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J. Biol. Chem., Vol. 278, Issue 36, 33839-33847, September 5, 2003

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

From the ^aHuman Molecular Genetics Laboratory, Prince Henry's Institute of Medical Research, Level 4 Block E, Monash Medical Centre, 246 Clayton Rd., Clayton, Victoria, 3168, Australia, the ^bDepartment of Anatomy and Cell Biology and Howard Florey Institute, the Departments of ^eGenetics and ^fBiochemistry and Molecular Biology, University of Melbourne, Victoria, 3010, Australia, the ^hDepartment of Biochemistry and Molecular Biology, Monash University, Clayton, Victoria, 3168, Australia, the ⁱAustralian Research Council Centre of Excellence for Biotechnology and Development, Monash University, Clayton 3168, Australia, and the ^gLudwig Institute for Cancer Research, P. O. Box 2008, Royal Melbourne Hospital, Parkville, Victoria 3050, Australia

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 SOX9·CaM 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.

Received for publication, February 27, 2003 , and in revised form, June 6, 2003.

^* This work was supported by Australian National Health and Medical Research Council Grants 983001 and 198713 (to V. R. H.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

^c Both authors contributed equally to this work.

^d Present address: MRC Molecular Haematology Unit, Institute of Molecular Medicine, John Radcliffe Hospital, Headington, Oxford OX3 9DU, United Kingdom.

^j To whom correspondence should be addressed: Vincent R. Harley, Prince Henry's Institute of Medical Research, Level 4 Block E, Monash Medical Centre, 246 Clayton Rd., Clayton, Victoria, 3168, Australia. Tel.: 61-395943244; Fax: 61-395946125; E-mail: Vincent.Harley{at}phimr.monash.edu.au.

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