Molecular characterization of an alpha interferon receptor 1 subunit (IFNaR1) domain required for TYK2 binding and signal transduction.

Binding of alpha interferon (IFNalpha) to its receptors induces rapid tyrosine phosphorylation of the receptor subunits IFNaR1 and IFNaR2, the TYK2 and JAK1 tyrosine kinases, and the Stat1 and Stat2 transcription factors. Previous studies have demonstrated that TYK2 directly and specifically binds to and tyrosine phosphorylates IFNaR1 in vitro. We now report a detailed analysis of the TYK2 binding domain on the IFNaR1 subunit. First, we used an in vitro binding assay to identify the TYK2 binding motif in IFNaR1 as well as the critical residues within this region. The most striking feature is the importance of a number of hydrophobic and acidic residues. A minor role is also ascribed to a region resembling the proline-rich "box 1" sequence. In addition, mutations which disrupt in vitro binding also disrupt the coimmunoprecipitation of the receptor and TYK2. We also provide direct evidence that the binding region is both necessary and sufficient to activate TYK2 in vivo. Specifically, mutations in the binding domain act in a dominant-negative fashion to inhibit the IFNalpha-induced tyrosine phosphorylation of TYK2 and Stat2. Further, introduction of dimerized glutathione S-transferase-IFNaR1 fusion proteins into permeabilized cells is sufficient to induce phosphorylation of TYK2 and the receptor, confirming the role of the binding domain in IFNalpha signal transduction. These studies provide clues to the sequences determining the specificity of the association between JAK family tyrosine kinases and cytokine receptors as well as the functional role of these kinases in cytokine signal transduction.

Direct binding to and tyrosine phosphorylation of the alpha subunit of the type I interferon receptor by p135tyk2 tyrosine kinase.

Binding of type I interferons (IFNs) to their receptors induces rapid tyrosine phosphorylation of multiple proteins, including the alpha and beta subunits of the receptor, the polypeptides that form the transcriptional activator ISGF3 alpha (Stat113, Stat84, and Stat91), and the p135tyk2 and Jak-1 tyrosine kinases. In this report, we demonstrate that the alpha subunit of the type I IFN receptor (IFN-R) corresponds to the product of a previously cloned receptor subunit cDNA and, further, that the p135tyk2 tyrosine kinase directly binds and tyrosine phosphorylates this receptor subunit. Glutathione S-transferase (GST) fusion proteins encoding the different regions of the cytoplasmic domain of the alpha subunit can bind the p135tyk2 contained in human cell lysates. The association between the alpha subunit and Tyk2 was demonstrated by immunoblotting with anti-Tyk2 and antiphosphotyrosine antibodies and by using an in vitro kinase assay. Analogous experiments were then performed with recombinant baculoviruses encoding constitutively active Jak family tyrosine kinases. In this case, p135tyk2, but not Jak-1 or Jak-2 protein, binds to the GST-IFN-R proteins, suggesting that the interaction between these two proteins is both direct and specific. We also demonstrate that Tyk2, from extracts of either IFN alpha-treated human cells or insect cells infected with the recombinant baculoviruses, can catalyze in vitro phosphorylation of GST-IFN-R protein in a specific manner. Deletion mutants of the GST-IFN-R protein were used to localize both the binding and tyrosine phosphorylation site(s) to a 46-amino-acid juxtamembrane region of the alpha subunit, which shows sequence homology to functionally similar regions of other cytokine receptor proteins. These data support the hypothesis that the Tyk2 protein functions as part of a receptor complex to initiate intracellular signaling in response to type I IFNs.

Direct interaction of Jak1 and v-Abl is required for v-Abl-induced activation of STATs and proliferation.

