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J Biol Chem, Vol. 274, Issue 27, 19261-19268, July 2, 1999


Novel Low Molecular Weight Microtubule-associated Protein-2 Isoforms Contain a Functional Nuclear Localization Sequence*

Kate Lakoski LovelandDagger §, Daniella HerszfeldDagger , Brendan ChuDagger , Emily RamesDagger , Elizabeth ChristyDagger , Lyndall J. Briggs, Rushdi Shakri, David M. de KretserDagger , and David A. Jans

From the Dagger  Institute of Reproduction & Development, Monash University, Clayton, Victoria 3168 and the  Nuclear Signalling Laboratory, Department of Biochemistry and Molecular Biology, John Curtin School of Medical Research, Canberra 2601, Australia

    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Known high and low molecular weight (LMW) MAP2 protein isoforms result from alternative splicing of the MAP2 gene. Contrary to previous reports that MAP2 is neural-specific, we recently identified MAP2 mRNA and protein in somatic and germ cells of rat testis, and showed the predominant testicular isoform is LMW. Although cytoplasmic in neural tissue, MAP2 appeared predominantly nuclear in germ cells using immunohistochemistry. We sought to determine whether this unexpected localization was due to the inclusion of exon 10 within novel LMW MAP2 isoforms. Normally excluded from the LMW MAP2c, exon 10 harbors a putative CcN motif, comprising a nuclear localization sequence (NLS) flanked by regulatory phosphorylation sites for protein kinase CK2 and cdc2 kinase. Characterization of MAP2 mRNA in adult and immature brain and testis, by reverse transcriptase-polymerase chain reaction/Southern analysis and Northern blot, identified novel LMW forms containing exons 10 and 11, previously detected only in high molecular weight MAP2a and 2b. The MAP2 NLS targeted a large heterologous protein to the nucleus, as demonstrated using bacterially expressed MAP2-CcN-beta -galactosidase fusion protein and an in vitro nuclear import assay. Antibodies raised against the fusion protein produced a testicular immunohistochemical staining pattern correlating with MAP2 protein distribution in the nucleus of most germ cells, and precipitated both ~70-kDa and >220-kDa proteins recognized by the commercial MAP2-specific HM2 monoclonal antibody, supporting our hypothesis of a novel LMW MAP2 isoform. These results demonstrate the presence of a functional NLS in MAP2 and indicate that novel LMW MAP2 isoforms may be targeted to the nucleus in both neural and non-neuronal tissues.

    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Microtubule-associated protein-2 (MAP2),1 the most abundant MAP in neurons (reviewed in Ref. 1), has recently been shown to be present in both somatic and germ cells of the rat testis (2). Several high molecular weight (HMW) and low molecular weight (LMW) forms are present in neural tissue, while in the adult rat testis the predominant MAP2 is a LMW form of ~74 kDa, which correlates with the prevalence of transcripts that lack the projection arm coding sequences of the HMW MAP2 isoforms (2). The testis contains several MAP2 transcripts, with 6-kb mRNAs in the Sertoli and Leydig cells, and transcripts of ~2.5 and 3.5 kb in the haploid spermatids. Each of these transcripts contain coding sequence from both the 5' and 3' ends of MAP2 (2).

The initial descriptions of MAP2 indicated that three isoforms existed, with the ~288- and 280-kDa MAP2a and MAP2b HMW isoforms containing a binding site for the regulatory RII subunit of cAMP-dependent protein kinase (PKA), a putative binding site for calmodulin, and three repeats of a tubulin binding motif (3) (reviewed in Ref. 1). Cloning of the cDNA encoding the ~70-kDa MAP2c LMW isoform demonstrated that the PKA binding site and three tubulin repeats were present, but the calmodulin binding site was absent (3). More recently, a phosphatidylinositol binding site has been identified on HMW MAP2 that is absent from MAP2c (4, 5). With the report of the gene sequence and exon structure of human MAP2 (6), it was shown that MAP2a and MAP2b differ from each other by the inclusion of exon 8 in MAP2a (7), while MAP2c lacks exons 9, 10, and 11, which encode the long projection arm of the HMW MAP2 isoforms.

There are now several reports describing additional MAP2 mRNA splice variants in neuronal tissues (reviewed in Ref. 8; see summary in Table I). The inclusion and exclusion of exons 1, 2, and 3, encoding 5'-untranslated regions, has been shown to vary (9), which may conceivably impact on mRNA stability, translation, or localization. The inclusion of exon 16 yields MAP2 isoforms that contain four rather than three repeats of the tubulin binding motif (10, 11, 12), but the effect of this on tubulin binding by MAP2 is unclear (13). An additional exon, exon 13, was identified through sequencing of the MAP2 gene (6).

                              
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Table I
Summary of known and hypothetical MAP2 isoforms
Data are collated from Refs. 2, 6, and 8.

