Conversion of threonine 757 to valine enhances Stat5a transactivation potential.
The growth hormone family of cytokines transduces intracellular signals through the Jak2-Stat5 pathway to activate the transcription of target genes. Amino acids within the C termini of Stats constitute the transactivation domain but also regulate the time course of tyrosine phosphorylation and extent of DNA binding. We mutated Thr(757) in the C-terminal of Stat5a (Thr-Stat5) to Val (Val-Stat5) and Asp (Asp-Stat5) and examined the effect on nuclear translocation, DNA binding, and prolactin-induced transcriptional activation of a Stat5-responsive luciferase reporter gene. Val-Stat5 produced a 5-fold higher increase in transcriptional activity relative to Thr-Stat5; Asp-Stat5 produced a similar response to Thr-Stat5. The increased transactivation was ligand induced and was not due to differences in basal expression of Val-Stat5 or to a constitutively activated Stat5 protein. Similar rates of loss of DNA binding ability and phosphorylation of Val- and Thr-Stat5 were observed following a single pulse of prolactin, indicating that the dephosphorylation pathways were unaltered. The serine-threonine kinase inhibitor H7 inhibited the transactivation potential of Thr-, Val-, and Asp-Stat5 to a similar extent, eliminating phosphorylation of Thr(757) as a regulatory mechanism. The results suggest that Thr(757) modulates the transactivation potential of Stat5 by a mechanism(s) that is dependent on the formation of Stat5 dimers and/or their nuclear translocation.
Gowri
P M
PM
Graduate Center for Toxicology, University of Kentucky, Lexington 40536-0305, USA.
Ganguly
T C
TC
Cao
J
J
Devalaraja
M N
MN
Groner
B
B
Vore
M
M
D001224:Aspartic Acid / Q000737:chemistry; D015153:Blotting, Western; D002460:Cell Line; D002467:Cell Nucleus / Q000378:metabolism; D004247:DNA / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000378:metabolism / Q000502:physiology*; D004305:Dose-Response Relationship, Drug; D006801:Humans; D008024:Ligands; D008156:Luciferases / Q000378:metabolism; D008894:Milk Proteins; D010766:Phosphorylation; D010957:Plasmids / Q000378:metabolism; D011388:Prolactin / Q000494:pharmacology; D017434:Protein Structure, Tertiary; D050799:STAT5 Transcription Factor; D015398:Signal Transduction; D013912:Threonine / Q000737:chemistry*; D013997:Time Factors; D015534:Trans-Activators / Q000737:chemistry / Q000378:metabolism / Q000502:physiology*; D015533:Transcriptional Activation; D014162:Transfection; D025521:Tumor Suppressor Proteins; D014633:Valine / Q000737:chemistry*; D001616:beta-Galactosidase / Q000378:metabolism
D004268:DNA-Binding Proteins; D008024:Ligands; D008894:Milk Proteins; D050799:STAT5 Transcription Factor; C494092:STAT5A protein, human; D015534:Trans-Activators; D025521:Tumor Suppressor Proteins; D013912:Threonine; D001224:Aspartic Acid; D011388:Prolactin; D004247:DNA; D008156:Luciferases; D001616:beta-Galactosidase; D014633:Valine