The Signal Transducer and Activator of Transcription (STAT) family of proteins was first discovered in the 1990's as key proteins in cytokine signaling. Since then, the field has greatly advanced in the past 15 years, providing significant insight into the structure, function, and regulation of STATs. STATs are latent cytoplasmic transcription factors consisting of seven mammalian members. They are Tyr phosphorylated upon activation, a post-translational modification critical for dimerization, nuclear import, DNA binding, and transcriptional activation. In recent years, unphosphorylated STATs have also been observed to dimerize and drive transcription, albeit by yet an obscure mechanism. In addition, the function of cytoplasmic STATs is beginning to emerge. Here, we describe the structure, function, and regulation of both unphosphorylated and phosphorylated STATs. STAT isoforms from alternative splicing or proteolytic processing, and post-translational modifications affecting STAT activities are also discussed.

Stat3 is a member of the signal transducer and activator of transcription family, which is important in cytokine signaling. Gene ablation studies have revealed a requirement for Stat3 in diverse biological processes (Akira, S. 2000. Oncogene. 19: 2607–2611; Levy, D.E., and C.K. Lee. 2002. J. Clin. Invest. 109:1143–1148). Previously, the function of Stat3 had been attributed exclusively to its transcriptional activity in the nucleus. In this study, we reveal an interaction between Stat3 and the microtubule (MT)-destabilizing protein stathmin. Stathmin did not overtly affect ligand-stimulated Stat3 activation. In contrast, the expression of Stat3 is required for the stabilization of MTs and cell migration. We further demonstrate that Stat3-containing cells are resistant to the MT-destabilizing effect of stathmin overexpression. In addition, down-regulation of stathmin protein levels in Stat3-deficient cells partially reversed the MT and migration deficiencies. Recombinant Stat3 was also capable of reversing stathmin inhibition of tubulin polymerization in vitro. Our results indicate that Stat3 modulates the MT network by binding to the COOH-terminal tubulin-interacting domain of stathmin and antagonizing its MT destabilization activity.

STATs are activated by various cytokines and growth factors via tyrosine phosphorylation, which leads to sequential dimer formation, nuclear translocation, binding to specific DNA sequences, and regulation of gene expression. Recently, serine phosphorylation of Stat3 on Ser-727 by ERK has been identified in response to epidermal growth factor (EGF). Here, we report that Ser-727 phosphorylation of Stat3 can also be induced by JNK and activated either by stress or by its upstream kinase and that various stress treatments induce serine phosphorylation of Stat3 in the absence of tyrosine phosphorylation. Inhibitors of ERK and p38 did not inhibit UVinduced Stat3 serine phosphorylation, suggesting that neither of them is involved. We further demonstrate that JNK1, activated by its upstream kinase MKK7, negatively regulated the tyrosine phosphorylation and DNA binding and transcriptional activities of Stat3 stimulated by EGF. Correspondingly, pretreatment of cells with UV reduced the EGF-stimulated tyrosine phosphorylation and phosphotyrosine-dependent activities of Stat3. The inhibitory effect was not observed for Stat1. Our results suggest that Stat3 is a target of JNK that may regulate Stat3 activity via both Ser-727 phosphorylation-dependent and -independent mechanisms. STATs1 are activated by various cytokines and several growth factors and function as important biological links between the cell surface and the transcriptional events in the nucleus. Seven STAT genes have been identified, which contain a conserved structure of SH2 and a DNA-binding domain (reviewed in Ref. 1). Binding of cytokines to their respective receptors stimulates the Janus kinase family, which phosphorylates STAT proteins on a specific tyrosine residue (Tyr-701 in Stat1 and Tyr-705 in Stat3) at the COOH terminus. Homo-or heterodimers are formed between the phosphorylated tyrosine and its partner's SH2 domain. These dimers translocate into the nucleus and function as transcription factors by binding to their recognition sequences and regulating the target gene expression (reviewed in Refs.

STAT proteins are activated by phosphorylation at speci®c tyrosine residue at the carboxy-terminus which is required for dimer-formation, nuclear translocation, DNA binding and transcriptional activity in cells treated with cytokines and growth factors. Recent studies have indicated that STATs are also phosphorylated by MAPK, or extracellular signal-regulated kinase (ERK) on serine. We investigated the role of ERK on the regulation of STAT activity. Here, we report that ERK2 activated by its upstream kinase, MEK1, represses Stat3 transcriptional activity induced by Src or Jak-2. To unravel the mechanism of repression, we further showed that Stat3 DNA binding activity and its tyrosine phosphorylation are also inhibited under the same conditions. ERK2 phosphorylates Stat3 on three serinecontaining peptides and decreases its tyrosine phosphorylation induced by EGF treatment. We also detected an association of ERK2 and Stat3 in vivo which is modulated positively by activation of ERK2, but negatively by Jak2. We propose that MAP kinase cascade may negatively regulate Stat3 activities by decreasing its tyrosine phosphorylation and also possibly by association.