In contrast to MAP2 in neural tissue, testicular MAP2 appears to be abundant in the nucleus of testicular cells when examined by immunohistochemistry (2). An explanation for this surprising result was obtained with the identification of a putative nuclear localization sequence (NLS) encoded in exon 10. This sequence (Fig. 1) has the features of a typical bipartite NLS characterized by two clusters of positively charged amino acids separated by a spacer of 10-12 amino acid residues (14), as well as being flanked by consensus phosphorylation sites for protein kinase CK2 (CK2) and the cyclin-dependent kinase (cdk) cdc2, both of which are known to regulate NLS-dependent nuclear import of proteins such as the simian virus SV40 large tumor-antigen (T-Ag) (15, 16), and together with the NLS constitute the T-Ag CcN motif (for CK2 site, cdk site, and NLS). Exon 10 of MAP2 thus harbors a putative CcN motif, which may confer regulated nuclear import on MAP2.

Our initial study of MAP2 in the testis demonstrated the presence of LMW mRNAs and protein, and so we hypothesized that this tissue may contain novel LMW MAP2 isoforms that include exon 10 containing the putative NLS. The present work describes the identification of novel transcripts encoding LMW MAP2 species, which contain either exon 11 or exon 10/11 in both juvenile and adult brain and testis. The functionality of this NLS is demonstrated using an in vitro assay system to measure nuclear import, and evidence for the expression of protein from these novel mRNAs based on immunohistochemical staining of tissue sections and immunoprecipitation is presented.

    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Animals and Cell Culture-- Male Sprague-Dawley rats and female New Zealand White rabbits (6-7 weeks) were obtained from the Monash University Central Animal House. These investigations were approved by the Monash University Standing Committee on Ethics in Animal Experimentation and conform to the NHMRC/CSIRO/AAC Code of Practice for the Care and Use of Animals for Experimental Purposes. Cells of the HTC rat hepatoma tissue culture (a derivative of Morris hepatoma 7288C) line were cultured in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum as described previously (17-19).

Chemicals and Reagents-- Chemicals and other reagents were obtained from sources previously described (17, 19-21). Isopropyl-beta -thiogalactoside was obtained from Roche Molecular Biochemicals, and the sulfhydryl labeling reagent 5-iodacetamido-fluorescein (IAF) from Molecular Probes.

Reverse Transcription and Polymerase Chain Reaction-- Reverse transcription (RT) was performed as described (22) using the avian myeloblastosis virus enzyme. First round polymerase chain reaction (PCR) samples were prepared in a final volume of 20 µl containing 5 µl of the RT sample with Tth Plus enzyme (Biotech International, Bentleigh, Western Australia) as previously reported (22). Nested PCR using 1 or 0.1 µl of the first round PCR product as the template was performed in a 20-µl volume with 0.2 mM dNTPs, 1.1 units of Tth Plus, 2 mM MgCl2, and 1× Tth buffer. PCR extensions were at 58 °C for 30 s.

The oligonucleotide primers and nucleotide positions listed below were based on the rat MAP2b sequence (RNMAP2R; Ref. 23), with 5' end modifications. The indicated exon position is based on the human sequence (6): 26.1 (bp 4413-4431, exon 10), 5'-GTGGATCCGAGGCAGAATTTCCACTCC-3'; 26.2 (bp 4427-4445, exon 10), 5'-TCGCCGGCAGAACTAGTACTCCTGAAAGAAAAGTAGC-3'; 27.1 (bp 4624-4643, exons 11 and 12), 5'-GTGGATCCTCACCACTTGTTGCTGTGG-3'; 27.2 (bp 4549-4567, exon 11), 5'-ATGCCGGCTCGAGAAATGAGTTTCCTGGAAGG-3'; 28.1 (bp 288-307, exon 6), 5'-GAAGATCTCACAGGGCACCTATTCAG-3'; 28.2 (bp 332-351, exon 6), 5'-ATGCCGGCATCTACGTAGAGCTGACCTCAGCTGAC-3'; 29 (bp 4252-4272, exon 9), 5'-GAAGATCTATTATGGATGCCGACAGCC-3'; 30 (bp 5282-5302, exon 18), 5'-GAAGATCTCCTTCTCCTTGAAATCCAGC-3'; and 31 (bp 4475-4494, exon 10), 5'-GTGGATCCGCTTTTTTCCTTCTCACTTC-3'.

Northern and Southern Blotting-- Northern blot analysis was performed as described previously (24), following transfer in 20× standard sodium citrate (SSC; Ref. 25) to MagnaCharge membrane (MSI, Westborough, MA) and fixation by UV treatment (5 min) and baking (80 °C for 30 min). Southern blot analysis followed transfer of DNA to MagnaCharge (as above) or Hybond N+ in 0.4 M NaOH (Amersham Pharmacia Biotech). Prehybridization and hybridization with cDNAs (65 °C) and oligonucleotides (42 °C) were performed in Rapid Hyb (Amersham Pharmacia Biotech) according to the manufacturer's protocol. Membranes were washed to 0.1× SSC (for Northern blots) or 0.2× SSC (for Southern blots) with 1% SDS at 65 °C.

