Defective calmodulin-mediated nuclear transport of the sex-determining region of the Y chromosome (SRY) in XY sex reversal

The sex-determining region of the Y chromosome (SRY) plays a key role in human sex determination, as mutations in SRY can cause XY sex reversal. Although some SRY missense mutations affect DNA binding and bending activities, it is unclear how others contribute to disease. The high mobility group domain of SRY has two nuclear localization signals (NLS). Sex-reversing mutations in the NLSs affect nuclear import in some patients, associated with defective importin-beta binding to the C-terminal NLS (c-NLS), whereas in others, importin-beta recognition is normal, suggesting the existence of an importin-beta-independent nuclear import pathway. The SRY N-terminal NLS (n-NLS) binds calmodulin (CaM) in vitro, and here we show that this protein interaction is reduced in vivo by calmidazolium, a CaM antagonist. In calmidazolium-treated cells, the dramatic reduction in nuclear entry of SRY and an SRY-c-NLS mutant was not observed for two SRY-n-NLS mutants. Fluorescence spectroscopy studies reveal an unusual conformation of SRY.CaM complexes formed by the two n-NLS mutants. Thus, CaM may be involved directly in SRY nuclear import during gonadal development, and disruption of SRY.CaM recognition could underlie XY sex reversal. Given that the CaM-binding region of SRY is well-conserved among high mobility group box proteins, CaM-dependent nuclear import may underlie additional disease states.     

Site-directed mutagenesis of serine 158 demonstrates its role in spinach leaf sucrose-phosphate synthase modulation

Site-directed mutagenesis of spinach sucrose-phosphate synthase (SPS) was performed to investigate the role of Ser158 in the modulation of spinach leaf SPS. Tobacco plants expressing the spinach wild-type (WT), S158A, S158T and S157F/S158E SPS transgenes were produced. Expression of transgenes appeared not to reduce expression of the tobacco host SPS. SPS activity in the WT and the S158T SPS transgenics showed light/dark modulation, whereas the S158A and S157F/S158E mutants were not similarly light/dark modulated: the S158A mutant enzyme was not inactivated in the dark, and the S157F/S158E was not activated in the light. The inability to modulate the activity of the S158A mutant enzyme by protein phosphorylation was demonstrated in vitro. The WT spinach enzyme immunopurified from dark transgenic tobacco leaves had a low initial activation state, and could be activated by PP2A and subsequently inactivated by SPS-kinase plus ATP. Rapid purification of the S158A mutant enzyme from dark leaves of transgenic plants using spinach-specific monoclonal antibodies yielded enzyme that had a high initial activation state, and pre-incubation with leaf PP2A or ATP plus SPS-kinase (the PKIII enzyme) caused little modulation of activity. The results demonstrate the regulatory significance of Ser158 as the major site responsible for dark inactivation of spinach SPS in vivo, and indicate that the significance of phosphorylation is the introduction of a negative charge at the Ser158 position.     

Loss of Fgfr2 leads to partial XY sex reversal

In mammals, sex is determined in the bipotential embryonic gonad by a balanced network of gene actions which when altered causes disorders of sexual development (DSD, formerly known as intersex). In the XY gonad, presumptive Sertoli cells begin to differentiate when SRY up-regulates SOX9, which in turn activates FGF9 and PGDS to maintain its own expression. This study identifies a new and essential component of FGF signaling in sex determination. Fgfr2 mutant XY mice on a mixed 129/C57BL6 genetic background had either normal testes, or developed ovotestes, with predominantly testicular tissue. However, backcrossing to C57BL6 mice resulted in a wide range of gonadal phenotypes, from hypoplastic testes to ovotestes with predominantly ovarian tissue, similar to Fgf9 knockout mice. Since typical male-specific FGF9-binding to the coelomic epithelium was abolished in Fgfr2 mutant XY gonads, these results suggest that FGFR2 acts as the receptor for FGF9. Pgds and SOX9 remained expressed within the testicular portions of Fgfr2 mutant ovotestes, suggesting that the Prostaglandin pathway acts independently of FGFR2 to maintain SOX9 expression. We could further demonstrate that double-heterozygous Fgfr2/Sox9 knockout mice developed ovotestes, demonstrating that both Fgfr2 and Sox9 can act as modifier intersex genes in the heterozygous state. In summary, we provide evidence that FGFR2 is important for male sex determination in mice, thereby rendering human FGFR2 a candidate gene for unsolved DSD cases such as 10q26 deletions.     

