STAT3 Serine Phosphorylation by ERK-Dependent and -Independent Pathways Negatively Modulates Its Tyrosine Phosphorylation

Abstract

Recent studies have indicated that serine phosphorylation regulates the activities of STAT1 and STAT3. However, the kinase(s) responsible and the role of serine phosphorylation in STAT function remain unresolved. In the present studies, we examined the growth factor-dependent serine phosphorylation of STAT1 and STAT3. We provide in vitro and in vivo evidence that the ERK family of mitogen-activated protein (MAP) kinases, but not JNK or p38, specifically phosphorylate STAT3 at serine 727 in response to growth factors. Evidence for additional mitogen-regulated serine phosphorylation is also provided. STAT1 is a relatively poor substrate for all MAP kinases tested both in vitro and in vivo. STAT3 serine phosphorylation, not its tyrosine phosphorylation, results in retarded mobility of the STAT3 protein on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Importantly, serine 727 phosphorylation negatively modulates STAT3 tyrosine phosphorylation, which is required for dimer formation, nuclear translocation, and the DNA binding activity of this transcriptional regulator. Interestingly, the cytokine interleukin-6 also stimulates STAT3 serine phosphorylation, but in contrast to growth factors, this occurs by an ERK-independent process.The JAK/STAT pathways are activated by various cytokines and growth factors such as interleukin-6 (IL-6), epidermal growth factor (EGF), and platelet-derived growth factor (PDGF) (reviewed in references 5 and 15). Once these growth factor or cytokine receptors are occupied by their ligands, tyrosine residues in the cytoplasmic domain of the receptors become phosphorylated by cytokine receptor-associated JAK family tyrosine kinases (19,32) or by the growth factor-activated receptor tyrosine kinases (16,26,34). The phosphorylated tyrosine residue, positioned within specific amino acid sequences, provides docking sites for the recruitment of specific STATs via their SH2 domains (13,33). STATs associated with the receptors are consequently phosphorylated at conserved tyrosine residues. This induces the STATs to dimerize via their own SH2 domains (29), translocate into the nucleus, and activate STAT target genes (30). To date, six STAT proteins have been characterized at the molecular level (reviewed in reference 15).EGF and PDGF activate primarily STAT1 and STAT3 (27,28,31,41). STAT1 and STAT3 can form three distinct dimers to activate target genes: STAT1 or STAT3 homodimers and STAT1-STAT3 heterodimers. Although STAT1 and STAT3 are simultaneously activated by various growth factors and cytokines, the results of knockout-mouse experiments suggest a specific role of STAT1 as a target for interferons (10, 21). However, STAT3 is expected to play a broader role. Recent results which show that STAT3 is activated by the expression of Src oncogenes (39) or infection by human T-cell leukemia virus type 1 (22) raise the possibility that STAT3 is involved in tumorigenic cell growth.Mitogen-activated protein (MAP) kinase pathways play important roles in the regulation of cell growth and differe...

Interleukin-6-induced STAT3 transactivation and Ser727 phosphorylation involves Vav, Rac-1 and the kinase SEK-1/MKK-4 as signal transduction components

“…As a control, blots were stripped and reprobed with gated. Previous reports have indicated that IL-6-induced STAT3 Ser(#( phosphorylation is an ERK-1-independent process [41,50], and we have been able to confirm these results in HepG2 cells. Blocking ERK activation by overexpression of dominant-negative Raf, N∆Raf [46], or by using the MEK inhibitor PD09859 did not alter STAT3 Ser(#( phosphorylation or transactivation (results not shown).…”

“…Possibly, p38 squelches the activity of an upstream kinase like SEK-1\MKK-4, which is blocked in the presence of SB203580. This hypothesis is supported by recent results, showing that SEK-1\MKK-4 is indeed capable of binding and activating p38 [29,50] and that p38 has an inhibiting effect on JNK-1-mediated Elk activation [33]. Whether the effects of SB203580 on STAT3 transactivation involve cross talk between the p38 and SAPK\JNK pathways requires further investigation.…”

Thyroid hormone promotes the phosphorylation of STAT3 and potentiates the action of epidermal growth factor in cultured cells

“…EGF alone also increased the phosphorylation and nuclear uptake of MAPK ; the effect of EGF on MAPK was enhanced by T % . STAT3 has been shown by others to undergo Serine-727 phosphorylation by the MAPK pathway [23], as well as by a mechanism independent of that pathway [7]. This serine phosphorylation has been regarded as essential for maximal transcriptional activity of STAT3 [6].…”

“…When stimulated by EGF, STAT3 is tyrosine-phosphorylated at residue 705 and translocates to the nucleus, either as a homodimer or as a heterodimer with the 91 kDa STAT1α, where it binds to selected EGF-responsive DNA sequences [5]. STAT3 and STAT1α both contain a C-terminal serine residue (residue 727) that is subject to phosphorylation by the mitogen-activated protein kinase (MAPK) pathway [6,7], as well as by other serine kinases [6][7][8].…”

STAT activation by the PDGF receptor requires juxtamembrane phosphorylation sites but not Src tyrosine kinase activation

“…Equal amounts of nuclear extracts were subjected to mobility shift analysis using the hSIE (upper panel) or Ie (lower panel) probe expressing CSF/PDGF receptors. MAP kinases have been implicated in phosphorylation of STAT proteins on serine and thereby regulating transactivation by and tyrosine phosphorylation of STATs (Wen et al, 1995;Chung et al, 1997). Activation of the MAP kinases ERK1 and ERK2 was inferred from the appearance of electrophoretically-retarded, phosphorylated forms (Anderson et al, 1990).…”