A Tripartite Nuclear Localization Signal in the PDZ-domain Protein L-periaxin

The murine Periaxin gene encodes two PDZ-domain proteins in myelin-forming Schwann cells of the vertebrate peripheral nervous system (Dytrych, L., Sherman, D. L., Gillespie, C. S., and Brophy, P. J. (1998) J. Biol. Chem. 273, 5794–5800). Here we show that L-periaxin is targeted to the nucleus of embryonic Schwann cells. Subsequently, the protein redistributes to the plasma membrane processes of the myelinating Schwann cell where it is believed to function in a signaling complex. In contrast, L-periaxin remains in the nucleus when expressed ectopically in oligodendrocytes, the myelin-forming glia of the central nervous system. The nuclear localization signal (NLS) is basic and tripartite and comprises three signals that act synergistically. Nuclear targeting of L-periaxin is energy-dependent and is inhibited by cell-cell contact. These data show that L-periaxin is a member of a growing family of proteins that can shuttle between the nucleus and cortical signaling/adherence complexes. The murine Periaxin gene encodes two PDZ-domain proteins in myelin-forming Schwann cells of the vertebrate peripheral nervous system (Dytrych, L., Sherman, D. L., Gillespie, C. S., and Brophy, P. J. (1998) J. Biol. Chem. 273, 5794–5800). Here we show that L-periaxin is targeted to the nucleus of embryonic Schwann cells. Subsequently, the protein redistributes to the plasma membrane processes of the myelinating Schwann cell where it is believed to function in a signaling complex. In contrast, L-periaxin remains in the nucleus when expressed ectopically in oligodendrocytes, the myelin-forming glia of the central nervous system. The nuclear localization signal (NLS) is basic and tripartite and comprises three signals that act synergistically. Nuclear targeting of L-periaxin is energy-dependent and is inhibited by cell-cell contact. These data show that L-periaxin is a member of a growing family of proteins that can shuttle between the nucleus and cortical signaling/adherence complexes. peripheral nervous system nuclear localization signal fetal calf serum green fluorescent protein polymerase chain reaction base pair myelin basic protein central nervous system L-periaxin was identified in a screen for cytoskeleton-associated proteins in myelinating Schwann cells of the peripheral nervous system (PNS)1 (1.Gillespie C.S. Sherman D.L. Blair G.E. Brophy P.J. Neuron. 1994; 12: 497-508Abstract Full Text PDF PubMed Scopus (146) Google Scholar). Subsequently it was shown that the murine Periaxin (Prx) gene encodes L-periaxin and a truncated isoform, S-periaxin (2.Dytrych L. Sherman D.L. Gillespie C.S. Brophy P.J. J. Biol. Chem. 1998; 273: 5794-5800Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar). Both proteins have N-terminal PDZ protein-binding domains (2.Dytrych L. Sherman D.L. Gillespie C.S. Brophy P.J. J. Biol. Chem. 1998; 273: 5794-5800Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar), a motif which was named after the three proteins in which it was first identified, namely post-synaptic density protein PSD-95,Drosophila d iscs large(dlg) tumor suppressor gene and the tight junction-associated protein ZO-1. This motif consists of an approximately 90-amino acid protein-binding module found in a growing family of proteins that are believed to have an organizing and signaling function at sites of cell-cell contact (3.Sheng M. Neuron. 1996; 17: 575-578Abstract Full Text Full Text PDF PubMed Scopus (296) Google Scholar) Some PDZ-domain proteins such as ZO-1, MAGI-1c, and LIM-kinase 1 can redistribute between the cell cortex and the nucleus which is believed to reflect their role in transmitting regulatory signals between the cell surface and the nucleus (4.Dobrosotskaya I. Guy R.K. James G.L. J. Biol. Chem. 1997; 272: 31589-31597Abstract Full Text Full Text PDF PubMed Scopus (175) Google Scholar, 5.Gottardi C.J. Arpin M. Fanning A.S. Louvard D. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 10779-10784Crossref PubMed Scopus (309) Google Scholar, 6.Yang N. Mizuno K. Biochem. J. 1999; 338: 793-798Crossref PubMed Scopus (47) Google Scholar). Nuclear targeting of MAGI-1c is thought to be regulated by a nuclear localization signal (NLS) (4.Dobrosotskaya I. Guy R.K. James G.L. J. Biol. Chem. 1997; 272: 31589-31597Abstract Full Text Full Text PDF PubMed Scopus (175) Google Scholar). NLSs are relatively short sequences which may be identified by their capacity to transport heterologous proteins into the nucleus (7.Nigg E.A. Nature. 1997; 386: 779-787Crossref PubMed Scopus (925) Google Scholar). Classical NLSs comprise either one or two (mono- or bipartite) basic sequences (7.Nigg E.A. Nature. 1997; 386: 779-787Crossref PubMed Scopus (925) Google Scholar), although new types of non-basic targeting domains seem to function as NLSs in nuclear ribonucleoprotein particle proteins (8.Michael W.M. Eder P.S. Dreyfuss G. EMBO J. 1997; 16: 3587-3598Crossref PubMed Scopus (332) Google Scholar). In the case of the LIM-kinase 1, the PDZ domain itself has a direct role in regulating the nuclear export whereas a short, basic NLS appears to direct import (6.Yang N. Mizuno K. Biochem. J. 1999; 338: 793-798Crossref PubMed Scopus (47) Google Scholar). Classical NLSs function by binding to the importin/karyopherin complex prior to Ran-mediated nuclear uptake (7.Nigg E.A. Nature. 1997; 386: 779-787Crossref PubMed Scopus (925) Google Scholar). Here we show that although L-periaxin has a PDZ domain, which is believed to recruit proteins to a cortical structure involved in transmembrane signaling (1.Gillespie C.S. Sherman D.L. Blair G.E. Brophy P.J. Neuron. 1994; 12: 497-508Abstract Full Text PDF PubMed Scopus (146) Google Scholar, 9.Scherer S.S. Xu Y.T. Bannerman P.G.C. Sherman D.L. Brophy P.J. Development. 1995; 121: 4265-4273Crossref PubMed Google Scholar), it also possesses an unusual tripartite NLS. Localization of L-periaxin to the Schwann cell nucleus when it is first expressed in the embryonic PNS indicates that the NLS is functional in vivo. The Schwann cell line, 33B, was a generous gift from Dr. Eric Blair, Leeds University, and was maintained in complete medium, 10% fetal calf serum (FCS), Dulbecco's modified Eagle's medium with penicillin (100 IU/ml), and streptomycin (100 μg/ml) (Sigma). Schwann cells were isolated from the sciatic nerves of P5 rats and placed in a 35-mm Petri dish containing pre-warmed L15 (Sigma). The epineurium was removed by dissection with fine forceps and the nerve fibers were cut into small pieces and digested with collagenase (0.24 mg/ml) for 45 min at 37 °C before the addition of trypsin (0.6 mg/ml) for 10 min. Trypsinization was terminated by the addition of complete medium. After trituration, the cells were centrifuged for 3 min at 1000 rpm, resuspended in complete medium and cultured on flasks pre-coated with poly-d-lysine (0.1 mg/ml). After exposure to 10 μm cytosine arabinoside (Ara C, Sigma) in complete medium for 7 days, fibroblasts were killed by complement lysis using Thy-1.1 monoclonal antibody diluted 1:10 (generous gift of Dr. A. F. Williams, Oxford University) and baby rabbit complement (Cedarlane Laboratories Ltd, Canada). Complement-mediated lysis was repeated. The cultures were then expanded in complete medium with 2 μm forskolin and seeded on coverslips coated with poly-d-lysine and laminin (Sigma). A modified protocol was used for embryonic Schwann cells. After collagenase treatment at 37 °C for 40 min, hyaluronidase (1 mg/ml, Sigma) was added to the nerve segments for 15 min after which an additional amount of collagenase was added (0.08 mg/ml) and the cells were transferred to complete medium. Cells were plated on laminin-coated coverslips that had been pre-coated with poly-d-lysine. The cultures were incubated for 6.5 h before fixation. L-periaxin cDNA (1.Gillespie C.S. Sherman D.L. Blair G.E. Brophy P.J. Neuron. 