Biomedical subjects

I M Kerr

Publications and source records attributed to I M Kerr.

At least 37 records · Page 2Linked to original sources

PubMed1996

Participation of JAK and STAT proteins in growth hormone-induced signaling.

The binding of growth hormone leads to dimerization of its receptor, accompanied by phosphorylation and activation of intracellular tyrosine kinases (JAKs) and the latent cytoplasmic transcriptions factors STAT1, STAT3, and STAT5. Both JAK1 and JAK2 are phosphorylated in response to growth hormone in mouse 3T3 F442A and human HT1080 cells. The roles of JAKs in growth hormone signal transduction were examined by using mutant HT1080 cells missing either JAK1 or JAK2. JAK2 is absolutely required for growth hormone-dependent phosphorylation of the receptor, STAT1 and STAT3, JAK1, and the SH2-containing adaptor molecule Shc. In contrast, JAK1 is not required for any of the above functions. These data indicate that JAK2 is both necessary and sufficient for the growth hormone-dependent phosphorylation events required to couple the receptor both to STAT-dependent signaling pathways and to pathways involving Shc. Furthermore, STAT5 is activated by growth hormone in 3T3 F442A cells, but not in HT1080 cells, revealing that the set of STATs activated by growth hormone can vary, possibly contributing to the specificity of the growth hormone response in different cell types.

3T3 Cells↗

PubMed1996

JAKs, STATs and signal transduction in response to the interferons and other cytokines.

The isolation and complementation of mutant human cell lines has established an essential role for the JAK (Janus kinase) family of protein tyrosine kinases and STAT (signal transduction and transcription) factors in the Interferon response pathways. Activation of STATs by JAKs occurs in receptor complexes at the cell membrane. Activated STATs form homo- or heterodimers and, with or without additional factors, migrate to the nucleus to initiate transcription. Different STAT combinations interact differentially with related DNA response elements. Signalling pathways of this novel type are likely utilized by a wide variety of polypeptide ligands. Data from the IL2, IL6 and IFN systems indicate a major role for the tyrosine phosphorylated receptor/JAK complexes (rather than substrate specificity of the JAKs per se) in STAT selection. The mutant cell lines lacking individual JAKs and STATs are being used together with kinase-negative JAK mutants which differentially affect the IFN-gamma, and IFN-alpha beta and IL-6 pathways in the further analysis of these and additional systems.

Cytokines↗

PubMed1996

Kinase-negative mutants of JAK1 can sustain interferon-gamma-inducible gene expression but not an antiviral state.

The receptor-associated protein tyrosine kinases JAK1 and JAK2 are both required for the interferon (IFN)-gamma response. The effects of expressing kinase-negative JAK mutant proteins on signal transduction in response to IFN-gamma in wild-type cells and in mutant cells lacking either JAK1 or JAK2 have been analysed. In cells lacking endogenous JAK1 the expression of a transfected kinase-negative JAK1 can sustain substantial IFN-gamma-inducible gene expression, consistent with a structural as well as an enzymic role for JAK1. Kinase-negative JAK2, expressed in cells lacking endogenous JAK2, cannot sustain IFN-gamma-inducible gene expression, despite low level activation of STAT1 DNA binding activity. When expressed in wild-type cells, kinase-negative JAK2 acts as a dominant-negative inhibitor of the IFN-gamma response. Further analysis of the JAK/STAT pathway suggests a model for the IFN-gamma response in which the initial phosphorylation of JAK1 and JAK2 is mediated by JAK2, whereas phosphorylation of the IFN-gamma receptor is normally carried out by JAK1. The efficient phosphorylation of STAT 1 in the receptor-JAK complex may again depend on JAK2. Interestingly, a JAK1-dependent signal, in addition to STAT1 activation, appears to be required for the expression of the antiviral state.

Animals↗

PubMed1996

Function of Stat2 protein in transcriptional activation by alpha interferon.