Radiolabeled cDNA was made from gel-purified PCR product (Wizard PCR Prep Promega Corp., Madison, WI) using a random primer kit (as in Ref. 22). The exon 10/11 MAP2 cDNA used corresponded to 227 base pairs of the rat coding sequence (RNMAP2R; Ref. 23), from 4413 to 4643 bp, corresponding to amino acids 1441-1475 of rat MAP2 (see Fig. 1). It was produced as a product of RT-PCR from adult rat testis RNA using primers 26.1 and 27.1 (see above) and sequenced using dye terminator PCR on an ABI model 330 Gene Sequencer (Monash University Department of Microbiology). Oligonucleotide labeling was performed using 0.36 ng of oligonucleotide in a 20-µl reaction mixture containing 20 units of terminal deoxynucleotidyltransferase enzyme (Promega) and 75 µCi of [32P]dCTP in 1× buffer supplied with the enzyme for 30 min at 37 °C.

beta -Galactosidase Fusion Protein-- The plasmid expressing the MAP2-CcN-beta -galactosidase fusion protein (MAP2-CcN-beta -Gal) was derived by ligating a PCR fragment, generated using oligonucleotides 26.2 and 27.2 and encoding parts of exons 10 and 11, into the unique SmaI site of plasmid vector pPR2 (26). The fusion protein contains MAP2 amino acids 1441-1475 fused N-terminal to the Escherichia coli beta -galactosidase (beta -Gal) sequence (amino acids 9-1023; Refs. 15, 16, and 26). The T-Ag-CcN-beta -Gal fusion protein used as a control contains SV40 T-Ag amino acids 111-135, including the CcN motif (including CK2 and cdk phosphorylation sites and NLS) fused N-terminal to the beta -Gal amino acids 9-1023. beta -Gal fusion proteins were expressed in E. coli, purified by affinity chromatography, and labeled with IAF as described previously (15, 16, 26). Protein concentrations were determined using the dye binding assay of Bradford (27), with bovine serum albumin as a standard.

Antibody Production-- Each of two rabbits received subcutaneous injections at multiple sites of approximately 400 µg of MAP2-CcN-beta -Gal in 0.5 ml of Freund's complete adjuvant (first immunization; Sigma) followed at 4 weeks in two weekly intervals with injections of approximately 100 µg of MAP2-CcN-beta -Gal in 0.5 ml of Freund's incomplete adjuvant (Sigma). Serum was collected for testing after the third and fourth injections, and a final collection of serum was taken by cardiac puncture under anesthesia after the fifth injection. The serum used for further studies was designated DH-1.

Immunohistochemistry-- Immunohistochemistry with DH-1 was performed as described previously (22) using rat tissues fixed in Bouin's fluid and embedded in paraffin. The DH-1 serum was applied at 1:2000 overnight at room temperature. Incubation with biotinylated sheep anti-rabbit (Silenus, Victoria, Australia; 1:500 dilution) for 1 h was followed by incubation with streptavidin-horseradish peroxidase (HRP; Silenus; 1:1000 dilution) for 30 min and development with 3,3'-diaminobenzidine tetrahydrochloride to detect primary antibody binding as a brown precipitate. Harris hematoxylin was used as a blue counterstain. Application of the same protocol to MAP2 localization with the HM-2 monoclonal antibody (Sigma; in ascites fluid; recognizes HMW and LMW MAP2s) at dilutions of 1:100 to 1:400 yielded only a faint signal on testis sections. To obtain a distinct signal with this antibody, the Dako Catalyzed Signal Amplification system was employed according to the manufacturer's specification, with HM-2 at 1:400. Development and counterstaining were performed as above. Results were analyzed using an Olympus BX50 microscope and photographed using Ultra-50 Agfacolor film (Agfa-Gevaert AG, Leverkusen, Germany).

Immunoprecipitation and Western Blotting-- A lysate of day 7 rat brain was prepared for immunoprecipitation by addition of radioimmune precipitation buffer (50 mM Tris, pH 7.4, 150 mM NaCl, 20 mM EDTA, 1% Triton X-100, 1% sodium deoxycholate, 0.1% SDS, 100 µm sodium vanadate, 0.01 mg/ml leupeptin, 100 kallikrien international units/ml Trasylol, 0.5 mM dithiothreitol, and 0.5 mM phenylmethylsulfonyl fluoride) to ~310 mg/ml, followed by Dounce homogenization on ice. An equal volume of radioimmune precipitation buffer was added, and insoluble material was removed by centrifugation (~13,000 rpm) for 30 min at 4 °C. The resulting supernatant (100 µl) was precleared with 20 µl of protein A-Sepharose (AMRAD Pharmacia Biotech, North Ryde, New South Wales, Australia) for 2 h at 4 °C with rotation, incubated with 10 µl of neat DH-1 immune or preimmune serum for 2 h, and then 10 µl of protein A-Sepharose added for another 90 min at 4 °C. Each sample was washed twice in 1 ml of 1% Tween 20, 150 mM NaCl, 1 mM EDTA in phosphate-buffered saline and once in 1 ml of phosphate-buffered saline. Each protein A-Sepharose pellet was boiled in 50 µl of 2× reducing sample buffer, and 10 µl of the supernatant loaded onto a 10% SDS-polyacrylamide gel with protein size standards (BenchMark; Life Technologies, Inc.).

Following electrophoresis, the proteins were transferred to Immobilon P polyvinylidene difluoride transfer membrane (Millipore, Bedford, MA), and blocked by incubation in 3% nonfat milk, 0.1% Tween 20, Tris-buffered saline for 1 h at room temperature. All washes were in 1% milk, 0.1% Tween 20, Tris-buffered saline. The HM-2 antibody was used at 1:10,000 at 37 °C for 90 min, and biotinylated anti-rabbit (1:10,000; 60 min at room temperature) and streptavidin-HRP (1:5000; 60 min at room temperature) as above. Antibody binding was detected using the ECL Plus chemiluminescence system (Amersham Pharmacia Biotech) with Kodak X-Omat film.

