Inhibition of protein phosphatase 2A induces serine/threonine phosphorylation, subcellular redistribution, and functional inhibition of STAT3

Abstract

Signal transducers and activators of transcription (STATs) are rapidly phosphorylated on tyrosine residues in response to cytokine and growth factor stimulation of cell surface receptors. STATs hereafter are translocated to the nucleus where they act as transcription factors. Recent reports suggest that serine phosphorylation of STATs also is involved in the regulation of STAT-mediated gene transcription. Here, we studied the role of serine͞threonine phosphatases in STAT3 signaling in human antigen-specific CD4 ؉ T cell lines and cutaneous T cell lymphoma lines, expressing a constitutively activated STAT3. We show that an inhibitor of protein phosphatases (PPs) PP1͞PP2A, calyculin A, induces (i) phosphorylation of STAT3 on serine and threonine residues, (ii) inhibition of STAT3 tyrosine phosphorylation and DNA binding activity, and (iii) relocation of STAT3 from the nucleus to the cytoplasm. Similar results were obtained with other PP2A inhibitors (okadaic acid, endothall thioanhydride) but not with inhibitors of PP1 (tautomycin) or PP2B (cyclosporine A). Pretreatment with the broad serine͞threonine kinase inhibitor staurosporine partly blocked the calyculin A-induced STAT3 phosphorylation, whereas inhibitors of serine͞threonine kinases, such as mitogen-activated protein kinase-1 extracellular-regulated kinase-kinase, mitogenactivated protein p38 kinase, and phosphatidylinositol 3-kinase, did not. In conclusion, we provide evidence that PP2A plays a crucial role in the regulation of STAT3 phosphorylation and subcellular distribution in T cells. Moreover, our findings suggest that the level of STAT3 phosphorylation is balanced between a staurosporine-sensitive kinase(s) and PP2A.STATs (signal transducers and activators of transcription) are latent cytoplasmic transcription factors that upon activation translocate into the nucleus where they activate target genes (reviewed in ref. 1). At present, seven STATs have been cloned, all of which have an Src homology 2 domain near their carboxyl terminus and a tyrosine residue near position 700 (e.g., Y705 in STAT3). Upon ligation, cytokine and growth factor receptor-associated Janus kinases (JAKs) become activated, possibly by transphosphorylation and͞or autophosphorylation. Once activated, JAKs phosphorylate the receptor on key tyrosine residues, which leads to recruitment of STAT proteins, which in turn are tyrosine-phosphorylated by JAKs. Phosphorylated STAT proteins homodimerize or heterodimerize through reciprocal Src homology 2-phosphotyrosine interactions and translocate to the nucleus where they bind specific DNA elements and regulate transcriptional activity of target genes (reviewed in refs. 1-3).STATs also are serine-phosphorylated in response to ligation of many cytokine and growth factor receptors (reviewed in ref. 4). The major site for serine phosphorylation in STAT1 and STAT3 is residue 727 (5), allthough additional serine phosphorylation sites have been proposed (6). Serine phosphorylation of STAT proteins modulate the DNA binding and͞or transcri...

In vivo activation of STAT3 in cutaneous T-cell lymphoma. Evidence for an antiapoptotic function of STAT3

“…22,23 In brief, cells (1 Â 10 6 ) were incubated for 12 h (SOCS3wt) or 24 h (STAT3wt and STAT3D) with 20 ng/ml Tetracycline (Tet) (Invitrogen, Taastrup, Denmark). Cells were then pelleted, lysed in ice-cold lysis buffer, and subjected to Western blotting using the indicated antibodies.…”

Constitutive SOCS-3 expression protects T-cell lymphoma against growth inhibition by IFNα

“…22,26 Myc-Tagged SOCS-3 was immunoprecipitated using 5 mg anti-Myc (9E10) (Santa Cruz Biotechnology, Santa Cruz, CA, USA) and Protein-A agarose beads (KemEn-Tec, Copenhagen, Denmark). Membranes were probed using antibodies specific for Myc (9E10), Anti-Stat1, anti-Stat6, and anti-SOCS-3 from Santa Cruz, anti-PY-Stat1 and anti-PY-Stat5 from New England Biolabs (Beverly, MA, USA), anti-PY-Stat3 from Nanotools (Denzlingen, Germany), anti-Stat3 and antiStat5 from Transduction Laboratories (Lexington, KY, USA), antiPhosphor-tyrosine (PY) 4G10 from Upstate Biotechnologies, and HRP-conjugated anti-rabbit Ig, HRP-conjugated anti-mouse Ig, or HRP-conjugated anti-goat Ig from Dako (Glostrup, Denmark) and evaluated using enhanced chemiluminescence (ECL) according to the manufacturers instructions (Amersham, Buckinghamshire, UK).…”

Inhibition of IL-6 and IL-10 Signaling and Stat Activation by Inflammatory and Stress Pathways

“…There is general agreement that phosphorylation of Stat1 and Stat3 on serine 727 enhances the transcriptional potency of tyrosinephosphorylated Stat dimers (46,61,74,76,78,79), and one study suggests that DNA binding is enhanced as well (72). However, several studies have suggested that serine phosphorylation of Stats actually suppresses tyrosine phosphorylation and DNA binding (58,60,66,80), although only one of these studies tested this directly using a mutated Stat (58). We have not excluded that serine phosphorylation of Stats may contribute to modulating Stat activity in our system, but several lines of evidence suggest that the predominant site of inhibition occurs upstream of Stats.…”