Serine Phosphorylation of GH-Activated Signal Transducer and Activator of Transcription 5a (STAT5a) and STAT5b: Impact on STAT5 Transcriptional Activity

Abstract

Signal transducer and activator of transcription 5b (STAT5b), the major liver-expressed STAT5 form, is phosphorylated on both tyrosine and serine in GH-stimulated cells. Although tyrosine phosphorylation is known to be critical for the dimerization, nuclear translocation, and activation of STAT5b DNA-binding and transcriptional activities, the effect of STAT5b serine phosphorylation is uncertain. Presently, we identify Ser730 as the site of STAT5b serine phosphorylation in GH-stimulated liver cells. We additionally show that the serine kinase inhibitor H7 partially blocks the GH-stimulated formation of (Ser,Tyr)-diphosphorylated STAT5b without inhibiting STAT5b nuclear translocation. Evaluation of the functional consequences of STAT5b serine phosphorylation by mutational analysis revealed an approximately 50% decrease in GH-stimulated luciferase reporter gene activity regulated by an isolated STAT5-binding site when STAT5b Ser730 was mutated to alanine and under conditions where STAT5 DNA-binding activity was not diminished. No decrease in GH-stimulated reporter activity was seen with the corresponding STAT5a-Ser725Ala mutant; however, a decrease in reporter activity occurred when the second established STAT5a serine phosphorylation site, serine 779, was additionally mutated to alanine. Unexpectedly, STAT5a-Ser725,779Ala and STAT5b-Ser730Ala displayed approximately 2-fold higher GH- or PRL-stimulated transcriptional activity compared with wild-type STAT5b when assayed using an intact beta-casein promoter-luciferase reporter. Finally, STAT5b-stimulated gene transcription was abolished in cells treated with H7, but in a manner unrelated to the inhibitory effects of H7 on STAT5b Ser730 phosphorylation. These findings suggest that the effects of STAT5b and STAT5a serine phosphorylation on STAT-stimulated gene transcription can be modulated by promoter context. Moreover, in the case of STAT5a, phosphorylation of serine 779, but not serine 725, may serve to regulate target gene transcriptional activity.

EGF receptor‐mediated, c‐Src‐dependent, activation of Stat5b is downregulated in mitogenically responsive hepatocytes

“…Ser-730 on Stat5b can be phosphorylated (Grimley et al, 1999). The functional importance of this is not clear but there is evidence that phosphorylation of Ser-730 modulates the effects on gene transcription of Stat5b (Park et al, 2001). We examined serine phosphorylation of Stat5b in the hepatocytes, using a specific antibody directed towards Ser-730 (Yamashita et al, 1998).…”

S731 in the transactivation domain modulates STAT5b activity

“…While serine phosphorylation of STAT1 and STAT3 has been shown to be necessary for full transcriptional activity, the regulation and importance of serine phosphorylation of STAT5b is poorly understood [11]. In some cell models, STAT5b is constitutively phosphorylated on S731, and this phosphorylation is enhanced upon Prl or GH stimulation [12][13][14]. Although the kinase that mediates phosphorylation of S731 has yet to be identified, mutation of S731 influences STAT5b transcriptional activity in response to PRL or GH [13][14][15][16][17][18].…”

“…In some cell models, STAT5b is constitutively phosphorylated on S731, and this phosphorylation is enhanced upon Prl or GH stimulation [12][13][14]. Although the kinase that mediates phosphorylation of S731 has yet to be identified, mutation of S731 influences STAT5b transcriptional activity in response to PRL or GH [13][14][15][16][17][18]. For example, transient transfection of the S731A STAT5b mutant into HepG2 liver cells decreases the transcriptional activity of the GH-induced NTCP-promoter luciferase reporter compared to the corresponding wild type (wt) STAT5b [14].…”

“…Although the kinase that mediates phosphorylation of S731 has yet to be identified, mutation of S731 influences STAT5b transcriptional activity in response to PRL or GH [13][14][15][16][17][18]. For example, transient transfection of the S731A STAT5b mutant into HepG2 liver cells decreases the transcriptional activity of the GH-induced NTCP-promoter luciferase reporter compared to the corresponding wild type (wt) STAT5b [14]. In contrast, transient transfection of the S731A STAT5b mutant in COS-1 cells increases the transcriptional activity of the β-casein promoter luciferase reporter compared to wtSTAT5b [14].…”

“…For example, transient transfection of the S731A STAT5b mutant into HepG2 liver cells decreases the transcriptional activity of the GH-induced NTCP-promoter luciferase reporter compared to the corresponding wild type (wt) STAT5b [14]. In contrast, transient transfection of the S731A STAT5b mutant in COS-1 cells increases the transcriptional activity of the β-casein promoter luciferase reporter compared to wtSTAT5b [14]. These data suggest that the effect of serine phosphorylation on STAT5b function may be cell type-and gene-specific.…”