In Vitro Nuclear Import Assay-- Analysis of nuclear import kinetics at the single cell level was performed using mechanically perforated HTC cells in conjunction with confocal laser scanning microscopy (CLSM) was as described previously (19-21). NLS-dependent nuclear protein import can be reconstituted in this system through the exogenous addition of cytosolic extract (reticulocyte lysate), an ATP-regenerating system, and transport substrate (0.2 mg/ml IAF-labeled fusion protein). In experiments where the ATP dependence of transport was tested, apyrase pretreatment was used to hydrolyze endogenous ATP in cytosolic extracts (10 min at room temperature with 800 units/ml) and perforated cells (15 min at 37 °C with 0.2 units/ml) (20, 28, 29), and transport assays were then performed in the absence of the ATP regenerating system (16, 20). Image analysis of CLSM files using the MacIntosh NIH Image 1.49 public domain software and curve fitting were performed as described (19, 21).

    RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Novel LMW MAP2 mRNAs Are Present in Rat Brain and Testis-- LMW and HMW isoforms of MAP2 arise from alternative mRNA splicing, with the HMW isoforms containing the ~3.7 kb of exon 9 sequence that encodes the large projection arm of about 150 kDa (Fig. 1). Thus, mRNAs encoding HMW and LMW isoforms can be distinguished on the basis of inclusion or exclusion of exon 9 sequences. The use of primer pairs designed to amplify mRNAs encoding HMW and LMW MAP2 isoforms (Fig. 2A) resulted in the production of multiple PCR products from both immature and mature brain and testis RNA samples (Fig. 2B). The identity of these products as MAP2 was confirmed by DNA blot analysis using a MAP2c cDNA.


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  		Fig. 1.   Presence of a CcN motif within known and novel MAP2 isoforms. The CcN motif within exon 10 of the MAP2 coding sequence is shown in single-letter amino acid code, indicated as a black bar above the diagram of HMW MAP2. It has the characteristic two clusters of positively charged amino acids (underlined) separated by a spacer of 10-12 amino acid residues of bipartite NLSs (14), flanked by consensus phosphorylation sites for protein kinase CK2 and the cyclin-dependent kinase cdc2 (phosphorylation site serine/threonines in bold type), both of which are known to regulate NLS-dependent nuclear import of proteins such as the SV40 large tumor-antigen (15, 16). Putative novel LMW isoforms based on the exon structure described for the human gene (6) are illustrated, with an asterisk to indicate the possibility of additional alternative exon splicing within the 5' and 3' coding sequences.


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  		Fig. 2.   RT/PCR and Southern blot analysis of brain and testis RNAs reveals multiple HMW and LMW MAP2 mRNAs. Panel A, schematic illustration of PCR rationale indicating primer position relative to known MAP2 coding sequence exons. Products are illustrated with boxes to represent primers, solid lines indicating regions expected to be present in amplified product, and dotted lines indicating regions predicted to be absent from amplified product due to limited extension time employed during amplification and alternative mRNA splicing. Panel B, left panels, amplification of HMW MAP2 mRNAs with primers 29 exon 9) and 30 (exon 18); right panels, amplification of LMW MAP2 mRNAs with primers 28.2 (exon 6) and 30 (exon 18). Input RNA was from adult (Ad) and day 15 (d15) testis and adult (Ad) and day 25 (d25) brain. Upper panels, ethidium bromide-stained gel; lower panels, autoradiographs corresponding to gels in upper panels following hybridization with cDNA encoding MAP2c sequence.

Selective amplification of HMW MAP2 mRNAs was achieved using a 5' primer in exon 9 (29). The predominant HMW MAP2 mRNA product sizes amplified varied between samples, with an ~800-bp product amplified from adult testis, two products of ~1120 and 1070 bp observed in day 15 testis, and a single prominent product of ~1070 bp amplified from both day 25 and adult brain RNA samples. As the focus of this study is on the identification of novel LMW isoforms, the identity of these products was not investigated beyond the use of Southern blot analysis to confirm hybridization with a MAP2 probe. The 1120-bp product could correspond to a product consisting of exons 9-12, 14, 15, 17, and 18 (corresponding to MAP2a or 2b), while the 800- and 1070-bp products could derive from variants lacking exons 10 and 11 (800-bp product) or exon 11 (1070-bp product).

Amplification of the LMW MAP2 transcripts using a 5' primer in exon 6 yielded a ~1040-bp product and a ~760-bp product in all but the adult brain mRNA sample. These products were shown to encode MAP2 products by DNA blot analysis and were used for the subsequent amplification and characterization of novel mRNA isoforms described below.

