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

Abstract

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-␤-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 ϳ70-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.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 ϳ74 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 ϳ2.5 and 3.5 kb in the haploid spermatids. Each of these transcripts contain coding sequence from both the 5Ј and 3Ј ends of MAP2 (2). The initial descriptions of MAP2 indicated that three isoforms existed, with the ϳ288-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 ϳ70-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 phosphatidy...

Analysis of activin/TGFB-signaling modulators within the normal and dysfunctional adult human testis reveals evidence of altered signaling capacity in a subset of seminomas

“…Protein loading was indicated by blotting for a-tubulin (Sigma Chemical Co). Briefly, lysates were prepared by homogenization at 4 8C in RIPA buffer (150 mM sodium chloride, 1% Nonidet P-40, 0.5% Tween-20, 0.1% SDS, 1 mM ethylenediaminetetraacetic acid) in the presence of protease inhibitors (Protease Inhibitor Cocktail Set III, Calbiochem, San Diego, CA, USA), as previously described (Loveland et al 1999). Protein concentration was determined using the BioRad DC protein assay (Bio-Rad).…”

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Expression of hedgehog signalling components in adult mouse testis

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“…Protein samples from mouse tissues were prepared by homogenization at 4°C in RIPA buffer (1% Nonidet P-40, 0.5% sodium deoxycholate, 0.1% sodium dodecyl chloride in PBS) in the presence of protease inhibitors (Protease Inhibitor Cocktail Set III, Calbiochem, NSW, Australia), essentially as described (Loveland et al, 1999). Sample protein concentration was determined using the Bio-Rad DC protein assay (Bio-Rad, NSW, Australia), and 20 g of protein per lane for importins ␤1, ␤3, and ␣3 and 80 g per lane for importin ␣4 were loaded onto 12% sodium dodecyl sulfate (SDS) -polyacrylamide gel electrophoresis gels with protein size standards (PageRuler Prestained Protein Ladder, Fermentas).…”