Alpha interferon (IFN-alpha)-induced transcriptional activation requires the induction of a complex of DNA-binding proteins, including tyrosine-phosphorylated Stat1 and Stat2, and of p48, a protein which is not phosphorylated on tyrosine and which comes from a separate family of DNA-binding proteins. The isolation and characterization of U6A cells, which lack Stat2, have allowed the introduction of normal and mutant forms of Stat2 so that various functions of the Stat2 protein can be examined. As reported earlier, Stat1, which is the second target of tyrosine phosphorylation in IFN-alpha-treated cells, is not phosphorylated in the absence of Stat2. We show that all mutations that block Stat2 phosphorylation also block Stat1 phosphorylation. These include not only the mutations of Y-690 and SH2 domain residues that are involved in tyrosine phosphorylation but also short deletions at the amino terminus of the protein. Two mutants of Stat2 that are not phosphorylated on tyrosine can act as dominant negative proteins in suppressing wild-type Stat2 phosphorylation, most likely by competition at the receptor-kinase interaction site(s). We also show that the COOH-terminal 50 amino acids are required for transcriptional activation in response to IFN-alpha. Mutants lacking these amino acids can be phosphorylated, form IFN-stimulated gene factor 3, and translocate to the nucleus but cannot stimulate IFN-alpha-dependent transcription. Seven acidic residues are present in the deleted COOH-terminal residues, but 24 acidic residues still remain in the 100 carboxy-terminal amino acids after deletion. Thus, transcriptional activation is unlikely to depend on acidic amino acids alone.

Amino Acid Sequence↗

PubMed1996

Roles of JAKs in activation of STATs and stimulation of c-fos gene expression by epidermal growth factor.

The tyrosine kinase JAK1 and the transcription factors STAT1 and STAT3 are phosphorylated in response to epidermal growth factor (EGF) and other growth factors. We have used EGF receptor-transfected cell lines defective in individual JAKs to assess the roles of these kinases in STAT activation and signal transduction in response to EGF. Although JAK1 is phosphorylated in response to EGF, it is not required for STAT activation or for induction of the c-fos gene. STAT activation in JAK2- and TYK2-defective cells is also normal, and the tyrosine phosphorylation of these two kinases does not increase upon EGF stimulation in wild-type or JAK1-negative cells. In cells transfected with a kinase-negative mutant EGF receptor, there is no STAT activation in response to EGF and c-fos is not induced, showing that the kinase activity of the receptor is required, directly or indirectly, for these two responses. The data do not support a role for any of the three JAK family members tested in STAT activation and are consistent with a JAK-independent pathway in which the intrinsic kinase domain of the EGF receptor is crucial. Furthermore, data from transient transfection experiments in HeLa cells, using c-fos promoters lacking the STAT regulatory element c-sis-inducible element, indicate that this element may play only a minor role in the induction of c-fos by EGF in these cells.

Base Sequence↗

PubMed1996

Interactions between STAT and non-STAT proteins in the interferon-stimulated gene factor 3 transcription complex.

The first STAT-containing transcription factor to be studied, the alpha-interferon-induced ISGF3, is composed of a Stat1:2 heterodimer and a weak DNA-binding protein, p48, that is a member of a growing family of proteins similar to the so-called interferon regulatory factor (IRF-1). The p48 and Stat1:2 heterodimer do not associate stably in the absence of DNA, but we show that amino acids approximately 150 to 250 of Stat1 and a COOH-terminal portion of p48 exhibit physical interaction, implying contact that stabilizes ISGF3. Moreover, amino acid exchanges within the Stat1 contact region diminish or abolish the functional activity of Stat1. This protein interaction domain may be important in other STAT proteins to recruit partners to multiprotein transcription factors.

DNA-Binding Proteins↗

PubMed1995

Combinatorial association and abundance of components of interferon-stimulated gene factor 3 dictate the selectivity of interferon responses.

Genes containing the interferon-stimulated response element (ISRE) enhancer have been characterized as transcriptionally responsive primarily to type I interferons (IFN alpha/beta). Induction is due to activation of a multimeric transcription factor, interferon-stimulated gene factor 3 (ISGF3), which is activated by IFN alpha/beta but not by IFN gamma. We found that ISRE-containing genes were induced by IFN gamma as well as by IFN alpha in Vero cells. The IFN gamma response was dependent on the ISRE and was accentuated by preexposure of cells to IFN alpha, a treatment that increases the abundance of ISGF3 components. Overexpression of ISGF3 polypeptides showed that the IFN gamma response depended on the DNA-binding protein ISGF3 gamma (p48) as well as on the 91-kDa protein STAT91 (Stat1 alpha). The transcriptional response to IFN alpha required the 113-kDa protein STAT113 (Stat2) in addition to STAT91 and p48. Mutant fibrosarcoma cells deficient in each component of ISGF3 were used to confirm that IFN gamma induction of an ISRE reporter required p48 and STAT91, but not STAT113. A complex containing p48 and phosphorylated STAT91 but lacking STAT113 bound the ISRE in vitro. IFN gamma-induced activation of this complex, preferentially formed at high concentrations of p48 and STAT91, may explain some of the overlapping responses to IFN alpha and IFN gamma.