Nested PCR to achieve selective amplification of novel LMW MAP2 mRNAs from the products shown in Fig. 2 used a 3' primer in exon 11 (Fig. 3A), an exon not previously reported to be present in LMW MAP2 isoforms. Two products of approximately 320 and 520 bp were observed in testis and brain samples from both immature and mature animals (Fig. 3B). These products were both recognized by the MAP2c cDNA in Southern hybridization experiments, but only the 520-bp band was bound by oligonucleotides specific to exon 10 (Fig. 3C). The 320-bp band was purified and sequenced, which further identified it the product of a MAP2 mRNA spanning exon 6, 7, and 11, and excluding exons 9 and 10 (data not shown). Thus, the 320-bp band would encode a LMW MAP2 isoform (Fig. 3D). The 200-bp size difference between the two PCR products corresponds to the size of exon 10 in the human MAP2 cDNA (6), consistent with the amplification of an mRNA spanning exons 6, 7, 10, and 11 (Fig. 3D).


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  		Fig. 3.   Nested PCR amplification of LMW MAP2 mRNAs yields two novel products from testis and brain containing exons 10 and 11. Panel A, schematic illustration of primer position and rationale used for nested PCR. Products are shown with boxes representing primers, solid lines indicating regions expected to be present in amplified product, and dotted lines indicating regions predicted to be absent from amplified product, due to limited extension time employed during amplification and alternative mRNA splicing. Panel B, nested PCR amplification of LMW MAP2 PCR products shown in Fig. 2, re-amplified with primers 28.2 (exon 6) and 27.2 (exon 11). Amplification with neat (N) and 1:10 dilution (D) of input cDNA from first round PCR. Input RNA was from adult testis (Ad T), day 15 testis (d15 T), adult brain (Ad B), and day 25 brain (d25 B). Upper panel, ethidium bromide-stained gel; lower panel, autoradiograph corresponding to gel in upper panel following hybridization with cDNA encoding MAP2c sequence. Panel C, lane 1, total PCR product from nested PCR amplification of LMW MAP2 from adult testis; lane 2, isolated 320-bp band from lane 1 product. Upper panel, ethidium bromide-stained gel; lower panel, autoradiograph corresponding to gel in upper panel following hybridization with oligonucleotide 26.1 specific to exon 10. The identity of the 320-bp product was confirmed by direct sequence analysis. Panel D, schematic illustration of MAP2 mRNAs amplified by RT-PCR. The open bars represent the full predicted coding sequences of the novel LMW MAP2 mRNAs, while the filled bars list the exons that were documented to be present in each product.

Northern blot analysis was used to examine the relative abundance of these novel LMW mRNAs in brain and testis using a cDNA probe containing sequences present in exons 10 and 11 (Fig. 4). This probe yielded a hybridization signal at >9 kb in the brain RNA samples at all ages, demonstrating, as expected, the presence of this sequence in HMW MAP2 mRNAs. The relative intensity of the >9-kb band appeared greatest in the day 9 post partum brain sample. In addition, the day 9 brain sample contained a ~6-kb mRNA signal, which would correspond to one or more of the novel LMW MAP2 mRNAs lacking the 3.7-kb exon 9 sequence (Fig. 4B). In the testis, a very faint hybridization signal at >9 kb was detected in the adult sample, but distinct hybridization signals at ~6 kb were observed in testis RNAs at several ages (days 10, 15, and 35 and adult), again corresponding to novel LMW MAP2 mRNA. The time required to visualize the MAP2 signal using autoradiography was reproducibly <24 h for brain samples and 5-6 days for testis samples.


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  		Fig. 4.   Northern blot analysis with cDNA encoding parts of exons 10 and 11 illustrates novel LMW MAP2 transcript abundance in rat brain and testis. Panel A, RNA samples are from day 9 (D9), day 15 (D15), and adult (Ad) brain, and from day 10 (D10), day 15 (D15), day 25 (D25), day 35 (D35), and adult (Ad) testis. Upper panel, autoradiograph with positions of 9 kb (HMW) and 6 kb (LMW) MAP2 mRNA positions marked. Lower panel, ethidium bromide gel showing 28 S rRNA. Panel B, schematic illustration of the predicted mRNAs encoded by the transcripts detected in panel A.

Cell-specific Localization of MAP2 in the Nucleus by Immunohistochemistry-- To address the question of whether these novel transcripts are translated into MAP2 proteins, a fusion protein containing parts of exons 10 and 11 (MAP2 CcN-beta -Gal) was derived as described under "Materials and Methods" and used as an antigen to produce rabbit polyclonal antisera. One rabbit serum (DH-1) demonstrated cell-specific patterns of immunoreactivity in the brain (data not shown) and in the testis (Fig. 5, D and E). Within the seminiferous epithelium of the testis, binding of DH-1 was detected in the nucleus of Sertoli cells, spermatogonia, late pachytene spermatocytes (stages VII-XIV), and spermatids up to stage X. In addition, the cytoplasm of Sertoli cells was intensely labeled. Staining was clearly absent from both the nucleus and cytoplasm of early meiotic germ cells (preleptotene spermatocytes at stages VII through to stage VI pachytene spermatocytes). This pattern of DH-1 binding to male germ cells correlates with that observed with the HM2 monoclonal antibody to MAP2 (Fig. 5, A and B). However, with HM2, the staining in spermatogonial nuclei was less intense than that observed in the nuclei of meiotic and post-meiotic germ cells, and the Sertoli cell cytoplasmic and nuclear staining was faint. Labeling of peritubular call cytoplasm could also be observed with HM-2 (Fig. 5, A and B).


