A SOX9 defect of calmodulin-dependent nuclear import in campomelic dysplasia/autosomal sex reversal.

Abstract

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 beta 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.

• Authors: Anthony Argentaro, Helena Sim, Sabine Kelly, Scott Preiss, Andrew Clayton, David A Jans, Vincent R Harley
• Journal: The Journal of biological chemistry (J Biol Chem) Vol. 278 Issue 36 Pg. 33839-47 (Sep 05 2003) ISSN: 0021-9258 [Print] United States
• PMID: 12810722 (Publication Type: Journal Article, Research Support, Non-U.S. Gov't)

Chemical References

• Calmodulin
• Cross-Linking Reagents
• High Mobility Group Proteins
• Imidazoles
• Nuclear Localization Signals
• Recombinant Proteins
• SOX9 Transcription Factor
• Transcription Factors
• beta Karyopherins
• Acrylamide
• calmidazolium
• Egtazic Acid
• Glutaral

Topics

• Acrylamide (pharmacology)
• Active Transport, Cell Nucleus
• Amino Acid Sequence
• Animals
• COS Cells
• Calmodulin (chemistry, metabolism)
• Cell Nucleus (metabolism)
• Cross-Linking Reagents (pharmacology)
• Disorders of Sex Development
• Dose-Response Relationship, Drug
• Egtazic Acid (pharmacology)
• Electrophoresis, Polyacrylamide Gel
• Escherichia coli (metabolism)
• Female
• Glutaral (pharmacology)
• High Mobility Group Proteins (genetics, physiology)
• Imidazoles (pharmacology)
• Immunohistochemistry
• Male
• Models, Biological
• Molecular Sequence Data
• Mutation
• Mutation, Missense
• Nuclear Localization Signals
• Protein Binding
• Protein Conformation
• Protein Structure, Tertiary
• Recombinant Proteins (metabolism)
• SOX9 Transcription Factor
• Sequence Homology, Amino Acid
• Sertoli Cells (metabolism)
• Spectrometry, Fluorescence
• Time Factors
• Transcription Factors (genetics, physiology)
• Transcription, Genetic
• Transfection
• beta Karyopherins (chemistry)