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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 Loveland, Daniella Herszfeld, Brendan Chu, Emily Rames, Elizabeth Christy, Lyndall J. Briggs^¶, Rushdi Shakri^¶, David M. de Kretser, and David A. Jans^¶

From the  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

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 ~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.

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