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  		Fig. 5.   Polyclonal antisera directed against the MAP2-CcN-beta -Gal fusion protein yields cell-specific pattern of immunoreactivity identical to that observed for MAP2 mRNAs in mature rat testis. A and B, HM-2 on day 60 rat testis at 1:400; C, no primary antibody on day 60 rat testis as control for HM-2 staining; D and E, DH-1 antiserum on day 40 rat testis at 1:2000; F, preimmune DH-1 antiserum on day 40 rat testis at 1:2000. Bar equals 100 µm in A and D, and 50 µm in B, C, E, and F. Stages of the rat cycle are indicated in A and D. Areas with primary spermatocytes (PS) and round spermatids (RS) are labeled, and spermatogonia are indicated in B and E.

Reactivity of the DH-1 polyclonal serum with MAP2 proteins in a lysate of day 7 rat brain (Fig. 6) was demonstrated using immunoprecipitation with the DH-1 antiserum, followed by Western blot analysis with the MAP2 HM2 monoclonal antibody. Reactivity of the DH-1 serum with both known HMW (>220 kDa) and novel LMW (~70 kDa) MAP2 proteins was observed.


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  		Fig. 6.   Immunoprecipitation and Western blot analysis documents reactivity of DH-1 with MAP2 proteins in day 7 rat brain lysate. Day 7 brain lysates were immunoprecipitated using preimmune serum or DH-1 and then examined by Western blot with HM-2 monoclonal antibody. Estimated sizes of precipitated proteins are indicated.

The NLS of MAP2 Is Capable of Targeting a Heterologous Protein to the Nucleus-- To test whether the putative CcN motif of MAP2 exon 10 (see Introduction and Fig. 1) is able to transport a heterologous and otherwise cytoplasmic carrier protein (beta -Gal from E. coli) to the nucleus, the fusion protein MAP2-CcN-beta -Gal was labeled with a fluorescent tag and its nuclear import kinetics measured in vitro using mechanically perforated HTC cells and CLSM (20, 28, 30) and compared with those for labeled beta -Gal alone. The MAP2-CcN motif was capable of targeting beta -Gal (476 kDa) to the nucleus (Fig. 7, A and B, left panel), maximally accumulating to levels over 2.5-fold (2.69 ± 0.08) those in the cytoplasm, with half-maximal levels being attained within 7.2 ± 0.6 min. As observed previously (16), beta -Gal was completely excluded from the nucleus in vitro (Fn/cmax = 0.48 ± 0.01; Fig. 7B, left panel). MAP2-CcN-beta -Gal was also found to localize in the nucleus of microinjected HTC cells (data not shown), in contrast to beta -Gal itself.


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  		Fig. 7.   Nuclear import of fusion protein MAP2-CcN-beta -Gal in vitro. A, CLSM images are shown for IAF-labeled MAP2-CcN-beta -Gal in the presence and absence of either exogenously added cytosol or an ATP-regenerating system as indicated after 30 min at room temperature (see "Materials and Methods"). B, nuclear import kinetics. Measurements represent the average of three separate experiments, where each point represents the average of 10-11 separate measurements for each of nuclear, cytoplasmic and background (autofluorescence) fluorescence. Data were fitted for the function Fn/c(t) = Fn/cmax × (1 - e-kt), where Fn/c is defined as the ratio of nuclear to cytoplasmic fluorescence after the subtraction of fluorescence due to autofluorescence (17, 20). Results for MAP2-CcN-beta -Gal are compared (left panel) to those for beta -galactosidase (beta -Gal).

Dependence of Nuclear Uptake Conferred by the MAP2-CcN Motif on ATP and Cytosolic Factors-- We tested the dependence of nuclear transport mediated by the MAP2-CcN motif on cellular factors. Conventional NLS-dependent nuclear protein import in vitro is known to be dependent both on ATP and on the addition of exogenous cytosol (16, 28, 29). The latter supplies the NLS-binding/nuclear pore complex-docking dimer of importin 58/97 (31) as well as the monomeric GTP-binding protein/GTPase Ran/TC4 (32, 33) and interacting proteins (see Ref. 34), all of which are essential for nuclear accumulation. Nuclear accumulation of MAP2-CcN-beta -Gal was found to be dependent on both ATP and exogenous cytosol (Fig. 7, A and B, right panel), whereby maximal accumulation was 1.0 ± 0.08 and 1.22 ± 0.06 in the absence of cytosol or the ATP-regenerating system, respectively. The MAP2-CcN motif can be concluded to confer all of the hallmarks of conventional nuclear protein import, being dependent on energy and cytosolic factors.

    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

This study makes a number of fundamentally new observations with respect to MAP2 expression and subcellular localization, raising the possibility that particular MAP2 isoforms may have a functional role in the nucleus. The results demonstrate: 1) the existence at both the mRNA and protein level of previously unidentified LMW MAP2 forms, which contain exon 10/11 encoding a phosphorylation-regulated targeting signal (CcN motif) for regulated nuclear protein import; 2) that this signal is functional in targeting a large heterologous carrier protein to the nucleus; and 3) that in contrast to currently widely accepted notions, MAP2 is expressed in non-neural tissue.

