Stat1 Serine Phosphorylation Occurs Independently of Tyrosine Phosphorylation and Requires an Activated Jak2 Kinase

Abstract

Gamma interferon (IFN-␥) induces both tyrosine and serine phosphorylation of Stat1. Stat1 serine phosphorylation is required for maximal transcriptional activity of Stat1. In this report, we present evidence that Stat1 tyrosine phosphorylation is not a prerequisite for Stat1 serine phosphorylation, although an active Jak2 kinase is required for both phosphorylation events. Stat1 serine phosphorylation occurs with a more delayed time course than tyrosine phosphorylation. The occurrence of serine phosphorylation without tyrosine phosphorylation suggests that serine phosphorylation takes place in the cytoplasm. Experiments performed with cells expressing either dominant-negative or constitutively active Ras protein indicated that the Ras-mitogenactivated protein kinase pathway is probably not involved in IFN-␥-induced Stat1 serine phosphorylation. Finally, a kinase capable of correct Stat1 serine phosphorylation was detected in partially purified cytoplasmic extracts from both IFN-␥-treated and untreated cells.Polypeptide signalling that changes gene expression often involves the STAT proteins (16). These latent cytoplasmic transcription factors become activated by phosphorylation of tyrosine residues, catalyzed either by receptor-associated Janus (Jak) kinases or by receptor tyrosine kinases. The STAT molecules then dimerize by reciprocal phosphotyrosine-SH2 interactions and enter the cell nucleus to effect transcriptional changes. It has been amply demonstrated that transcriptional activation is further amplified when the serine residues of STAT proteins are phosphorylated (22). A single serine in Stat3 and Stat1, residue 727 in both cases, appears to be the major, if not the only, serine kinase target site (21). In addition to these studies, a number of other findings suggestive of serine phosphorylation of STATs that are also tyrosine phosphorylated have been described. For example, Stat3 and Stat5 show a ligand-dependent, slower migration that is also time dependent and is inhibited by the serine kinase inhibitor H7 (2,3,24).The signalling pathway(s) involved in serine phosphorylation of the STATs is not known at present, although we do show here that Jak2 is required for STAT serine phosphorylation in response to gamma interferon (IFN-␥). In this study, we explored the time course and cellular locus of serine and tyrosine phosphorylation and the potential interdependence of the two. In addition, we examined the nature of the serine phosphorylation pathways, demonstrating the lack of evidence for participation of several prominent serine kinases. However, we did detect a kinase capable of catalyzing phosphorylation of Stat1, predominantly on residue 727. MATERIALS AND METHODSCell culture. U3A cells (13,14), ␥2A cells (12), and their derived cell lines were grown in Dulbecco modified Eagle medium (DMEM) supplemented with 10% cosmic serum (HyClone Laboratories Inc.). NIH 3T3 cells and cells stably transfected with either dominant-negative Ras (dnRas) or constitutively active Ras (CARas) were grown in DMEM supple...

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

“…Previous work on the interactions between MAPK and Jak-Stat pathways has focused on MAPK-dependent phosphorylation of a conserved carboxyl-terminal serine residue in the Stat proteins themselves (46, 58 -61, 66, 71-79). 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).…”

“…2) Inhibition of Stat3 mutated at serine 727 (Fig. 9); it is unlikely that inhibition of Stat3-S727A can be explained on the basis of phosphorylation of other serine residues in Stat3, since phosphopeptide mapping experiments have shown that serine 727 is the predominant site of serine phosphorylation (46,58,72,74). 3) Inhibition of IL-6 activation of ERKs (which are downstream of Jaks, but independent of Stats) occurred in primary fibroblasts, where, in contrast to hemopoietic cells, IL-6 activation of ERKs was detectable (L. Ivashkiv, unpublished observations).…”

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

“…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].…”

A Mutation in the SH2 Domain of STAT2 Prolongs Tyrosine Phosphorylation of STAT1 and Promotes Type I IFN-induced Apoptosis

“…A different study showed that the DNA-binding domain of STAT2 is functional, as two amino acid substitutions (V453I, V454I) affected IFN-␣-mediated biological responses that were STAT2 dependent, but ISGF3 independent (Brierley and Fish, 2005). A conserved serine residue found in the TAD of STAT1, STAT3, STAT4, and STAT5 must be phosphorylated to maximally drive gene expression Zhu et al, 1997;Morinobu et al, 2002;Xue et al, 2002;Pilz et al, 2003).…”

“…Mutation of STAT1 or STAT2 in the conserved arginine in the SH2 domain or mutation in the conserved tyrosine phosphorylation site abrogates dimerization or nuclear translocation (Gupta et al, 1996;Li et al, 1997;Mowen and David, 1998). Although a mutation in the conserved serine 727 found in the transactivation domain (TAD) of STAT1, as well as in STAT3 and STAT4, impairs transcriptional activity Zhu et al, 1997;Morinobu et al, 2002), a mutation in the N-terminal domain of STAT1 (F77A) impairs its oligomerization, tyrosine dephosphorylation, and gene transcription (Meyer et al, 2004). In contrast, forced dimerization of STAT1 by mutation of two conserved cysteine residues within the SH2 domain renders cells more responsive to the apoptotic effects of IFNs (Sironi and Ouchi, 2004).…”