Requirement of Ca²⁺and CaMKII for Stat1 Ser-727 phosphorylation in response to IFN-γ

Abstract

In response to IFN-γ, the latent cytoplasmic protein signal transducers and activators of transcription 1 (Stat1) becomes phosphorylated on Y701, dimerizes, and accumulates in the nucleus to activate transcription of IFN-γ-responsive genes. For maximal gene activation, S727 in the transcription activation domain of Stat1 also is inducibly phosphorylated by IFN-γ. We previously purified a group of nuclear proteins that interact specifically with the Stat1 transcription activation domain. In this report, we identified one of them as the multifunctional Ca 2+ /calmodulin-dependent kinase (CaMK) II. We demonstrate that IFN-γ mobilizes a Ca 2+ flux in cells and activates CaMKII. CaMKII can interact directly with Stat1 and phosphorylate Stat1 on S727 in vitro . Inhibition of Ca 2+ flux or CaMKII results in a lack of S727 phosphorylation and Stat1-dependent gene activation, suggesting in vivo phosphorylation of Stat1 S727 by CaMKII. Thus two different cellular signaling events, IFN-γ receptor occupation and Ca 2+ flux, are required for Stat1 to achieve maximal transcriptional activation through regulation of phosphorylation.

SET-induced calcium signaling and MAPK/ERK pathway activation mediate dendritic cell-like differentiation of U937 cells

“…U937 cells that overexpress SET undergo a number of changes similar to cells treated with IFN-g, that is, they exit the cell cycle; 36 upregulate the expression of GTP cyclohydrolase I, 37 CD11b 39 and CD86; 38 and activate the calcium signaling/CaMKII 40 and MAPK/ERK pathways, 41 but fail to activate STAT1, the main mediator of IFN-g signaling. 42 IFN-g signaling orchestrates different cellular programs, including antimicrobial and antitumor mechanisms as well as cell proliferation and differentiation, through transcriptional regulation of target genes.…”

“…[36][37][38][39][40][41] Since the signal transducing activator of transcription-1 (STAT1) moderates IFN-g-induced gene expression, 42 we analyzed whether ectopic expression of SET activates STAT1 DNA binding, which is a function of its phosphorylation at tyrosine SET induces dendritic cell-like differentiation of U937 cells A Kandilci and GC Grosveld (Y701). Western blot analysis after 8, 11, 12, 14, 16 and 24 h of FS induction showed no Y701 phosphorylation of STAT1 and ELISA-based transcription factor-binding assays showed no difference in the DNA-binding activity of STAT1 after FS induction at the same time points (data not shown).…”

p38 MAPK enhances STAT1-dependent transcription independently of Ser-727 phosphorylation

“…On the basis of recent studies, a scenario for the phosphorylation of STAT1 at Ser-727 is emerging: (i) in the IFN pathway, Tyr-701 phosphorylation of STAT1 is necessary for subsequent Ser-727 phosphorylation to occur; and (ii) in the IFN-independent pathway, only Ser-727 and no Tyr-701 phosphorylation is induced (14,15). Recently, PKC␦ and CaMKII have been described as IFN-␣-and -␥-activated, respectively, STAT1 Ser-727 kinases (16,17). The IFNindependent (or stress-induced) serine kinase is most likely the p38 MAPK (10,13,15,18).…”

“…Although the residue Ser-727 lies within a good mitogen-activated protein kinase (MAPK) consensus sequence (PMS 727 P), most experimental data do not support an involvement of MAPKs in the IFN-stimulated Ser-727 phosphorylation of STAT1 (10,(13)(14)(15). Recently, Ca(2ϩ)͞calmodulin-dependent kinase (CaMK) II and protein kinase C (PKC)␦ were reported to phosphorylate STAT1 at Ser-727 on treatment with IFN-␥ and -␣, respectively (16,17), acting possibly via the PI3K͞Akt pathway (15). In addition, the IFN-induced phosphorylation of STAT1 at Tyr-701 is needed for subsequent Ser-727 phosphorylation (14).…”

Serine-phosphorylated STAT1 is a prosurvival factor in Wilms' tumor pathogenesis

“…In this case, the role of the protein kinase activating STAT1 on serine becomes critical. Previously different kinases were implicated in S727 STAT1 phosphorylation, including p38 kinase in response to lipopolysaccharide or ultraviolet (UV); p38 kinase, ERKs, c-Jun NH 2 -terminal kinases (JNKs), PI3K, 3-phosphoinositide-dependent protein kinase I (PDK1) and p90 ribosomal S6 kinase (p90RSK2) in the cellular response to UVB; p38 kinase, JNK, PKCd, CaMKII in response to INFg (Kovarik et al, 1999;Nair et al, 2002;Xu et al, 2005;Zykova et al, 2005); and PKC in B-CCL patients (Frank et al, 1997). In this report, for the first time, we provide evidence that CK2 is involved in STAT1 phosphorylation.…”