Abstract
A predicted alanine to proline substitution in Stat5b that results in profound short stature, growth hormone insensitivity, and immunodeficiency represents the first natural mutation of this transcription factor in a human. To understand the mechanisms responsible for these pathophysiological abnormalities, we have studied the biochemical and biophysical properties of the mutant Stat5b molecule. In a cellular reconstitution model growth hormone robustly stimulated tyrosine phosphorylation and transcriptional activity of wild-type Stat5b while Stat5bA630P was minimally modified and did not promote reporter gene expression. Steady state levels of Stat5bWT were approximately 3-fold higher than Stat5bA630P in cell extracts prepared with nonionic detergents. Although initial rates of biosynthesis of both proteins were similar, pulse-chase experiments established that the apparent half-life of newly synthesized soluble Stat5bA630P was <15% of Stat5bWT (3.5 h versus >24 h). Stat5bA630P accumulated in cells primarily in cytoplasmic inclusion bodies. Structural analysis of the isolated SH2 domain containing the A630P mutation showed that it resembled the wild-type SH2 segment but that it exhibited reduced thermodynamic stability and slower folding kinetics, displayed an increased hydrophobic surface, and was prone to aggregation in solution. Our results are compatible with a model in which Stat5bA630P is an inactive transcription factor by virtue of its aberrant folding and diminished solubility triggered by a misfolded SH2 domain. The potential for aggregation and formation of cytoplasmic inclusions raises the possibility that Stat5bA630P could produce additional defects through inhibition of proteasome function.
Highlights
Initiated tyrosine phosphorylation, dimerization, transport into the nucleus, binding to DNA response elements on target genes, and recruitment of a complex of co-activator proteins that stimulate transcription [2, 3]
Many of the growth promoting actions of Growth hormone (GH) are mediated by the peptide growth factor, IGF-I (6 – 8), and IGF gene expression is under the control of GH [9]
Our observations demonstrate that this individual has a protein folding disorder where the aberrantly folded SH2 domain of the mutant Stat5b molecule triggers primary transcription factor deficiency and protein aggregation
Summary
Recombinant Plasmids and Adenoviruses—An expression plasmid in pcDNA3 and an adenovirus encoding NH2-terminal FLAG-tagged rat Stat5bWT have been described previously [10], as have an expression plasmid for the mouse GH receptor and for FLAG-tagged rat Stat5bY699F and reporter plasmids HS7-TK-Luc and HS7-IGF P1-Luc [21]. Whole cell protein extracts were prepared starting 1 h after infection with the Stat5b adenovirus and assessed by immunoblotting with primary antibodies to FLAG M2 and tubulin. Studies of Protein Stability—Cos-7 cells in 60-mm dishes were transiently transfected with expression plasmids encoding either Stat5bWT or Stat5bA630P (500 ng). Equilibrium folding and unfolding were monitored by CD absorbance at 222 nm upon increasing guanidine HCl concentrations from 0 to 6 M at 0.25 M intervals to obtain free energy differences for each transition as described previously [23]. Stopped flow kinetics were measured on a Hi-Tech SF61-DX2 fluorometer using excitation of a 295- and 310-nm band pass filter For these analyses, unfolded proteins (1– 4 M) in 6 M guanidine HCl were rapidly diluted (1:10 ratio) into renaturation buffer. Molecular Modeling—Modeling was performed using Swiss Model, a program that predicts structures reliably (root mean square deviation Ͻ 2 Å) for sequences with at least 50 – 60% identity, using coordinates from the crystal structure of human Stat (Swiss Protein Database 1YVL) as the template [24]
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