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J. Biol. Chem., Vol. 281, Issue 10, 6552-6558, March 10, 2006

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Aberrant Folding of a Mutant Stat5b Causes Growth Hormone Insensitivity and Proteasomal Dysfunction^*

From the Department of Biochemistry and Molecular Biology, and Department of Pediatrics, Oregon Health & Science University, Portland, Oregon 97239

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 Stat5b^A630P was minimally modified and did not promote reporter gene expression. Steady state levels of Stat5b^WT were 3-fold higher than Stat5b^A630P 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 Stat5b^A630P was <15% of Stat5b^WT (3.5 h versus >24 h). Stat5b^A630P 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 Stat5b^A630P 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 Stat5b^A630P could produce additional defects through inhibition of proteasome function.

Received for publication, October 5, 2005 , and in revised form, November 14, 2005.

^* This work was supported in part by National Institutes of Health Grants F32 DK070447 (to D. J. C.), R01 DK063073 (to P. R.), and R01 CA58110 (to R. G. R.). 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.

¹ To whom correspondence should be addressed: Dept. of Biochemistry and Molecular Biology, Oregon Health & Science University, 3181 SW Sam Jackson Rd., Portland, OR 97239. Tel.: 503-494-0536; Fax: 503-494-8393; E-mail: rotweinp{at}ohsu.edu.

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