Cytokines such as interleukin-6 induce tyrosine and serine phosphorylation of Stat3 that results in activation of Stat3-responsive genes. We provide evidence that Stat3 is present in the mitochondria of cultured cells and primary tissues, including the liver and heart. In Stat3−/− cells, the activities of complexes I and II of the electron transport chain (ETC) were significantly decreased. We identified Stat3 mutants that selectively restored the protein's function as a transcription factor or its functions within the ETC. In mice that do not express Stat3 in the heart, there were also selective defects in the activities of complexes I and II of the ETC. These data indicate that Stat3 is required for optimal function of the ETC, which may allow it to orchestrate responses to cellular homeostasis.

STAT3 is a latent cytoplasmic transcription factor responsive to cytokine signaling and tyrosine kinase oncoproteins by nuclear translocation when tyrosine phosphorylated. We report that malignant transformation by activated Ras is impaired without STAT3, in spite of the inability of Ras to drive STAT3 tyrosine phosphorylation or nuclear translocation. Moreover, STAT3 mutants that cannot be tyrosine phosphorylated, are retained in the cytoplasm, or cannot bind DNA nonetheless supported Ras-mediated transformation. Unexpectedly, STAT3 was detected within mitochondria, and exclusive targeting of STAT3 to mitochondria without nuclear accumulation facilitated Ras transformation. Mitochondrial STAT3 sustained altered glycolytic and oxidative phosphorylation activities characteristic of cancer cells. Thus, in addition to its nuclear transcriptional role, STAT3 regulates a metabolic function in mitochondria, supporting Ras-dependent malignant transformation.

Cytokines and growth factors induce tyrosine phosphorylation of signal transducers and activators of transcription (STATs) that directly activate gene expression. Cells stably transformed by the Src oncogene tyrosine kinase were examined for STAT protein activation. Assays of electrophoretic mobility, DNA-binding specificity, and antigenicity indicated that Stat3 or a closely related STAT family member was constitutively activated by the Src oncoprotein. Induction of this DNA-binding activity was accompanied by tyrosine phosphorylation of Stat3 and correlated with Src transformation. These findings demonstrate that Src can activate STAT signaling pathways and raise the possibility that Stat3 contributes to oncogenesis by Src.

Activation of early response genes by interferons (IFNs) requires tyrosine phosphorylation of STAT (signal transducers and activators of transcription) proteins. It was found that the serine-threonine kinase mitogen-activated protein kinase (MAPK) [specifically, the 42-kilodalton MAPK or extracellular signal-regulated kinase 2 (ERK2)] interacted with the alpha subunit of IFN-alpha/beta receptor in vitro and in vivo. Treatment of cells with IFN-beta induced tyrosine phosphorylation and activation of MAPK and caused MAPK and Stat1 alpha to coimmunoprecipitate. Furthermore, expression of dominant negative MAPK inhibited IFN-beta-induced transcription. Therefore, MAPK appears to regulate IFN-alpha and IFN-beta activation of early response genes by modifying the Jak-STAT signaling cascade.

Interferons (IFNs) induce early-response genes by stimulating Janus family (Jak) tyrosine kinases, leading to tyrosine phosphorylation of Stat transcription factors. Previous studies implicated protein-tyrosine phosphatase (PTP) activity in the control of IFN-regulated Jak/Stat signaling, but the specific PTPs responsible remained unidentified. We have found that SH2 domain-containing PTP1 (SHPTP1; also called PTP1C, HCP, or SHP) reversibly associates with the IFN-␣ receptor complex upon IFN addition. Compared with macrophages from normal littermate controls, macrophages from motheaten mice, which lack SHPTP1, show dramatically increased Jak1 and Stat1␣ tyrosine phosphorylation, whereas Tyk2 and Stat2 activation is largely unaffected. These findings correlate with selectively increased complex formation on a gamma response element, but not an IFN-stimulated response element, in motheaten macrophages. Our results establish that SHPTP1 selectively regulates distinct components of Jak/Stat signal transduction pathways in vivo.Interferons (IFNs) were the first of many cytokines and growth factors that were found to stimulate the expression of early-response genes by inducing the tyrosine phosphorylation of SH2 domain-containing transcription factors (subsequently termed Stats) (20, 21). Tyrosine phosphorylation of Stat proteins induces them to form homodimeric and/or heterodimeric complexes, which translocate to the nucleus and interact with similar elements in a variety of enhancers to promote transcription. Janus family protein-tyrosine kinases (PTKs) are integral components of the signaling cascades regulated by cytokines that activate Stats (17).Recent work in many laboratories has identified several downstream components of IFN-␣/␤ signaling. Two subunits (␣ and ␤) of the IFN-␣/␤ receptor (IFN␣/␤R) have been molecularly cloned (28,44). Binding of IFN-␣/␤ to its receptor results in rapid activation of the Janus PTKs Jak1 and Tyk2. This results in tyrosyl phosphorylation of both Stat1␣ (p91) and Stat2 (p113) and the formation of at least two transcription factor complexes. One complex, composed of a heterotrimer of Stat1␣, Stat2, and the DNA-binding component p48, binds to IFN-stimulated response elements (ISREs) (12,13,30

