In vitro activation of a transcription factor by gamma interferon requires a membrane-associated tyrosine kinase and is mimicked by vanadate.

In response to interferon ␥ (IFN␥), intercellular adhesion molecule-1 (ICAM-1) is expressed on human keratinocytes, a cell type that is critically involved in cutaneous inflammation. An ICAM-1 5 regulatory region palindromic response element, pI␥RE, has been shown to confer IFN␥-dependent transcription enhancement. By electrophoretic mobility shift assays (EMSA), pI␥RE forms a distinct complex with proteins from IFN␥-treated human keratinocytes, termed ␥ response factor (GRF). Binding of GRF is tyrosine phosphorylation-dependent, and mutations of pI␥RE that disrupt the palindromic sequence or alter its spatial relationship abrogate GRF binding. Supershift EMSAs using antibodies to characterized STAT proteins suggest that GRF contains a Stat1␣-like protein; however, non-ICAM-1 IFN␥-responsive elements (REs) known to bind Stat1␣ homodimers fail to compete for GRF binding in EMSA, and pI␥RE does not cross-compete with these REs that complex with homodimeric stat1␣. The pI␥RE⅐GRF complex also displays a distinctly different electrophoretic mobility compared to that of IFN␥REs complexed to homodimeric Stat1␣. These findings indicate that a distinct complex containing a Stat1␣-like protein mediates IFN␥-induced ICAM-1 gene transcription and identifies a subset of IFN␥-responsive genes that appear to be regulated by this complex.

Section: Icam-1 Gene Induction By Ifn␥ Via a Distinct Stat Complexsupporting

confidence: 55%

Section: Icam-1 Gene Induction By Ifn␥ Via a Distinct Stat Complexmentioning

confidence: 99%

Interferon γ-dependent Induction of Human Intercellular Adhesion Molecule-1 Gene Expression Involves Activation of a Distinct STAT Protein Complex

Naik

Shibagaki²,

Li³

et al. 1997

“…Tyrosine phosphorylation of STAT1 is regulated both by Jak1/Jak2 protein-tyrosine kinases and by protein-tyrosine phosphatase. Significantly, treatment with sodium vanadate, a protein-tyrosine phosphatase inhibitor, can sustain the phosphorylation of Tyr-701, preventing down-regulation of STAT1 transcriptional activity (18,19). Biochemical analysis has demonstrated that dephosphorylated STAT1 is rapidly exported back into the cytoplasm and takes part in subsequent activation-inactivation cycles and that loss of dephosphorylation leads to prolonged STAT1 activation in the nucleus (20 -27).…”

Section: Stat1mentioning

confidence: 99%

STAT1-induced Apoptosis Is Mediated by Caspases 2, 3, and 7

Sironi

Ouchi

2004

108

STAT1(signal transducer and activator of transcription 1) has been implicated as a mediator of a variety of biological responses in response to stimulation by specific growth factors and cytokines. To understand better the role of STAT1 in the interferon-␥ (IFN-␥)-induced phenotype, we generated an active form of STAT1 (STAT1C) by substituting Cys residues for both Arg-656 and Asn-658 within the C-terminal loop of the STAT1 SH2 domain. The IFN-␥ activation site element was stimulated and bound efficiently by STAT1C without IFN-␥ treatment. STAT1C was found to be tyrosine-phosphorylated in the nucleus for more than 30 h after IFN-␥ stimulation. STAT1-negative U3A cells reexpressing STAT1C showed retarded cell growth and underwent apoptosis when treated with IFN-␥. Further analysis demonstrated that apoptosis was preceded by proteolytic cleavage of caspases 2, 3, and 7, and wild type STAT1 also induced cleavage of caspase 7 when expressed in STAT1-negative U3A cells, indicating that STAT1C augments potential activity of wild type STAT1. Studies with cycloheximide treatment showed that protein synthesis induced in the first 24 h after IFN-␥ treatment was required for apoptosis under these conditions. Finally, we found that STAT1C-induced apoptosis was, in part, mediated by caspase 2, 3, and 7 because benzyloxycarbonyl-valyl-aspartyl-valyl-alanyl-aspartic acid fluoromethyl ketone (Z-VDVAD-FMK) treatment partially blocked apoptosis. These results suggest that prolonged nuclear localization of activated STAT1 results in apoptosis involving specific regulation of caspase pathway.Signal transducer and activator of transcription (STAT) 1 1 plays an important role in such IFN-␥-induced biological responses as tumor surveillance (1-4), the immune response (5, 6), and cell growth control (7,8). It has been suggested that it serves as a tumor suppressor by activating proapoptotic pathways (3, 9 -14).In response to IFN-␥ stimulation, STAT1 becomes phosphorylated on Tyr-701 and forms a homodimer through reciprocal SH2-phosphotyrosine interactions. Phosphorylation on Tyr-701 is required for subsequent nuclear translocation, DNA binding, and gene activation (15)(16)(17). Tyrosine phosphorylation of STAT1 is regulated both by Jak1/Jak2 protein-tyrosine kinases and by protein-tyrosine phosphatase. Significantly, treatment with sodium vanadate, a protein-tyrosine phosphatase inhibitor, can sustain the phosphorylation of Tyr-701, preventing down-regulation of STAT1 transcriptional activity (18,19). Biochemical analysis has demonstrated that dephosphorylated STAT1 is rapidly exported back into the cytoplasm and takes part in subsequent activation-inactivation cycles and that loss of dephosphorylation leads to prolonged STAT1 activation in the nucleus (20 -27).STAT1 has been implicated in apoptosis in response to IFN-␥ stimulation. Mutant forms of STAT1 lacking the N-terminal 62 amino acid residues or carrying a single amino acid substitution of Ala-31 for Arg-31 inhibited STAT1 tyrosine dephosphorylation and greatly enhanced the anti...

