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

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 Stat5b A630P was minimally modified and did not promote reporter gene expression. Steady state levels of Stat5bWT were ϳ3-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). 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 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.Sequence-specific transcription factors are modular proteins containing distinct domains that mediate their actions, including the ability to move among different subcellular compartments, to bind to DNA in chromatin in the nucleus, and to interact with transcriptional co-activators, co-repressors, and other regulatory molecules (1). The Stat 2 family consists of seven related proteins (Stats 1, 2, 3, 4, 5a, 5b, and 6) that are responsible for many of the transcriptional effects of cytokines, growth factors, and hormones (2, 3). Stat proteins are found in latent form in the cytoplasm of unstimulated cells. They are activated upon ligand binding to its receptor by a series of steps consisting of receptorinitiated 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).Growth hormone (GH) plays a central role in regulating somatic growth and intermediary metabolism in many vertebrate species, including humans (4). Upon binding to its transmembrane receptor, GH triggers the activation of the receptor-associated tyrosine-protein kinase Jak2, which in addition to recruiting a variety of other signaling molecules leads to the activation of Stats 1, 3, 5a, and 5b (4, 5). Many ...

Phosphorylation of a Conserved Serine in the Deoxyribonucleic Acid Binding Domain of Nuclear Receptors Alters Intracellular Localization

“…Refs. [71][72][73] and because in some of our images we observed what appeared to be aggregates of the receptors (Figs. 5 and 6), we examined the solubility of the receptors and the 'S78D' mutants in the transfected COS-7 cells.…”

Cutting Edge: Decreased Accumulation and Regulatory Function of CD4+CD25high T Cells in Human STAT5b Deficiency

“…1A) had undetectable levels of STAT5b protein and normal levels of STAT5a, similar to those of STAT5b A630P/A630P fibroblasts or EBV-transformed B cell lines (15,16). Immunological evaluation between 3 and 19 years of age (Fig.…”

STAT5B (signal transducer and activator of transcription 5B)

“…These reports are from homozygous patients while the parents are heterozygous for the STAT5b mutation and display a normal phenotype. The phenotype of each STAT5b mutant is similar: pronounced short stature, growth hormone insensitivity despite normal to high levels of GH in the serum, and extremely low IGF-I and IGFBP-3 levels (Chia et al, 2006;Hwa et al, 2007;Nadeau et al, 2011).…”

“…Laron type dwarfism II (LTD2) is mediated by defects in STAT5b (Nadeau et al, 2011;Freeth et al, 1998;Chia et al, 2006).…”