In Abelson murine leukemia virus (A-MuLV)-transformed cells, members of the Janus kinase (Jak) family of non-receptor tyrosine kinases and the signal transducers and activators of transcription (STAT) family of signaling proteins are constitutively activated. In these cells, the v-Abl oncoprotein and the Jak proteins physically associate. To define the molecular mechanism of constitutive Jak-STAT signaling in these cells, the functional significance of the v-Abl-Jak association was examined. Mapping the Jak1 interaction domain in v-Abl demonstrates that amino acids 858 to 1080 within the carboxyl-terminal region of v-Abl bind Jak1 through a direct interaction. A mutant of v-Abl lacking this region exhibits a significant defect in Jak1 binding in vivo, fails to activate Jak1 and STAT proteins, and does not support either the proliferation or the survival of BAF/3 cells in the absence of cytokine. Cells expressing this v-Abl mutant show extended latency and decreased frequency in generating tumors in nude mice. In addition, inducible expression of a kinase-inactive mutant of Jak1 protein inhibits the ability of v-Abl to activate STATs and to induce cytokine-independent proliferation, indicating that an active Jak1 is required for these v-Abl-induced signaling pathways in vivo. We propose that Jak1 is a mediator of v-Abl-induced STAT activation and v-Abl induced proliferation in BAF/3 cells, and may be important for efficient transformation of immature B cells by the v-abl oncogene.

Beta interferon and oncostatin M activate Raf-1 and mitogen-activated protein kinase through a JAK1-dependent pathway.

Activation of early response genes by interferons (IFNs) and other cytokines requires tyrosine phosphorylation of a family of transcription factors termed signal transducers and activators of transcription (Stats). The Janus family of tyrosine kinases (Jak1, Jak2, Jak3, and Tyk2) is required for cytokine-induced tyrosine phosphorylation and dimerization of the Stat proteins. In order for IFNs to stimulate maximal expression of Stat1alpha-regulated genes, phosphorylation of a serine residue in the carboxy terminus by mitogen-activated protein kinase (MAPK) is also required. In HeLa cells, both IFN-beta and oncostatin M (OSM) stimulated MAPK and Raf-1 enzyme activity, in addition to Stat1 and Stat3 tyrosine phosphorylation. OSM stimulation of Raf-1 correlated with GTP loading of Ras, whereas IFN-beta activation of Raf-1 was Ras independent. IFN-beta- and OSM-induced Raf-1 activity could be coimmunoprecipitated with either Jak1 or Tyk2. Furthermore, HeLa cells lacking Jak1 displayed no activation of STAT1alpha, STAT3, and Raf-1 by IFN-beta or OSM and also demonstrated no increase in the relative level of GTP-bound p21ras in response to OSM. The requirement for Jak1 for IFN-beta- and OSM-induced activation of Raf-1 was also seen in Jak1-deficient U4A fibrosarcoma cells. Interestingly, basal MAPK, but not Raf-1, activity was constitutively enhanced in Jak1-deficient HeLa cells. Transient expression of Jak1 in both Jak-deficient HeLa cells and U4A cells reconstituted the ability of IFN-beta and OSM to activate Raf-1 and decreased the basal activity of MAPK, while expression of a kinase-inactive form of the protein showed no effect. Moreover, U4A cells selected for stable expression of Jak1, or COS cells transiently expressing Jak1 or Tyk2 but not Jak3, exhibited enhanced Raf-1 activity. Therefore, it appears that Jak1 is required for Raf-1 activation by both IFN-beta and OSM. These results provide evidence for a link between the Jaks and the Raf/MAPK signaling pathways.

Dimerization of a chimeric CD4-interferon-alpha receptor reconstitutes the signaling events preceding STAT phosphorylation.

Interferon-alpha induces the rapid tyrosine phosphorylation of a number of molecules, including the cognate receptors, JAK-family kinases (Jak1 and tyk2), and latent transcription factors (STATs 1 and 2). Here, we describe the use of chimeric molecules composed of the extracellular domain of CD4 fused to the intracellular domain of the interferon-alpha receptor subunit 1 (IFNaR1). Antibody mediated crosslinking dimerizes the transfected chimeras, activates tyk2 and induces a tyk2-dependent tyrosine phosphorylation of the intracellular domain of the chimera. We further define the major site of IFNaR1 phosphorylation, and show that phosphorylation of this site is required for association with STAT2. Finally, we show that homodimerization of IFNaR1 is not sufficient to activate the STATs, suggesting a role for the IFNaR2 subunit and Jak1 in the transduction of the interferon-alpha signal.

tyk2, prototype of a novel class of non-receptor tyrosine kinase genes.