Interleukin 12 (IL-12) is an important immunoregulatory cytokine whose receptor is a member of the hematopoietin receptor superfamily. Interleukin 12 (IL-12) is a monocyte/macrophage-derived cytokine (1, 2), which, through its many effects on natural killer (NK) and T lymphocytes, plays a central role in the initiation and control of cell-mediated immune responses (3,4). The receptor for IL-12 (IL-12R) is incompletely characterized, although a low-affinity subunit has recently been cloned (5). This subunit is a member of the hematopoietin receptor family, closely related to gpl3O. Like other family members, binding of IL-12 to the IL-12R induces rapid tyrosine phosphorylation of a range of intracellular substrates (6, 7). However, hematopoietin receptors do not possess intrinsic tyrosine kinase activity but instead associate with and activate members of the Janus (JAK) family of cytoplasmic protein tyrosine kinases (8-12). We have recently demonstrated that IL-12 treatment of human T and NK cells leads to the rapid tyrosine phosphorylation of both JAK2 and Tyk2 kinases, implicating these kinases in the immediate biochemical response to IL-12 (6).The biological effects of IL-12 include the rapid activation of early-response genes such as interferon y (IFN-,y) and perforin (3, 4), but the molecular mechanisms by which IL-12 might stimulate transcription are not known. A number of recent studies have identified a family of transcription factors called STATs (signal transducers and activators of transcription), which are involved in the signal-transduction cascades of many cytokines known to activate JAK kinases (8, 13-15). Originally described as mediators of IFN-induced transcription, STATs are latent cytoplasmic transcription factors that, after tyrosine phosphorylation, translocate to the nucleus and bind specific, but related, DNA sequences to promote transcription of cytokine-responsive genes (13). IFN-a induces tyrosine phosphorylation of STAT1 and STAT2, which associate with a nuclear 48-kDa DNA-binding protein to form a multiprotein transcriptional activator known as interferonstimulated gene factor 3 (16, 17). In contrast, IFN-,y promotes tyrosine phosphorylation and homodimerization of STAT1, which translocates to the nucleus and directly binds to a conserved sequence motif termed the IFN-y-activation site (18). Ligand binding to many hematopoietin receptors has now been shown to induce tyrosine phosphorylation of STAT family proteins, thereby promoting their ability to bind IFNy-activation site-related DNA sequences: the IL-6 family of cytokines activate both STAT1 and STAT3 (19,20), prolactin activates STAT1 (21) and STAT5 (22), and IL-4 activates a STAT protein designated IL-4-STAT (STAT6) (23). STAT4 is another family member with restricted distribution, being expressed mainly by myeloid cells and developing spermatogonia and also in thymus and spleen (24,25). To date, no STAT4-activating ligand has been identified.In this study we sought to determine whether STAT proteins could be identified ...

Expression of the STAT3 transcription factor in the heart is cardioprotective and decreases the levels of reactive oxygen species. Recent studies indicate that a pool of STAT3 resides in the mitochondria where it is necessary for the maximal activity of complexes I and II of the electron transport chain. However, it has not been explored whether mitochondrial STAT3 modulates cardiac function under conditions of stress. Transgenic mice with cardiomyocyte-specific overexpression of mitochondria-targeted STAT3 with a mutation in the DNA-binding domain (MLS-STAT3E) were generated. We evaluated the role of mitochondrial STAT3 in the preservation of mitochondrial function during ischemia. Under conditions of ischemia heart mitochondria expressing MLS-STAT3E exhibited modest decreases in basal activities of complexes I and II of the electron transport chain. In contrast to WT hearts, complex I-dependent respiratory rates were protected against ischemic damage in MLS-STAT3E hearts. MLS-STAT3E prevented the release of cytochrome c into the cytosol during ischemia. In contrast to WT mitochondria, ischemia did not augment reactive oxygen species production in MLS-STAT3E mitochondria likely due to an MLS-STAT3E-mediated partial blockade of electron transport through complex I. Given the caveat of STAT3 overexpression, these results suggest a novel protective mechanism mediated by mitochondrial STAT3 that is independent of its canonical activity as a nuclear transcription factor. STAT3 was originally identified as an IL-6-induced transcriptional activator of acute phase genes (1). However, other members of the IL-6 family, which utilize gp-130 receptor, as well as leptin, IL-12, IFN␣/␤, IL-10, GM-CSF, several growth factors, oncogenes, and stress such as hypoxia, also activate STAT3 (1). STAT3 is vital to embryonic development and STAT3-null mice are embryonic lethal (2). Analysis of tissuespecific conditional STAT3 knock-out mice has provided strong evidence that transcriptional activity of STAT3 plays a central role in the control of cell growth and host responses to inflammation and cellular stress (1). STAT3 positively regulates expression of anti-apoptotic (Bcl-2 and Bcl-xL) (1) and antioxidative proteins (MnSOD and metallothionein-1 and -2) (3, 4).Expression of STAT3 in the heart is associated with cardiac survival (5). When STAT3 is selectively deleted in cardiomyocytes, mice develop enhanced cardiac inflammation with fibrosis, dilated cardiomyopathy, and die prematurely due to congestive heart failure (5). Female mice, where STAT3 is not expressed in cardiomyocytes, develop post-partum cardiomyopathy, which is also seen in humans with reduced STAT3 expression in the myocardium (6). Ventricles from STAT3-null hearts show elevated levels of reactive oxygen species (ROS) 2 (6). Ischemic and pharmacologic preconditioning protected the viability of wild type but not STAT3 Ϫ/Ϫ cardiomyocytes (5). When STAT3 is overexpressed in cardiomyocytes, mice are less sensitive to the cardiotoxic effects of doxorubicin, which exerts i...

Background: Apart from its mitochondrial localization, mechanistic details of STAT3 import and assembly in mitochondria remain elusive. Results: Using an in vitro import assay, we show that STAT3 associates with the mitochondrial inner membrane in a GRIM-19-dependent manner. Conclusion: GRIM-19 chaperones the recruitment of STAT3 into mitochondrial inner membrane complexes. Significance: This study identifies a novel function of GRIM-19 and a mechanism for STAT3 import into mitochondria.