Calmodulin content, Ca2‐dependent calmodulin binding proteins, and testis growth: Identification of Ca2‐dependent calmodulin binding proteins in primary spermatocytes

Trejo, Raquel; Delhumeau, Graciela

doi:10.1002/(sici)1098-2795(199709)48:1<127::aid-mrd15>3.3.co;2-t

Cited by 2 publications

(2 citation statements)

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“…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.…”

Section: Discussionsupporting

confidence: 54%

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

Loveland

Herszfeld

Chu

et al. 1999

Journal of Biological Chemistry

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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...

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Section: Discussionsupporting

confidence: 54%

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

Loveland

Herszfeld

Chu

et al. 1999

Journal of Biological Chemistry

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“…Hence, components of the importin/Ran GTP-or CaM-dependent pathways may act to regulate nuclear accumulation of SOX9 into Sertoli cells to allow a sufficient amount of SOX9 protein to activate the anti-Mullerian hormone gene, MIS, SF1, and other genes. The subcellular localization and expression of CaM varies during testis development with CaM levels increasing just prior to the proliferation of Sertoli cells and spermatagonia (46,47). The calcium-CaM status of the Sertoli cells could be critical at this time when cells in the XY gonad are being committed as Sertoli cells.…”

Section: Discussionmentioning

confidence: 99%

A SOX9 Defect of Calmodulin-dependent Nuclear Import in Campomelic Dysplasia/Autosomal Sex Reversal

Argentaro

Sim

Kelly

et al. 2003

Journal of Biological Chemistry

102

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During mammalian sex determination, SOX9 is translocated into the nuclei of Sertoli cells within the developing XY gonad. The N-terminal nuclear localization signal (NLS) is contained within a SOX consensus calmodulin (CaM) binding region, thereby implicating CaM in nuclear import of SOX9. By fluorescence spectroscopy and glutaraldehyde cross-linking, we show that the SOX9 HMG domain and CaM interact in vitro. The formation of a SOX9⅐CaM binary complex is calcium-dependent and is accompanied by a conformational change in SOX9. A CaM antagonist, calmidazolium chloride (CDZ), was observed to block CaM recognition of SOX9 in vitro and inhibit both nuclear import and consequent transcriptional activity of SOX9 in treated cells. The significance of the SOX9-CaM interaction was highlighted by analysis of a missense SOX9 mutation, A158T, identified from a XY female with campomelic dysplasia/ autosomal sex reversal (CD/SRA). This mutant binds importin ␤ normally despite defective nuclear import. Fluorescence and quenching studies indicate that in the unbound state, the A158T mutant shows a similar conformation to that of the WT SOX9, but in the presence of CaM, the mutant undergoes unusual conformational changes. Furthermore, SOX9-mediated transcriptional activation by cells expressing the A158T mutant is more sensitive to CDZ than cells expressing WT SOX9. These results suggest first that CaM is involved in the nuclear transport of SOX9 in a process likely to involve direct interaction and second, that CD/SRA can arise, at least in part, from a defect in CaM recognition, ultimately leading to reduced ability of SOX9 to activate transcription of cartilage and testes-forming genes. SOX1 (Sry-related HMG box) proteins are a large family of transcription factors that play critical roles in cell fate, differentiation, and development and show diverse, overlapping expression profiles (1, 2). SOX proteins activate the transcription of target genes by binding to DNA in a sequence-specific manner through their HMG box and by interacting with specific partner proteins (1, 2).SOX9, an early embryonically expressed gene, has a role in binding to and regulating a number of genes in the chondrogenesis pathway (3-5) and testis formation pathway (6). The influence of SOX9 upon these pathways is evident where mutations in SOX9 result in the disease campomelic dysplasia/ autosomal sex reversal (CD/SRA), a severe bone malformation syndrome in which most XY individuals show male-to-female sex reversal (7,8). Unlike frameshift and nonsense mutations that occur throughout the open reading frame of human SOX9, all known missense point mutations in SOX9 that cause CD have been localized at the HMG box (9).During early human and mouse embryogenesis, SOX9 is expressed in the cytoplasm of Sertoli cells in both sexes, but by gestational week 7, at the onset of SRY expression in male embryos, SOX9 moves into the nucleus (10, 11). Here, SOX9 activates the gene for mullerian inhibitory substance, which is required for the regression of the female re...

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Calmodulin content, Ca2‐dependent calmodulin binding proteins, and testis growth: Identification of Ca2‐dependent calmodulin binding proteins in primary spermatocytes

Cited by 2 publications

References 44 publications

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

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

A SOX9 Defect of Calmodulin-dependent Nuclear Import in Campomelic Dysplasia/Autosomal Sex Reversal

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