“…Evidence from both in vitro and in vivo studies indicated that tyrosine phosphatases can act as positive as well as negative regulators of the IFN␣/␤-induced Jak/Stat pathway (17,21,22). Recently, we were able to demonstrate that the SH2 domain containing tyrosine phosphatase PTP1C (SHPTP1, HCP, or SHP) functions as a suppressor of IFN␣/␤ signal transduction in hematopoietic cells by specific down-modulation of Jak1 tyrosine phosphorylation (23).…”

mentioning

confidence: 99%

The SH2 Domain-containing Tyrosine Phosphatase PTP1D Is Required for Interferon α/β-induced Gene Expression

David

Zhou

Pine³

et al. 1996

100

Interferons (IFNs) induce early response genes by stimulating Janus family (Jak) tyrosine kinases, leading to tyrosine phosphorylation of Stat (signal transducer and activator of transcription) proteins. Previous studies demonstrated that a protein-tyrosine phosphatase (PTP) is required for activation of the ISGF3 transcription complex by IFN␣/␤, but the specific PTP responsible remained unidentified. We now show that the SH2 domain containing tyrosine phosphatase PTP1D (also designated as SHPTP2, SHPTP3, PTP2C, or Syp) is constitutively associated with the IFN␣/␤ receptor and becomes tyrosine-phosphorylated in response to ligand. Furthermore, transient expression of a phosphatase-inactive mutant or the COOH-terminal SH2 domain of PTP1D causes a dominant negative effect on IFN␣/␤-induced early response gene expression. These results provide strong evidence that PTP1D functions as a positive regulator of the IFN␣/␤-induced Jak/Stat signal transduction pathway.Interferons as well as many other cytokines and growth factors stimulate the expression of early response genes by inducing the tyrosine phosphorylation of SH2-containing transcription factors termed Stats (1-5), a process that involves the activation and tyrosine phosphorylation of members of the Janus family of protein-tyrosine kinases (Jaks) (6 -9). Tyrosine phosphorylation of the Stat proteins causes them to either homo-or heterodimerize and to translocate to the nucleus, where they interact with enhancer elements in a variety of promoters to initiate transcription (10,11). Serine phosphorylation of Stat1 by mitogen-activated protein kinase (MAPK, ERK2) is required in addition to tyrosine phosphorylation for maximum transcriptional activation (12, 13).Several components of the IFN␣/␤ 1 signaling pathway have been identified and molecularly cloned, such as the two subunits (␣ and ␤) of the IFN␣/␤ receptor (14 -16). Binding of IFN␣/␤ to its receptor causes the rapid activation of the Jak tyrosine kinases Jak1 and Tyk2 (6, 7) which results in tyrosine phosphorylation of both Stat1␣ (p91) and Stat2 (p113) (1,17) 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 (ISGF3␥), binds to interferon-stimulated response elements (ISREs) (18,19), whereas Stat1␣ homodimers bind to ␥ response region (GRR) elements (20).Although much attention has been devoted to the tyrosine kinases involved in Stat activation, the role of tyrosine phosphatases in IFN␣/␤ signal transduction is less understood. Evidence from both in vitro and in vivo studies indicated that tyrosine phosphatases can act as positive as well as negative regulators of the IFN␣/␤-induced Jak/Stat pathway (17,21,22). Recently, we were able to demonstrate that the SH2 domain containing tyrosine phosphatase PTP1C (SHPTP1, HCP, or SHP) functions as a suppressor of IFN␣/␤ signal transduction in hematopoietic cells by specific down-modulation of Jak1 tyrosine phosphorylation (23). Studies of Stat activation by...