We previously identified a novel protein tyrosine kinase gene, tyk2, by screening a human lymphoid cDNA library with a tyrosine kinase domain specific c-fms restriction fragment under low stringency hybridization conditions. We have now isolated and sequenced a full length tyk2 cDNA clone; demonstrated that this gene is widely expressed in hematopoietic and non-hematopoietic cell lines; and mapped it to chromosome 19p13.2. The cDNA clone is 4176 nucleotides long and codes for a putative protein with a molecular weight of 134 kilodaltons. Hydrophobicity analysis of our sequence does not identify a transmembrane domain, which is found in all members of the receptor class of protein tyrosine kinases; nor can we detect an SH2 domain, found in all previously identified non-receptor protein kinases. We therefore propose that tyk2 is the prototype of a new class of non-receptor protein tyrosine kinases.

Identification and chromosomal mapping of new human tyrosine kinase genes.

To identify novel protein tyrosine kinase (PTK) genes expressed in human lymphoid cells, we have screened B- and T-cell cDNA libraries at low stringency using a c-fms tyrosine kinase domain probe. Three new PTK genes were identified, based on the presence of conserved amino acid sequence motifs characteristic of the catalytic domain of tyrosine kinases. Of these three genes, one (tyk1) appears to be the human homologue of a previously cloned murine gene (ltk), which has been reported to encode a tyrosine kinase with a unique structure; while the second gene, tyk2 cannot be clearly assigned to any of the known PTK subfamilies, and therefore may be the prototype of a new PTK gene subfamily. The third gene (tyk3/fer) has been very recently cloned by others; we present additional characterization in this report. We have performed Northern blots to establish the size of the mRNA encoded for by these genes, and to confirm their expression in lymphoid cells. Finally, we have determined the chromosomal location of all three genes by analyzing human-mouse somatic cell hybrids.

Some extrachromosomal circular DNAs containing the Alu family of dispersed repetitive sequences may be reverse transcripts.

A 300 base-pair (bp) size class of small polydisperse circular DNA (spcDNA) isolated from the BSC-1 line of African Green monkey kidney cells was cleaved with the restriction endonuclease Sau3A, and the resulting fragments (100 to 200 bp) were cloned in bacteriophage M13 mp7. The nucleotide sequence of each of 24 clones containing DNA sequences homologous to the Alu family of mobile, dispersed, repetitive elements was then determined. Analysis of these sequences revealed that many, and perhaps all, of the 300 bp Alu-containing spcDNAs had regions in which the 5' and 3' ends of the normal Alu element were juxtaposed and covalently joined. Although more than one model can explain the generation of such circular molecules, the most attractive one at this time involves their generation from reverse transcripts of Alu-specific RNAs.

Structure of extrachromosomal circular DNAs containing both the Alu family of dispersed repetitive sequences and other regions of chromosomal DNA.

Small polydisperse circular DNA (spcDNA) isolated from the BSC-1 line of African Green monkey kidney cells was digested with the restriction endonuclease BamHI and cloned in bacteriophage lambda. The resulting library of 25,000 phage was then screened for the presence of the Alu family of short interspersed nucleotide sequences, and four of the 100 Alu-positive clones were characterized. In summary: (1) all four clones contained regions other than Alu that were homologous to the BSC-1 chromosome. Two contained Alu plus unique chromosomal DNA, one contained Alu plus an uncharacterized repetitive chromosomal DNA, and one contained Alu plus both unique and a specific tandemly repeated chromosomal DNA (alpha-satellite); (2) all four clones were derived from extrachromosomal circular DNAs and not from the accidental cloning of a very small amount of contaminating chromosomal material assumed to be present in spcDNA preparations; and (3) one clone represented an intact circular DNA with a restriction endonuclease cleavage map that was a circularly permuted version of its chromosomal homologue.