1994; 12: 497-508Abstract Full Text PDF PubMed Scopus (146) Google Scholar) was directionally subcloned into pCB6 expression vector (a gift from Dr. David Russell, Southwestern Medical Center, Dallas, TX) which includes a neomycin-resistance gene for selection. BD-GFP, in which the basic domain (244 bp) of L-periaxin (residues 118–196) (1.Gillespie C.S. Sherman D.L. Blair G.E. Brophy P.J. Neuron. 1994; 12: 497-508Abstract Full Text PDF PubMed Scopus (146) Google Scholar) was fused to green fluorescent protein (GFP), was generated by PCR. The enzyme Pfu polymerase (Stratagene) was used for all the PCR reactions. The forward primer was designed with an EcoRI site followed by an initiation codon with a strong Kozak consensus sequence. The reverse primer contained a BamHI site. The PCR product was digested with EcoRI and BamHI and cloned into the expression vector pEGFP-N1 (CLONTECH). This vector encodes a GFP which fluoresces with an enhanced quantum yield compared with wild-type GFP. The basic domain of L-periaxin was then expressed as an N-terminal fusion with GFP. DNA for transfection was prepared by Qiagen Midi-prep. A similar strategy was adopted to generate GFP fusion constructs with the basic sub-domains BD1 (residues 116–145, BD1-GFP), BD2 (residues 140–159, BD2-GFP), BD1 and BD2 (residues 116–176, BD1.2-GFP), and BD2 and BD3 (residues 140–196, BD2.3-GFP). The basic domain of L-periaxin cDNA (residues 118–196) was deleted using a PCR-ligation-PCR technique to generate PrxΔBD (10.Ali S.A. Steinkasserer A. BioTechniques. 1995; 18: 746-750PubMed Google Scholar). The template for the PCR was wild-type L-periaxin cDNA in pSPORT (Life Technologies, Inc.), and the final product was subcloned into the expression vector pSVSPORT (Life Technologies, Inc.). 33B cells grown in 100-mm plates were transfected with DNA (24 μg) and LipofectAMINE (64 μg, Life Technologies, Inc.) in Optimem (Life Technologies, Inc.) for 4 h at 37 °C in a humidified CO2 incubator. They were then washed and complete medium was added. To prepare permanent L-periaxin transfectants, 600 μg/ml G418 was added to the medium, and the cultures were maintained until single G418-resistant colonies appeared. These colonies were cloned out. Schwann cells were seeded on poly-d-lysine-coated coverslips in 35-mm dishes, and plasmid DNA (3 μg) with LipofectAMINE was added to the cells for 4 h after which they were grown for 24 to 48 h in DMEM with 10% FCS. The cells were either processed for immunocytochemistry or they were viewed directly for GFP fluorescence after brief fixation with 4% paraformaldehyde in 0.1 m sodium phosphate buffer, pH 7.4, at room temperature. Rabbit anti-periaxin (anti-170pep1) has been described (1.Gillespie C.S. Sherman D.L. Blair G.E. Brophy P.J. Neuron. 1994; 12: 497-508Abstract Full Text PDF PubMed Scopus (146) Google Scholar) and was diluted 1:3000 for immunofluorescence, and affinity-purified anti-170pep1 was used at 1:50 for immunoelectron microscopy. Anti-CNP (Chemicon) was used for immunocytochemistry at a dilution of 1:200. Sciatic nerves from mice at embryonic ages were fixed by immersion in freshly prepared 4% paraformaldehyde in 0.1 m sodium phosphate buffer, pH 7.4, at room temperature for 2 h. The nerves were washed in three changes of 0.1 m phosphate buffer for 10 min each and prepared for immunofluorescence microscopy as described previously (2.Dytrych L. Sherman D.L. Gillespie C.S. Brophy