The Ras/Raf/MAP kinase (ERK) pathway is a major signaling pathway induced by growth factors in mammalian cells. Two other types of mammalian MAP kinases, JNK (SAPK) and p38 (RK, CSBP), are induced by environmental stress. Although the immediate-early gene, egr-1, is induced by growth factors, cytokines, differentiation signals and DNA damaging agents, less is known about its induction by environmental stress and the mechanism involved. Here we report that in NIH3T3 cells, egr-1 is induced by various stress treatments such as heat shock, sodium arsenite, ultraviolet (U.V.) radiation, and anisomycin. p38 and JNK1, but not ERK2, were activated by these stress treatments. Induction of egr-1 by anisomycin is inhibited by a specific inhibitor of p38, SB 203580. We also show that p38 and JNK1 activated by their upstream kinases induce egr-1 promoter activity through activation of the ternary complex factor, Elk-1. The stress treatments also lead to an increase in Egr-1 protein phosphorylation and its DNA binding activity. Together, our data suggest that induction of egr-1 gene by growth factors and stress are mediated through different subgroups of MAP kinases which may also differentially affect egr-1 function on its target genes.

We previously reported an increase in signal transducer and activator of transcription 3 (Stat3) activation in keloid fibroblasts, which contributes to collagen production, cell proliferation, and migration. We further investigated the effect of epithelial-mesenchymal interaction on Stat3 in normal and keloid fibroblasts in noncoculture and coculture conditions. pY705 Stat3 was higher in keloid fibroblasts compared to normal fibroblasts in noncoculture. However, a more drastic decrease in pY705 Stat3 was observed in keloid fibroblasts compared to normal fibroblasts when cocultured with their respective keratinocytes over 5 days. To explore this paracrine effect, we examined the secretion of cytokines by cytokine arrays. Altered cytokine production was detected in keloid fibroblasts and keratinocytes, either in noncoculture or coculture conditions. IL-6, IL-8, monocyte chemoattractant protein-1, tissue inhibitor of metalloproteinases (TIMPs)-1, and TIMP-2 were major cytokines detected. Angiogenin, oncostatin M (OSM), vascular endothelial cell growth factor, IGF-binding protein-1, osteoprotegerin, and transforming growth factor-beta2 were present in keloid keratinocyte-fibroblast coculture, but absent in normal keratinocyte-fibroblast coculture. Only IL-6 and OSM stimulated strong pY705 Stat3 and cell proliferation in both normal and keloid fibroblasts. Other cytokines increased proliferation of keloid fibroblasts, but not normal fibroblasts, suggesting an altered state in keloid fibroblasts. Multiple cytokines likely contribute to keloid pathogenesis and a combinatorial neutralizing antibody/cytokine therapy may be effective in ameliorating keloid scars.

Stat3 is a latent transcription factor activated by various cytokines and growth factors. Phosphorylation on Tyr-705 is a prerequisite for dimer formation, nuclear translocation, binding to its cognate DNA sequences, and regulation of the target gene transcription. Ser-727 phosphorylation of Stat3 plays an additional role in the regulation of transcription. MEK kinase 1 (MEKK1) is a mitogen-activated protein kinase (MAPK) kinase kinase (MAPKKK) that activates the c-Jun NH 2 -terminal kinase signaling pathway. Here we report that MEKK1 is involved in the regulation of Stat3 activation by growth factors. Kinase-inactive MEKK1 inhibits Stat3 phosphorylation on tyrosine and serine, and its transcriptional activity stimulated by epidermal growth factor and platelet-derived growth factor in different cell types. In contrast, active MEKK1 induces Stat3 tyrosine and serine phosphorylation leading to a functionally active Stat3 capable of binding DNA and enhancing transcription. Ser-727 is phosphorylated by MEKK1 in vitro, whereas Tyr-705 phosphorylation induced by MEKK1 involves Src and Janus kinases in vivo. These data demonstrate for the first time a novel role of MEKK1 to modulate tyrosine kinases that results in the activation of specific members of STAT family. STAT1 family was first identified in the regulation of interferon-inducible gene transcription (1). These transcription factors were named Signal Transducers and Activators of Transcription, by virtue of their novel and unique dual functions as signaling molecules in the cytoplasm and as transcription factors following nuclear translocation. Concurrent to the explosion of STAT family was the discovery of a group of tyrosine kinases called Janus kinases (JAKs) in interferon and cytokine signaling (2, 3). Upon cytokine stimulation, these receptorassociated kinases transphosphorylate themselves and the receptors, creating recruitment sites for binding proteins containing the Src homology 2 (SH2) domains, such as STATs. The STATs are subsequently phosphorylated by JAKs on a single tyrosine site at the COOH terminus, form homo-or heterodimers via the reciprocal interactions between SH2 domain and the phosphorylated tyrosine, and translocate into the nucleus where they bind to DNA and regulate the transcription of target genes (reviewed in Refs. 4 -6).There are seven known mammalian STAT proteins, denoted by Stat1, Stat2, Stat3, Stat4, Stat5a, Stat5b, and Stat6, that are activated in various cytokine signaling. Stat3 was identified as an acute-phase response factor activated by interleukin-6 (IL-6) in mouse liver and by homology to Stat1 (7,8). In addition to cytokines, Stat3 is also strongly activated by growth factors, such as EGF, platelet-derived growth factor (PDGF), and colony-stimulating factor-1 (8-11). Although the mechanisms of Stat3 activation by growth factors are less defined compared with cytokines, the intrinsic tyrosine kinase activity of the EGF receptor (EGF-R) and the nonreceptor tyrosine kinases, Src and JAKs, are implicated in its tyrosine ph...

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