Aberrant folding of a mutant Stat5b causes growth hormone insensitivity and proteasomal dysfunction.
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.
Chia
Dennis J
DJ
Department of Biochemistry and Molecular Biology, Oregon Health & Science University, Portland, Oregon 97239, USA.
Subbian
Ezhilkani
E
Buck
Teresa M
TM
Hwa
Vivian
V
Rosenfeld
Ron G
RG
Skach
William R
WR
Shinde
Ujwal
U
Rotwein
Peter
P
D000328:Adult; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000818:Animals; D019556:COS Cells; D002478:Cells, Cultured; D002522:Chlorocebus aethiops; D005260:Female; D013006:Growth Hormone / Q000502:physiology; D006801:Humans; D046150:Laron Syndrome / Q000201:enzymology / Q000235:genetics; D008969:Molecular Sequence Data; D010766:Phosphorylation; D046988:Proteasome Endopeptidase Complex / Q000502:physiology*; D061988:Proteasome Inhibitors; D017510:Protein Folding; D017434:Protein Structure, Tertiary; D051381:Rats; D050799:STAT5 Transcription Factor / Q000737:chemistry / Q000235:genetics / Q000502:physiology*; D014443:Tyrosine / Q000378:metabolism
D061988:Proteasome Inhibitors; D050799:STAT5 Transcription Factor; C494095:STAT5B protein, human; C494097:Stat5b protein, rat; D014443:Tyrosine; D013006:Growth Hormone; D046988:Proteasome Endopeptidase Complex