Animals↗

PubMed1995

Contribution of STAT SH2 groups to specific interferon signaling by the Jak-STAT pathway.

In response to specific ligands, various STAT proteins (signal transducers and activators of transcription) are phosphorylated on tyrosine by Jak protein kinases and translocated to the nucleus to direct gene transcription. Selection of a STAT at the interferon gamma receptor as well as specific STAT dimer formation depended on the presence of particular SH2 groups (phosphotyrosine-binding domains), whereas the amino acid sequence surrounding the phosphorylated tyrosine on the STAT could vary. Thus, SH2 groups in STAT proteins may play crucial roles in specificity at the receptor kinase complex and in subsequent dimerization, whereas the kinases are relatively nonspecific.

Animals↗

PubMed1995

Jaks and Stats in signaling by the cytokine receptor superfamily.

Many cytokines mediate their biological effects through interaction with a distinct family of receptors termed the cytokine receptor superfamily. Although members of this family lack catalytic domains, they couple ligand binding to tyrosine phosphorylation. Recent studies have shown that a novel family of cytoplasmic protein tyrosine kinases, termed the Janus kinases (Jaks), associate with the cytokine receptors and are catalytically activated after ligand binding. The activated Jaks phosphorylate and activate members of a novel family of transcription factors termed signal transducers and activators of transcription (Stats). In addition, many cytokines induce the phosphorylation of SHC, Vav and the p85 subunit of PI-3 kinase. The region of the receptors proximal to the cytoplasmic membrane is required for Jak association, mitogenesis, Stat activation and Vav phosphorylation. The membrane-distal region, which contains the major sites of tyrosine phosphorylation, is required for phosphorylation of SHC and p85, not for mitogenesis, thus allowing functional dissection of the signaling pathways activated by cytokines.

Amino Acid Sequence↗

PubMed1995

Role of STAT2 in the alpha interferon signaling pathway.

We have isolated U6A, a mutant cell line which lacks the STAT2 subunit of the transcription factor interferon (IFN)-stimulated gene factor 3 (ISGF3). The response of U6A cells to IFN-alpha is almost completely defective, but the response to IFN-gamma is normal. Complementation of U6A cells with a cDNA encoding STAT2 restores the IFN-alpha response, proving that STAT2 is required in this pathway. Binding of IFNs to their receptors triggers tyrosine phosphorylation and activation of the receptors, JAK family kinases, STAT1, and STAT2. In IFN-alpha-treated U6A cells, phosphorylation of the essential tyrosine kinases TYK2 and JAK1 is normal, but the phosphorylation of STAT1 is weak. A mutant STAT2 protein in which the phosphorylated tyrosine at position 690 is changed to phenylalanine does not restore normal phosphorylation of STAT1 in response to IFN-alpha. The dependence of STAT1 phosphorylation on the presence of STAT2 but not vice versa (T. Improta, C. Schindler, C. M. Horvath, I. M. Kerr, G. R. Stark, and J. E. Darnell, Jr., Proc. Natl. Acad. Sci. USA 91:4776-4780, 1994) indicates that in the formation of ISGF3, these two proteins may be phosphorylated sequentially in response to IFN-alpha and that phosphorylated STAT2 may be required to allow unphosphorylated STAT1 to bind to the activated IFN-alpha receptor.

Antibodies↗

PubMed1994

Activation of JAK kinases and STAT proteins by interleukin-2 and interferon alpha, but not the T cell antigen receptor, in human T lymphocytes.