P.J. J. Biol. Chem. 1998; 273: 5794-5800Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar). Cultured cells on 13-mm glass coverslips (BDH) were fixed with 4% paraformaldehyde in 0.1 m sodium phosphate buffer for 30 min at room temperature. The cells were washed four times for 5 min each with PBS and blocked with 4% goat serum, 0.2% gelatin, 0.1% Triton X-100 in phosphate-buffered saline for 30 min. For both sections and cultured cells, immunofluorescence labeling was as described previously (2.Dytrych L. Sherman D.L. Gillespie C.S. Brophy P.J. J. Biol. Chem. 1998; 273: 5794-5800Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar). In some experiments, propidium iodide staining was combined with immunolabeling. Sections were incubated with propidium iodide (100 μg/ml; Sigma) for 2 min after immunolabeling was completed. The slides were viewed on an Olympus BX60 microscope or a Leica TCS 4D scanning confocal microscope when a maximum projection was made from a series of optical scans through the z axis of the tissue. The digitized images were processed using Adobe PhotoshopTM 3.0 software on a Power Macintosh 8600/200. Western blotting was performed as described previously (1.Gillespie C.S. Sherman D.L. Blair G.E. Brophy P.J. Neuron. 1994; 12: 497-508Abstract Full Text PDF PubMed Scopus (146) Google Scholar). P1 rat sciatic nerves were removed and fixed by immersion in 4% formaldehyde (freshly prepared from paraformaldehyde) in a 0.01 m sodium periodate, 0.075 m lysine, 0.1 m phosphate buffer containing 3% sucrose at pH 7.4 (11.McLean I.W. Nakane P.K. J. Histochem. Cytochem. 1974; 22: 1077-1083Crossref PubMed Scopus (3304) Google Scholar) for 2 h at room temperature. Tissue was processed, infiltrated with LR Gold (Agar Scientific Ltd., Stanstead, Essex, UK) containing 0.5% benzoin methyl ether, and then embedded in gelatin capsules as described previously (1.Gillespie C.S. Sherman D.L. Blair G.E. Brophy P.J. Neuron. 1994; 12: 497-508Abstract Full Text PDF PubMed Scopus (146) Google Scholar, 12.Berryman M.A. Porter W.R. Rodewa;d R.D. Hubbard A.L. J. Histochem. Cytochem. 1992; 40: 845-857Crossref PubMed Scopus (30) Google Scholar). Sections on formvar carbon-coated nickel grids were blocked with 1% bovine serum albumin, 0.5% fish skin gelatin, 0.05% Triton X-100, 0.05% Tween 20, 10 mm Tris, 500 mmNaCl, pH 7.4 for 30 min at room temperature, and incubated with affinity-purified anti-170pep1 antibody in the same buffer overnight at 4 °C. Grids were washed with the same buffer and incubated for 1 h with goat anti-rabbit IgG conjugated to 10 nm gold (1:20, Amersham Pharmacia Biotech). The grids were then processed for electron microscopy as described (1.Gillespie C.S. Sherman D.L. Blair G.E. Brophy P.J. Neuron. 1994; 12: 497-508Abstract Full Text PDF PubMed Scopus (146) Google Scholar) and examined at 80 kV in a Phillips 400 electron microscope. Rat L-periaxin cDNA was ligated downstream of the 1.9-kilobase myelin basic protein (MBP) promoter (generous gift of Drs. A. Gow and R. A. Lazzarini, Mt. Sinai School of Medicine, NY) at a BamHI site in the pMG2 vector (13.Gow A. Friedrich V.L. Lazzarini R.A. J. Cell Biol. 1992; 119: 605-616Crossref PubMed Scopus (134) Google Scholar). Purified DNA was diluted to a concentration of 2.5 μg/ml and introduced into the C57BL6 mouse genome by standard pronuclear microinjection of fertilized eggs from superovulated F1 (C57BL6 × CBA hybrid mice) hybrids (14.Hogan B. Beddington R. Costantini F. Lacy E. Manipulating the Mouse Embryo: A Laboratory Manual. 