20). However, unlike PTP ${sigma}$ , its close relatives PTP ${delta}$ andLAR were proposed to provide stimulatory cues for neurite growthin forebrain neurons and in hippocampal and DRG neurons, respectively(73, 82, 83). Since LAR can also employ the cadherin-cateninpathway and can trap N-cadherin in a substrate trapping assay(our unpublished data), it is possible that the final outcome(stimulation or inhibition of neurite growth) may depend onlevels of expression or activity of the competing PTPs on theregulation of the cadherin-catenin complex.

N-cadherin is strongly expressed in the growth cone, and thegrowth cone has been shown to have intense phosphotyrosine staining(79). By disrupting N-cadherin connection to the actin cytoskeleton,thereby reducing adhesive strength, this might allow the growthcone to advance. Based on this model, phosphorylation of N-cadherinmight result in its disconnection from the actin cytoskeletonby several possible means, which are not mutually exclusive.(i) It may lead to reduction in binding to ß-catenin.(ii) It may impair cadherin homodimerization, a step that isbelieved to be essential in driving cadherin adhesion function.(iii) It may alter interactions with other signaling molecules.

Our work shows that PTP ${sigma}$ has a role in the signaling events activatedby laminin. Interestingly, the PTP ${sigma}$ close relative LAR was shownto bind laminin via its fifth FNIII repeat, an interaction requiringthe splicing of a small exon within this domain (41). Sinceit appears that the alternative splicing of this exon is conservedamong the LAR family of PTPs, it suggests that laminin mightalso serve as a ligand for PTP ${sigma}$ . Moreover, ß1 integrinhas been shown to associate with tyrosine-phosphorylated proteinsat the growth cone (80), and ${alpha}$ 1ß1 and ${alpha}$ 3ß1integrin heterodimers are the laminin receptors in DRG neurons(66), further suggesting that tyrosine phosphorylation accompanieslaminin signaling and that PTP ${sigma}$ might be involved in regulatingthis signal. It was interesting that the growth of the DRG neuronson laminin in our studies was affected by an N-cadherin peptideinhibitor. This suggests overlapping responses in the signalingevents triggered by these two molecules. Indeed, there has beensome evidence for cross talk between the N-cadherin and lamininsignaling pathways (5, 33, 44, 48). Our observed acceleratedneurite growth on laminin (of sibling controls) in the presenceof the N-cadherin inhibitory peptide or upon reduction of calciumin the medium may be explained by an inhibition of N-cadherinlateral dimerization/clustering. Indeed, the monomeric inhibitorypeptide we used here was previously suggested to interfere withN-cadherin clustering (77). Such inhibition leads to reducedadhesiveness (64) by affecting the actin cytoskeleton (whichis shared with the laminin-integrin pathway), allowing fasterneurite growth. This faster neurite outgrowth is also mimickedby loss of PTP ${sigma}$ , which we propose normally dephosphorylates N-cadherin,leading to increased adhesiveness.

While we show here that N-cadherin (and ß-catenin)is a substrate for PTP ${sigma}$ , we cannot currently preclude the possibilitythat PTP ${sigma}$ has other substrates. Studies with flies have revealedgenetic interaction between the tyrosine kinase Abl and DLAR(78), although it is not known if Abl is a direct substratefor this phosphatase, and our unpublished studies failed todemonstrate physical interactions between mammalian PTP ${sigma}$ andAbl. Several LAR interacting proteins have been identified todate, including liprins and the Rac exchange factor Trio (16,40, 46, 54, 55), both collaborating genetically with DLAR inflies, although they are not its substrates (28). The existenceof additional PTP ${sigma}$ substrates in mammals is suggested by thepresence of several hyper-Tyr-phosphorylated proteins/bandsin the brain lysates of knockout mice in addition to N-cadherin(Fig. 1). Their identification and characterization await futureinvestigations.

ACKNOWLEDGMENTS

We thank P. Doherty for the Ncad-Fc construct and A. Griffithfor technical support.

This work was supported by the Canadian Institute of HealthResearch (CIHR). D.R. was a CIHR Investigator and currentlyholds a CRC chair from the Canadian Foundation for Innovation.

FOOTNOTES

* Corresponding author. Mailing address: Program in Cell Biology, The Hospital for Sick Children, 555 University Ave., Toronto M5G 1X8, Ontario, Canada. Phone: (416) 813-5098. Fax: (416) 813-5771. E-mail: drotin{at}sickkids.ca.

Published ahead of print on 23 October 2006.

Supplemental material for this article may be found at http://mcb.asm.org/.

REFERENCES

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