Our initial RT-PCR experiments demonstrated the presence of multiple mRNAs that would encode both HMW and LMW MAP2 (Fig. 2). We then performed nested PCR with primers designed to amplify selectively novel LMW MAP2 mRNAs, choosing a 3' primer that contains nucleotide sequences absent in MAP2c mRNA. The PCR products that resulted were shown to correspond to amplification of novel MAP2 LMW splice variants in brain and testis that both contain and exclude exon 10 (Fig. 3). One or both of these novel mRNAs was detected as a 6-kb transcript by Northern blot in the day 9 rat brain (Fig. 4). This 6-kb signal was not detected under the same conditions in the older brain samples, suggesting that the level of these novel LMW mRNAs declines as the brain matures. As previously documented for MAP2c, the novel LMW MAP2 transcripts containing and excluding exon 10 appear to be developmentally regulated in brain, with LMW MAP2 transcripts down-regulated during synaptogenesis (36, 37). The novel LMW mRNAs were detected in testis RNA samples ranging from day 10 postpartum to adult. The relatively lower signal intensity observed in the day 15 and 25 testis RNA samples may reflect emergence during this period of the early meiotic germ cell population, which does not contain MAP2 protein and mRNA (2). In addition, the difference in exposure times required to obtain a signal on Northern blots with brain versus testis RNA indicated that the relative abundance of all MAP2 mRNAs is much higher in the brain than in the testis at all postnatal ages examined. Stabilization of the MAP2 transcript has been described to facilitate accumulation of protein from low levels of mRNA in neural tissue (35), and a similar process may occur in testis cells. The current observation suggestive of down-regulation of other LMW MAP2 isoform(s) indicates that they may also perform developmentally regulated functions, possibly within the nucleus (see below). Alternatively, the population of cells containing the novel splice variants may reduce in proportion to the total number of cells as development proceeds.

In the testis, the pattern of immunohistochemical staining by the DH-1 antiserum, prepared with the exon 10/11-containing MAP2-CcN-beta -Gal fusion protein (Fig. 5, D and E), was consistent with that predicted from previous Northern blot analysis of MAP2 in RNA from enriched cell preparations (2). It also corresponded to the pattern observed using the commercially available MAP2-specific HM2 antibody (Fig. 5, A and B). Taken together, these results suggest that the pattern observed reflects the expression of LMW forms of MAP2 protein that contain exon 10/11. The precipitation of a LMW MAP2 protein from a rat brain lysate (Fig. 6) and from preparations of rat brain microtubules (data not shown) using the DH-1 serum further confirms that MAP2 exon 10/11 sequences are present in a LMW MAP2 isoform(s). Immunoprecipitation with the rabbit polyclonal antiserum raised against exon 10 and 11 sequences (absent from MAP2c) extracts a low molecular weight protein that binds the HM2 monoclonal antibody that is used to identify MAP2 protein. Detection of a ~70-kDa protein with these reagents indicates that there is a LMW MAP2 isoform that contains sequences present in exons 10 and/or 11. This protein would be encoded by the novel 6-kb transcript identified in the Northern analysis (Fig. 4), and could encode the protein we hypothesize to exist in the testis, which is a novel LMW isoform containing the nuclear localization sequence we have shown to be functional (Fig. 7).

In immunohistochemical analyses, the DH-1 serum detected protein in Sertoli cells, Leydig cells, spermatogonia, and post-meiotic germ cells (Fig. 5, D and E), but failed to detect protein in a large proportion of meiotic germ cells, consistent with the staining pattern obtained using the commercially available MAP2-specific HM2 antibody (Fig. 5, A and B). While this indicates that the DH-1 serum recognizes MAP2 in tissue sections, one cannot definitively conclude that the staining due to the DH-1 serum is exclusively attributable to the novel LMW MAP2 form, since the serum can clearly recognize proteins, additional to those recognized by the HM2 antibody, of sizes not previously documented for MAP2 (data not shown). We are currently investigating whether the additional proteins recognized by DH-1, are further novel MAP2 forms possibly lacking N- or C-terminal MAP2 exons that would not be recognized by HM2.

From the data presented above, it seems reasonable to attribute the nuclear staining observed in particular testis cells (Fig. 5) to the predominant LMW MAP2 form containing the CcN motif, and perhaps also to the CcN motif containing HMW forms. The MAP2 CcN motif within exon 10 is clearly able to target the heterologous protein beta -galactosidase into the nucleus in vitro, dependent on both ATP and exogenous cytosol, as shown here (Fig. 7), with the kinetics of nuclear import comparable to those of other CcN motif carrying proteins such as T-Ag and SWI5 (16, 17). Measurements (data not shown) of the binding affinity of the NLS-recognizing importin alpha /beta subunits for the MAP2 CcN motif using a direct binding assay (21) indicate an apparent dissociation constant of approximately 50 nM, typical of that for other bipartite NLS-containing proteins (e.g. Ref. 28). All of these results strongly imply that MAP2 exon 10 contains a conventional NLS. Preliminary results (data not shown) suggest that both CK2 and cdc2 can specifically phosphorylate the MAP2 CcN motif, which implies that these kinase sites may regulate MAP2 localization in similar fashion to T-Ag, SWI5, and nucleoplasmin (38). Intriguingly, also within the spacer of the bipartite NLS of the MAP2 CcN motif is the PSTV sequence, which closely resembles sites for Ser/Thr-O-linked N-acetylglucosamine glycosylation from a number of different proteins, including rat neurofilament (NF-M), alpha B crystallin, and nuclear proteins such as the estrogen receptor and serum response factor (39, 40). The possibility that dynamic glycosylation at this site may mask or alter NLS recognition in conjunction with CK2 and cdk phosphorylation, provides a number of fascinating permutations in terms of possible regulatory mechanisms for regulation of the MAP2 NLS and the focus of future work in this laboratory is to assess these possibilities directly. We predict that an explanation for the absence of MAP2 nuclear localization in neuronal tissue, despite the presence of exon 10 in the HMW isoforms, MAP2a and MAP2b, will emerge as our understanding of the regulatory mechanisms of MAP2 localization and function of its NLS is further expanded. Phosphorylation, glycosylation, and masking by the binding of other molecules such as calmodulin may each have a role to play in neural tissue.