2nd Ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY1994: 226-250Google Scholar). A total of 13 Founders were identified by PCR of tail-tip DNA using forward and reverse primers specific to the MBP promoter and L-periaxin cDNA, respectively. Schwann cells were transiently transfected with BD-GFP. After 24 h, the cells were washed twice with glucose-free minimal essential medium (Life Technologies, Inc.) containing 10% dialyzed FCS, penicillin (100 IU/ml) and streptomycin (100 μg/ml), and 2 mm glutamine and then incubated with 6 mm 2-deoxyglucose (Sigma) and 10 mm sodium azide in glucose-free medium containing dialyzed FCS for 2 h. Some cultures were allowed to recover for 2 h in complete medium at 37 °C. At the end of the incubation times, the cells were fixed with 4% paraformaldehyde, and images were captured on a Leica TCS 4D scanning confocal microscope. Schwann cells are first generated in mouse peripheral nerve between embryonic days 13.5 and 14.5 (E13.5 and E14.5) (15.Mirsky R. Jessen K.R. Curr. Opin. Neurobiol. 1996; 6: 89-96Crossref PubMed Scopus (210) Google Scholar). This is the earliest time at which L-periaxin is detectable by immunocytochemistry in Schwann cells of the mouse PNS, but, surprisingly, the protein is localized to the nucleus of these cells 1 and nuclear expression is also in freshly isolated Schwann cells in 1 Western blotting of embryonic peripheral nerve that the protein by the antibody was L-periaxin Subsequently, of L-periaxin to the processes of myelin-forming Schwann cells of murine sciatic nerve 1 and of rat sciatic nerve 1 with the plasma membrane and myelin (1.Gillespie C.S. Sherman D.L. Blair G.E. Brophy P.J. Neuron. 1994; 12: 497-508Abstract Full Text PDF PubMed Scopus (146) Google Scholar). The of L-periaxin to the plasma membrane appears to be specific to Schwann cells it is to the nucleus when ectopically expressed by in the myelin-forming glia of the central nervous system nuclear expression of L-periaxin in the L-periaxin is in the nucleus of myelinating when expressed in the the of the MBP A of rat was by immunofluorescence to L-periaxin and the and myelin A was also for L-periaxin and with propidium iodide to cells and which are 10 has a basic domain between and (1.Gillespie C.S. Sherman D.L. Blair G.E. Brophy P.J. Neuron. 1994; 12: 497-508Abstract Full Text PDF PubMed Scopus (146) Google Scholar). basic domains are to function as nuclear targeting we to basic was an NLS. L-periaxin expression in Schwann cells detectable in the of contact S.S. Xu Y.T. Bannerman P.G.C. Sherman D.L. Brophy P.J. Development. 1995; 121: 4265-4273Crossref PubMed Google Scholar), to the targeting of L-periaxin in cultured Schwann cells they were transfected with a of cDNA Schwann cells were transfected with wild-type L-periaxin cDNA, the protein was in the nucleus 4 of domain the nuclear uptake of L-periaxin in Schwann was the case that the basic domain was an NLS was by the targeting of a green fluorescent protein basic domain fusion protein to the Schwann cell nucleus and The same was used to the of nuclear of Schwann cells nuclear that nuclear was an 4 cells of were allowed to they their to the NLS of L-periaxin targeting by the basic domain is localization of wild-type L-periaxin and of a the basic domain in transfected Schwann cells. The of GFP and an L-periaxin basic to in the of transfected Schwann cells was The of nuclear of was by transfected cells in glucose-free medium containing 6 mm 2-deoxyglucose (Sigma) and 10 mm sodium azide for 2 h after which the cells were allowed to recover in complete medium for a 2 h for L-periaxin was performed as described the nuclear uptake of the tight PDZ domain protein is inhibited by we were to the of cell the localization of In of 33B cells the protein was targeted to the nucleus in cultures nuclear was 5 The of was in cultures by the with a 5 At the of the where the cell density is is a in the of cells that nuclear labeling 5 basic sub-domains are the NLS NLSs are we that at one of these sub-domains