The activation of Janus protein tyrosine kinases (JAKs) and signal transducer and activator of transcription (STAT) proteins by interleukin (IL)-2, the T cell antigen receptor (TCR) and interferon (IFN) alpha was explored in human peripheral blood-derived T cells and the leukemic T cell line Kit225. An IL-2-induced increase in JAK1 and JAK3, but not JAK2 or Tyk2, tyrosine phosphorylation was observed. In contrast, no induction of tyrosine phosphorylation of JAKs was detected upon stimulation of the TCR. IFN alpha induced the tyrosine phosphorylation of JAK1 and Tyk2, but not JAK2 or JAK3. IFN alpha activated STAT1, STAT2 and STAT3 in T cells, but no detectable activation of these STATs was induced by IL-2. However, IL-2 regulates the DNA binding and tyrosine phosphorylation of two STAT-like protein complexes which do not include STAT1, STAT2 or STAT3. STAT4 is not activated by IL-2. The activation of STAT5 cannot be excluded, so the IL-2-activated complexes most probably include at least one novel STAT. No STAT activity was detected in TCR-stimulated lymphocytes, indicating that the JAK/STAT pathway defined in this study constitutes an IL-2R-mediated signaling event which is not shared by the TCR. Finally, in other cell types the correlation between JAK1 activation and the induction of STAT1 has suggested that JAK1 may activate STAT1. The observation that IL-2 and IFN alpha activate JAK1 to a comparable degree, but only IFN alpha activates STAT1, indicates that JAK1 activation is not the only determining factor for STAT1 activation. Moreover, the data show that JAK1 stimulation is also not sufficient for STAT3 activation.

Base Sequence↗

PubMed1994

Jak-STAT pathways and transcriptional activation in response to IFNs and other extracellular signaling proteins.

Through the study of transcriptional activation in response to interferon alpha (IFN-alpha) and interferon gamma (IFN-gamma), a previously unrecognized direct signal transduction pathway to the nucleus has been uncovered: IFN-receptor interaction at the cell surface leads to the activation of kinases of the Jak family that then phosphorylate substrate proteins called STATs (signal transducers and activators of transcription). The phosphorylated STAT proteins move to the nucleus, bind specific DNA elements, and direct transcription. Recognition of the molecules involved in the IFN-alpha and IFN-gamma pathway has led to discoveries that a number of STAT family members exist and that other polypeptide ligands also use the Jak-STAT molecules in signal transduction.

Amino Acid Sequence↗

PubMed1994

Transcription factor ISGF-3 formation requires phosphorylated Stat91 protein, but Stat113 protein is phosphorylated independently of Stat91 protein.

Transcription factor ISGF-3 is a multiprotein, interferon alpha-activated transcription complex consisting of a 48-kDa DNA-binding protein and two proteins termed Stats (for signal transducers and activators of transcription) that become phosphorylated on tyrosine in the cell cytoplasm, a 113-kDa and either a 91- or 84-kDa polypeptide, the latter two of which arise from differentially spliced mRNAs. Using cell lines lacking the Stat91 or Stat84 proteins, we show that mutations in several different sites in the 91-kDa protein block the interferon alpha-induced phosphorylation of the 91-kDa protein and subsequent ISGF-3 formation. Although correct tyrosine phosphorylation on residue 690 of the Stat113 protein occurs independent of the Stat91/84 protein, the Stat113 phosphoprotein by itself moves to the cell nucleus much less efficiently in the absence of phosphorylated Stat91/84 protein.

Amino Acid Sequence↗

PubMed1994

Functional differences in the promoters of the interferon-inducible (2'-5')A oligoadenylate synthetase and 6-16 genes in interferon-resistant Daudi cells.

A clone of interferon-alpha-resistant (IFNr) Daudi cells retained much greater transcriptional inducibility of the (2'-5') oligoadenylate synthetase than the 6-16 gene despite the fact that the response of both genes is mediated by highly similar interferon-stimulable DNA response elements (ISRE). The primary IFN-alpha activatable transcription factor E (ISGF3) and the additional IFN-alpha-inducible ISRE-binding complex M were greatly reduced in the IFNr cells. The defect in E was in the E alpha subunit. In electrophoretic mobility-shift assays the 6-16 and (2'-5') oligoadenylate synthetase ISRE competed approximately equivalently for E and M. Moreover although active in wild-type cells the (2'-5') oligoadenylate synthetase ISRE was no more capable of conferring inducibility on a reporter gene in the IFNr cells than was the 6-16 ISRE. The contrasting response of the endogenous (2'-5') oligoadenylate synthetase and 6-16 genes in the IFNr cells is, therefore, unlikely simply to reflect the slight difference in the sequence of their ISRE. Consistent with this, in addition to the ISRE, sequences 5' to the ISRE in the (2'-5') oligoadenylate synthetase promoter appeared necessary for good induction by IFN alpha in the IFNr cells. Subtle quantitative changes in the phenotype of the IFNr cells have, however, precluded a more precise definition of the DNA element(s) involved.

