Abstract
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.
Highlights
Stat proteins constitute a family of latent cytoplasmic transcription factors that are activated by a large number of cytokines and growth factors [1,2,3]
We mutated the Thr757 in Stat5a to a Val or an Asp to determine the effect of loss of this putative phosphorylation site and replacement with an acidic amino acid residue, respectively, on the transactivation potential of ThrStat5
Signal transduction via the Stat5 pathway requires the recruitment of the latent cytoplasmic transcription factor to the phosphorylated tyrosine residue in the cytoplasmic domain of the receptor, phosphorylation of a key tyrosine (Tyr694) in Stat5 by the receptor-associated Janus kinases (Jak), followed by the interaction of the Src homology 2 (SH2) domain, and formation of dimers by each of the phosphotyrosines in the two Stats in a reciprocal manner
Summary
Janus kinases; SH, Src homology; PRL, prolactin; PRLRL, long form of the prolactin receptor; GAS, ␥-interferon activated sequences; H7, 1-(5-isoquinolinesulfonyl)-2methylpiperazine1⁄72HCl; PBS, phosphate-buffered saline; DTT, dithiothreitol; PMSF, phenylmethylsulfonyl fluoride; EMSA, electromobility shift assays. Deletion of the C terminus of Stat resulted in a dominant negative phenotype with sustained tyrosine phosphorylation and DNA binding relative to the wild type but with no transcriptional activation. We have demonstrated that PRL acts via the long form of the PRL receptor (PRLRL) to facilitate the binding of Stat multimers to two interferon-␥-activated sequences (GAS)-like elements in the promoter of the rat liver ntcp (Naϩ/taurocholate cotransporting polypeptide) gene to increase its transcription [15]. The amino acids within the C-terminal region in Stat5a and Stat5b isoforms from various species are conserved and contain a serine/threonine at position 757, which could serve as a potential phosphorylation site. We studied the effect of the serine/threonine kinase inhibitor, H7, on the transactivation potential of wild type Stat and its mutants. The present studies show that the mutation of Thr757 to Val markedly increased the transactivation potential of Stat, whereas mutation to Asp did not significantly alter this potential
Sign-in/Register to view highlights & summary Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