be for nuclear we found that all three domains were for nuclear localization when they were fused to the GFP nuclear localization was as in 4 D. The first of nuclear labeling of the protein which were enhanced when the was the first was for nuclear that the two sub-domains are of the NLS. The NLS of an essential sequence. of the and in the in the first of L-periaxin the function of the NLS which the of the NLS in basic sub-domains comprise the complete cells were transfected with the pEGFP-N1 vector or with fusion constructs with the basic domain of L-periaxin or sub-domains as described The localization of GFP fluorescence was 48 h after transfection and was to be nuclear similar to the shown in 4 D. For each three coverslips were and a of cells were in a new 33B cells were transfected with the pEGFP-N1 vector or with fusion constructs with the basic domain of L-periaxin or sub-domains as described The localization of GFP fluorescence was 48 h after transfection and was to be nuclear similar to the shown in 4 D. For each three coverslips were and a of cells were have shown that L-periaxin has a functional NLS of an unusual tripartite The NLS is by the single signal of the or the NLS of C. R.A. Biochem. Sci. 16: Full Text PDF PubMed Scopus Google Scholar). These signals with the importin/karyopherin complex which is into the nucleus in an energy-dependent by a that and the K. Biochem. Sci. 1998; Full Text Full Text PDF PubMed Scopus Google Scholar). The of proteins in cells has the that reflect the for in NLS N. K. EMBO J. 1997; 16: PubMed Scopus Google Scholar). The that the of the PDZ domain protein is cell-cell contact C.J. Arpin M. Fanning A.S. Louvard D. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 10779-10784Crossref PubMed Scopus (309) Google Scholar), whereas a domain appears to shuttle between the nucleus and in to J. Cell Biol. 1997; PubMed Scopus Google Scholar). data that the of proteins that nuclear uptake is by cell-cell contact L. J. 1997; PubMed Scopus Google Scholar). it is the nuclear function of L-periaxin it a growing of proteins that redistribute between the nucleus and cortical signaling and complexes. The is which between and the nucleus by of a complex in the Curr. Opin. 1998; PubMed Scopus Google Scholar). of the of L-periaxin be to transport through the nuclear complex into the nucleus Biochem. 1995; PubMed Scopus Google Scholar). the for and the of cell-cell it that nuclear of L-periaxin by the it a NLS. is of a tripartite signal be by the import it is that one of a basic NLS can the of nuclear uptake L. C. Biol. PubMed Scopus Google Scholar, J. Cell Biol. PubMed Scopus Google Scholar). The to the tripartite NLS of L-periaxin is that of the protein which has three basic two of which are to function as a strong NLS PubMed Scopus Google Scholar). The regulated of L-periaxin in embryonic Schwann cells is the first such for a PDZ-domain The of binding at the cell surface of the Schwann cell may be the for the of L-periaxin from the nucleus to myelinating processes as they the that the same are expressed in the myelin-forming glia of the such from the nucleus when L-periaxin is ectopically expressed in the myelinating of Some PDZ such as the PDZ domain of the protein have been shown to have the to to sequences M. A. Neuron. 1998; Full Text Full Text PDF PubMed Scopus Google Scholar, M. M. K. A. K. 1997; PubMed Scopus Google Scholar, D. Neuron. 1998; Full Text Full Text PDF PubMed Scopus Google Scholar, M. M. Neuron. 1998; Full Text Full Text PDF PubMed Scopus Google Scholar). we that nuclear targeting of L-periaxin in embryonic Schwann cells may the PDZ domain from in the until the at the cell cortex of the myelin-forming Schwann was by and in

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