2',5'-Oligoadenylate Synthetase↗

PubMed1994

Hepatitis delta virus replication in vitro is not affected by interferon-alpha or -gamma despite intact cellular responses to interferon and dsRNA.

The hepatitis delta virus (HDV) genome consists of circular ssRNA which has extensive intramolecular complementarity and can form a dsRNA rod-like structure. If such RNA species were to exist in an unmasked form in cells, they would be expected to induce interferon (IFN) expression and activate two IFN-inducible dsRNA-dependent enzymes with anti-viral activity, namely the dsRNA-dependent protein kinase (PKR) and 2',5' oligoadenylate (2',5' A) synthetase. Since the virus replicates to high copy number for prolonged periods in infected cells it is apparently able to evade these antiviral mechanisms. The RNA genome may be masked and fail to induce or activate the antiviral response, or the virus may inhibit such a response. Treatment of a hepatoma cell line, Huh7, and a fibrosarcoma cell line, HT1080, stably transfected with a trimeric HDV cDNA construct, with IFN-alpha or IFN-gamma for up to seven days failed to influence the level of expression of genomic or antigenomic HDV RNA, or delta antigen (Ag). This is consistent with either failure of activation or inhibition of the IFN response. However the induction of several IFN-responsive genes, including PKR, 2',5' A synthetase and class I MHC is normal and cotransfection of a construct expressing delta Ag did not affect expression from an IFN-inducible chloramphenicol acetyltransferase construct. In addition, the activation of PKR is not inhibited in HDV-expressing cells and antiviral assays suggest that the ability of these cells to mount an antiviral response to at least two cytopathic viruses is unaffected. IFN-beta is inducible normally by dsRNA in cells transfected with the delta cDNA trimer. We conclude that HDV replication is not inhibited by IFN-alpha or IFN-gamma, even though the responses of cells expressing HDV RNA and antigen to IFN and dsRNA are intact.

2',5'-Oligoadenylate Synthetase↗

PubMed1994

Interferon response pathways--a paradigm for cytokine signalling?

Considerable progress has been made in the past few years elucidating the molecular mechanisms of cytokine signalling. The interferons (IFNs) have provided a singular system allowing the detailed characterization of a specific cytokine signalling pathway. Studies on the IFN signal transduction pathway have identified protein tyrosine kinases (PTKs) that phosphorylate signal transducers (STATs) which then bind to DNA promoter sequences and activate gene transcription. Related work has shown that JAKs and STATs are also activated in response to a variety of the cytokines. Thus the novel type of signal transduction pathway identified for the IFNs promises to be more widely utilized than anticipated.

Animals↗

PubMed1994

Constitutive production of alpha and beta interferons in mutant human cell lines.

Alpha and beta interferons control expression of a selectable marker in the human hypoxanthine phosphoribosyltransferase-negative cell line 2fTGH, in which transcription of gpt is regulated by the upstream region of an interferon-responsive human gene. Selection of mutagenized 2fTGH cells in hypoxanthine-aminopterin-thymidine medium yielded mutants in one recessive (C1) and two dominant (C2 and C3) complementation groups. The mutants constitutively expressed low levels of beta interferon (C1), alpha interferon (C2), or both (C3).

Base Sequence↗

PubMed1993

A single phosphotyrosine residue of Stat91 required for gene activation by interferon-gamma.

Interferon-gamma (IFN-gamma) stimulates transcription of specific genes by inducing tyrosine phosphorylation of a 91-kilodalton cytoplasmic protein (termed STAT for signal transducer and activator of transcription). Stat91 was phosphorylated on a single site (Tyr701), and phosphorylation of this site was required for nuclear translocation, DNA binding, and gene activation. Stat84, a differentially spliced product of the same gene that lacks the 38 carboxyl-terminal amino acids of Stat91, did not activate transcription, although it was phosphorylated and translocated to the nucleus and bound DNA. Thus, Stat91 mediates activation of transcription in response to IFN-gamma.

Amino Acid Sequence↗