A mutant form of p135tyk2, an interferon-alpha inducible tyrosine kinase, suppresses the transformed phenotype of Daudi cells.

The type I interferons induce an anti-viral state and suppress cell growth. The p135tyk2 non-receptor tyrosine kinase appears to initiate, at least in part, the type I interferon signal transduction pathway, and thereby activates type I interferon-dependent gene expression. To determine if p135tyk2 can suppress growth and/or tumorigenesis, derivatives of the tyk2 gene were introduced into the tumorigenic cell line Daudi. Transfectants expressing a tyk2 construct missing the carboxy-terminal 22 amino acids cloned with a greatly reduced efficiency in soft agar and displayed a partial decrease in the ability to form tumors in athymic mice. In addition, transfectants producing a kinase deficient version of tyk2 show an increase in both growth rate and agar cloning efficiency, suggesting that the inactive kinase can act in a dominant-negative manner. Surprisingly, the carboxyl-terminal deleted protein lacks both auto-kinase activity, and activity towards a putative substrate, even though it induces a phenotype which is precisely the opposite of that produced by another kinase-deficient tyk2 mutant containing an altered ATP binding site. Thus, while these results add tyk2 to a growing list of interferon-alpha regulated proteins that might be able to suppress tumor formation, the biochemical basis of this activity remains unknown.

Elevation of CD5+ B lymphocytes in schizophrenia.

BACKGROUND: A variety of immunologic alterations have been observed in patients with schizophrenia. These findings have lent support to theories that autoimmune mechanisms may be important in some patients with the illness. The CD5+ B lymphocyte, a B-cell subset associated with autoimmune disease, has been the subject of two previously published studies yielding disparate results. METHODS: In this study, we used immunofluorescent flow cytometry to measure CD5+ B cells, total B and T cells, and CD4 and CD8 subsets in patients with schizophrenia and in normal control subjects. RESULTS: A significantly higher percentage of patients with schizophrenia, relative to normal control subjects, exhibited an elevated level of CD5+ B cells (27.6% vs 6.7%). Antipsychotic withdrawal had no effect on CD5+ B-cell levels, suggesting that medication effects were not the cause of this difference. No other studied lymphocyte subsets differed between the two groups. CONCLUSIONS: A subset of patients with schizophrenia have elevated levels of CD5+ B cells. This finding replicates an earlier study by another group and provides further evidence suggestive of autoimmune manifestations in schizophrenia.

Homodimerization and intermolecular tyrosine phosphorylation of the Tyk-2 tyrosine kinase.

The Jak kinases and Stat transcription factors play a major role in signaling of various cytokines including IFN alpha. In this report we show a ligand-independent interaction between Tyk-2 and Jak-1 kinases. We also demonstrate that the Tyk-2 kinase forms a homodimer that has the ability to undergo intermolecular tyrosine phosphorylation. The formation of the Tyk-2 homodimer is independent of both tyrosine phosphorylation and the presence of the tyrosine kinase domain.

The vav proto-oncogene product (p95vav) interacts with the Tyk-2 protein tyrosine kinase.

The vav proto-oncogene product participates in the signaling pathways activated by various cell-surface receptors, including the type I IFN receptor. During engagement of the type I IFN receptor, p95vav is phosphorylated on tyrosine residues, but the kinase regulating its phosphorylation has not been identified to date. Our studies demonstrate that p95vav forms a stable complex with the IFN-receptor-associated Tyk-2 kinase in vivo, and strongly suggest that this kinase regulates its phosphorylation on tyrosine. Thus, p95vav is engaged in IFN-signaling by a direct interaction with the functional type I IFN receptor complex to transduce downstream signals.

Convenient, nonradioactive, heteroduplex-based methods for identifying recurrent mutations in the BRCA1 and BRCA2 genes.