The role of nuclear localized MAP2 can only be surmised at this stage; that cytoskeletally associated elements can localize in the nucleus has been shown for Tau in a neuroblastoma line (41), the gamma -tubulin complex in yeast in cell cycle-dependent fashion (42), and actin binding proteins such as cofilin, whose nuclear translocation is regulated by stress-induced dephosphorylation (43, 44). Significantly, the regulated pattern of MAP2 mRNA expression in the testis coincides with the progression of the germ cells into their post-mitotic phase of development, and using immunohistochemistry, MAP2 protein was detected in this study in the nucleus of germ cells in the later stages of meiosis and in the nuclei of round spermatids. This pattern correlates with the time at which calmodulin is reported to move from the cytoplasm into the nucleus of germ cells (45, 46). Significantly, the latter study identified a number of calmodulin-binding proteins that were nuclear under these conditions, including one of 75 kDa, which was associated with the nuclear matrix of pachytene spermatocytes. The calmodulin binding site has been mapped to the region that would be encoded by exons 10 and 11 (3), raising the intriguing possibility that cell cycle-regulated movement into the nucleus of the novel CcN-motif-containing LMW MAP2 form may directly mediate calmodulin movement, through a type of piggy-back transport mechanism. That NLS-lacking proteins can accumulate in the nucleus through specific binding to NLS-containing partner proteins has been shown for several proteins, including adenovirus DNA polymerase (47), and interleukin-5 and its receptor subunits (48), while the function of calmodulin in regulating nuclear functions has been documented (49). The role of the functional CcN motif within novel LMW MAP2 forms, able to confer regulated nuclear import on a heterologous protein, may thus primarily be to effect regulated nuclear translocation of other molecules, including calmodulin as indicated, various RII isoforms (see Ref. 50), and tubulin, all of which bind to MAP2c. Other possibilities for the potential function of MAP2 in the nucleus are implied by studies linking MAP2 to changes in stimulation of DNA synthesis (51) to reports demonstrating the binding of MAP2 to centromeric DNA (52, 53) and a proposed structural role in the nuclear remodeling that takes place in the meiotic and haploid germ cells (54). The removal of histones and their replacement by transition proteins and subsequently by protamines is a predominant feature of these stages of spermatogenesis (55), and the mechanisms by which this occurs in concert with nuclear reshaping, possibly with the involvement of MAP2 up until step 7, are not fully understood. In the case of all of these possible roles of nuclear LMW MAP2, the CcN motif would enable nucleocytoplasmic localization of MAP2 forms and any proteins bound to it to be regulated precisely according to the proliferation state and stage of the cell cycle. Definitive determination of the precise composition of MAP2 forms in the testis, and of the signals regulating their nuclear import, should assist in elucidating the specific functions of the different forms in the nucleus and elsewhere in testicular cell development.

    ACKNOWLEDGEMENTS

We gratefully acknowledge the technical assistance of Terri Meehan in performing some of the immunohistochemical work, as well as Mary-Jane Gething and Bridget Shafit-Zagardo for critical reading of the manuscript, and Bruce Loveland and Ian van Driel for valuable discussions and assistance in analysis of the data.

    FOOTNOTES

* This work was supported by grants from the National Health and Medical Research Council of Australia and the Institute for Advanced Studies collaborative program (to A. N. U.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§ To whom correspondence should be addressed: Inst. of Reproduction and Development, Monash Medical Centre, 246 Clayton Rd., Clayton, Victoria 3168, Australia. Tel.: 613-9594-7125; Fax: 613-9594-7111; E-mail: kate.loveland{at}med.monash.edu.au.

    ABBREVIATIONS

The abbreviations used are: MAP, microtubule-associated protein; CcN, T-Ag CcN motif (for CK2 site, cdk site, and NLS); CK2, protein kinase CK2; cdc2, cyclin-dependent kinase (cdk); CLSM, confocal laser scanning microscopy; LMW, low molecular weight; HMW, high molecular weight; MAP2-CcN-beta -Gal, MAP2-CcN beta -galactosidase fusion protein; NLS, nuclear localization sequence; PKA, cAMP-dependent protein kinase; T-Ag, simian virus SV40 large tumor-antigen; bp, base pair(s); kb, kilobase pair(s); RT, reverse transcriptase; PCR, polymerase chain reaction; IAF, 5-iodacetamido-fluorescein.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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