Nuclear protein import: specificity for transport across the nuclear pore

Transport of proteins into the cell nucleus is thought to require specific localization sequences and may be mediated by nuclear pores. Following microinjection into fused cultured cells, nuclear protein import was directly monitored by fluorescence microscopy using B-phycoerythrin (PE; Mr 240,000) coupled to synthetic peptides corresponding to the simian virus 40 (SV-40) large T antigen nuclear localization signal. Peptides with a single amino acid replacement found in a cytoplasmic mutant of T antigen (cT) failed to promote uptake. Further studies with deletion peptides revealed the minimum sequence requirements for efficient nuclear import of PE conjugates to be similar to those previously defined genetically for large T antigen itself. No competitive inhibition of uptake was observed in cells expressing nuclear or cytoplasmic T antigen. Nuclear import was time- and temperature-dependent. The lectin wheat germ agglutinin (WGA) binds to glycoproteins bearing O-linked GlcNAc on the cytoplasmic face of the nuclear pore in vitro [J.A. Hanover et al. (1987) J. Biol. Chem. 262, 9887-9894] and in vivo. Microinjection of WGA into the cytoplasm of living cells did not alter the diffusion of dextran (Mr 10,000) into the nucleus, but blocked the uptake of PE conjugates. This inhibition was reversed when a competing saccharide was introduced into the cytoplasm.     

A cytoplasmically anchored nuclear protein interferes specifically with the import of nuclear proteins but not U1 snRNA

A cytoplasmically anchored mutant SV40 T antigen, FS T antigen, was shown previously to interfere specifically with the nuclear import of a heterologous nuclear protein, adenovirus 5 fiber protein, in cultured monkey cells (Schneider, J., C. Schindewolf, K. van Zee, and E. Fanning. 1988. Cell. 54:117-125; van Zee, K., F. Appel, and E. Fanning. 1991. Mol. Cell. Biol. 11:5137-5146). In this report, we demonstrate that FS T antigen also interferes with the nuclear import of adenovirus E1A and a peptide-albumin conjugate bearing multiple copies of the T antigen nuclear localization signal, but not with the import of U1 snRNA. A kinetic analysis indicates that nuclear import of the albumin- peptide conjugate is inhibited only when high intracellular concentrations of FS T antigen are reached. After microinjection into the cytoplasm of cultured cells, purified FS T antigen protein does not accumulate at the nuclear periphery, but rather is distributed in a punctate pattern throughout the cytoplasm. These data support a model in which cytoplasmic anchoring of FS T antigen enables the mutant protein to sequester and titrate out a cellular factor which is required for nuclear protein but not U1 snRNA import.     

Spatial changes in calcium signaling during the establishment of neuronal polarity and synaptogenesis

Calmodulin (CaM) potentiates Ca(2+)-dependent signaling pathways in both the cytoplasm and nucleus. We have investigated the mechanism of CaM nuclear transport using tissue culture cell microinjection and a permeabilized cell import assay. The inhibition of CaM import by the translocation inhibitor wheat germ agglutinin (WGA) and by chilling, indicates that CaM import is facilitated, but because ATP depletion does not affect CaM import, the mechanism does not appear to be active. Chilling and WGA arrest persist in ATP-depleted cells, indicating that CaM is not retained in the cytoplasm by an ATP-dependent mechanism. In permeabilized cells, both Ca(2+)-CaM and Ca(2+)-free CaM are sensitive to extract-dependent WGA and chilling import inhibition. Titration experiments in microinjected and permeabilized cells indicate that a saturable cytosolic factor(s) mediates chilling and WGA arrest.