The ability to identify individuals who are predisposed to specific malignant tumors is a promising molecular diagnostic by-product of over two decades of intensive research into the genetic pathogenesis of human cancer. Approximately 2% of Ashkenazi Jews carry recurrent germline mutations in either the BRCA1 or BRCA2 genes that may predispose these individuals to the development of breast and ovarian cancer. We have developed a nonisotopic method, based on the formation of heteroduplexes between polymerase chain reaction (PCR) amplified wild-type and mutant alleles, which can be used to identify the BRCA1 185delAG and the BRCA2 6174delT mutations. The same assay can also be used to verify the loss of heterozygosity in a tumor sample arising in an individual with a germline mutation. The four steps described in this report (PCR amplification, heteroduplex formation, acrylamide gel electrophoresis, and ethidium bromide staining/UV-fluorescence photography) can be readily and reproducibly performed in the course of a single day, making this a useful method for the routine identification of these mutations.

Comparative mutagenicity of ICR-191 to S. typhimurium and diploid human lymphoblasts.

Concentration-dependent mutagenicity of ICR-191 has been measured in Salmonella typhimurium strain TA98 and in a diploid human cell line. In both cell systems, approximately equigenerational exposure produced mutation linearly related to concentration in the lower range of ICR-191 concentrations tested. Saturation behavior was observed in the human cell assay but not in the bacterial assay. However, a 25-fold greater concentration of ICR-191 was required to induce a significant rise in the mutant fraction in the S. typhimurium assay than in the human cell assay. These differences may be linked to the differences in the biochemical events required for mutation or in the time of exposure to ICR-191.

[Radiculographic symptoms of changes in the adjacent intervertebral disks in the course of lumbar disk prolapse]. / Objawy radikulograficzne zmian sasiednich kraåków miedzykregowych w przebiegu przepuklin jader miazdzystych odcinka ledzwiowego kregoslupa.

From a group of 400 radiculographic examinations the authors selected 34 cases operated upon for disc prolapse. These patients had additional signs of prolapse of the neighbouring intervertebral discs. The analysis showed that the latter signs were present mostly in the discs situated above the level of disc prolapse. In only two cases these changes were found in the disc situated below the level of prolapse. The sign of bulging-in dural sac in radiculography may be an impression of bulging disc and, much less frequently, it can be caused by a slight prolapse of the disc. Demonstration of these changes in contrast-medium investigations of the vertebral canal should be taken into account during the planned operation. The importance of disturbances of spinal biomechanics in the development of multilevel discopathy is stressed.

FOXO3a mediates the androgen-dependent regulation of FLIP and contributes to TRAIL-induced apoptosis of LNCaP cells.

Androgen-withdrawal-induced apoptosis (AWIA) is deregulated in androgen refractory prostate cancer. Androgens have been shown to positively regulate expression of the antiapoptotic FADD-like interleukin-1beta-converting enzyme (FLICE)-like inhibitory protein (FLIP), and reduced FLIP expression precedes apoptosis after androgen withdrawal. Here, we show that FLIP protein expression is downregulated in castrated rats, while in LNCaP cells, androgens regulate FLIP in a manner that is dependent on phosphoinositol-3-kinase (PI3K) and Akt signaling. Specifically, treatment of LNCaP cells with LY294002, or expression of either PTEN or a non-phosphorylatable form of FOXO3a (FOXO3aTM), downregulates FLIP protein and mRNA. Conversely, treatment with androgens in the absence of PI3/Akt signaling, or following expression of FOXO3aTM, leads to increased FLIP expression. A FOXO3a binding site was identified in the FLIP promoter and shown necessary for the combined effects of androgens and FOXO3a on FLIP transcription. FOXO3a binds the androgen receptor, suggesting that the transcriptional synergy depends on an interaction between these proteins. Finally, LNCaP cells are sensitized to TRAIL-induced apoptosis by PTEN or LY294002, and rescued by androgens. FOXO3aTM also sensitizes cells to androgen-inhibited TRAIL apoptosis. Androgen rescue was diminished when either FOXO3a or FLIP was reduced by siRNA. These data support a role for FOXO3a in AWIA.