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We have investigated the interaction between GH (growth hormone) and GHR (GH receptor). We previously demonstrated that a truncated GHR that possesses a transmembrane domain but no cytoplasmic domain blocks receptor signalling. Based on this observation we investigated the impact of tethering the receptor's extracellular domain to the cell surface using a native lipid GPI (glycosylphosphatidylinositol) anchor. We also investigated the effect of tethering GH, the ligand itself, to the cell surface and demonstrated that tethering either the ecGHR (extracellular domain of GHR) or the ligand itself to the cell membrane via a GPI anchor greatly attenuates signalling. To elucidate the mechanism for this antagonist activity, we used confocal microscopy to examine the fluorescently modified ligand and receptor. GH-GPI was expressed on the cell surface and formed inactive receptor complexes that failed to internalize and blocked receptor activation. In conclusion, contrary to expectation, tethering an agonist to the cell surface can generate an inactive hormone receptor complex that fails to internalize.

Several studies have implicated the NF-kappaB inhibitor-like protein 1 (NFkBIL1) gene located in the class III region of the major histocompatibility complex (MHC) as a possible susceptibility locus for rheumatoid arthritis (RA). Based on limited homology, it has been suggested to be a member of the inhibitor of NF-kappaB (IkappaB) family of proteins, but a role in mRNA processing has also been proposed. We have investigated the expression of NFkBIL1 in RA synovial tissue and characterized its function. Real-time PCR showed the two NFkBIL1 mRNA splice variants are expressed in a tissue-specific manner. Dual immunofluorescent staining of human RA synovium with polyclonal anti-NFkBIL1 antibodies and anti-CD68, anti-CD3 or anti-factor VIII showed that NFkBIL1 was expressed in the rheumatoid synovial lining and sub-lining layers and co-localized in CD68+ and CD3+, but not Factor VIII+ cells. Confocal microscopy of cultured synovial fibroblasts revealed expression in speckled nuclear and homogenous cytoplasmic distributions, suggesting shuttling between the cytoplasmic and nuclear compartments. Functional tests showed that NFkBIL1 isoforms were incapable of associating with NF-kappaB and did not inhibit it, thus disproving the hypothesis that NFkBIL1 functions as an IkappaB. Affinity purification of endogenous NFkBIL1 proteins and co-immunoprecipitation experiments showed that NFkBIL1 can associate with mRNA and with three protein partners, identified by mass spectrometry as leukophysin, translation elongation factor 1 alpha and CTP synthase I. These data support a potential role for NFkBL1 in the pathogenesis of RA and indicates that it may be involved in mRNA processing or the regulation of translation.

We have mutated a conserved residue of the death domain of the interleukin-1 (IL-1) receptor-associated kinase (IRAK), threonine 66. The substitution of Thr-66 with alanine or glutamate prevented spontaneous activation of NF-kappaB by overexpressed IRAK but enhanced IL-1-induced activation of the factor. Like the kinase-inactivating mutation, K239S, the T66A and T66E mutations interfered with the ability of IRAK to autophosphorylate and facilitated the interactions of IRAK with TRAF6 and with the IL-1 receptor accessory protein, AcP. Wild-type IRAK constructs tagged with fluorescent proteins formed complexes that adopted a punctate distribution in the cytoplasm. The Thr-66 mutations prevented the formation of these complexes. Measurements of fluorescence resonance energy transfer among fluorescent constructs showed that the Thr-66 mutations abolished the capacity of IRAK to dimerize. In contrast, the K239S mutation did not inhibit dimerization of IRAK as evidenced by fluorescence resonance energy transfer measurements, even though microscopy showed that it prevented the formation of punctate complexes. Our results show that Thr-66 plays a crucial role in the ability of IRAK to form homodimers and that its kinase activity regulates its ability to form high molecular weight complexes. These properties in turn determine key aspects of the signaling function of IRAK.

We used a series of deletion mutations in the 5' untranslated region of the prototype D type retrovirus, Mason-Pfizer Monkey Virus (MPMV), to analyse RNA encapsidation. A region was identified upstream of the major splice donor which reduced particle production but had a proportionally greater effect on RNA packaging. A small deletion downstream of the splice donor had little effect on RNA production and caused no significant packaging defect. A large deletion encompassing the end of the primer binding site down to the splice donor had a dramatic effect, disrupting viral protein synthesis. Stable cell lines were produced containing packaging-defective virus. These first-generation packaging cell lines were used to package and transfer an MPMV-based vector., (Copyright 2001 Academic Press.)

We have identified a fourth splice variant of the TGF beta-activated kinase (TAK1), called TAK1-d, and identified an error in the previously published TAK1-c sequence. Our data shows that the c and d variants encode proteins whose carboxyl ends differ markedly from those of variants a and b. Analysis of the human TAK1 gene sequence, located at 6q16.1-q16.3, shows that the coding sequence is organised in 17 exons. The four splice variants result from alternative splicing of exons 12 and 16, the reading frame of exon 17 being determined by the presence or absence of exon 16. Study of the relative levels of expression of the four splice variants showed significant variations between tissues. Our evidence suggests that the alternative splicing of the TAK1 mRNA may have important functional implications.

Interleukin 1 (IL-1) and tumour necrosis factor (TNF) activate a novel protein kinase, TIP kinase, which phosphorylates beta-casein in vitro. We have identified and purified to homogeneity a tryptic fragment of beta-casein, called T1, which was phosphorylated by TIP kinase with kinetics similar to those of the intact protein (K[m] = 27 +/- 6 microM). Phosphopeptide maps of in vitro phosphorylated T1 and beta-casein were identical, confirming that T1 contained the main phosphorylation site of the protein. T1 corresponded to residues 114 to 169 of beta-casein. It was phosphorylated by constitutively active protein kinases to a much lesser extent than beta-casein and thus constituted a specific substrate of the cytokine-activated enzyme. This made possible the detection of TIP kinase in extracts of IL-1-stimulated HeLa and KB cells, which had been hampered by high background phosphorylation when beta-casein was used as substrate. Our results show that the use of fragment T1 allows detection of low levels of TIP kinase in crude samples. They also suggest that its activation, which had previously been observed only in connective tissue cells, may be a general response of many cell types to IL-1 or TNF.

The inflammatory cytokines interleukin 1 (IL1) and tumour necrosis factor (TNF) have a broad range of physiological effects. Whereas their immediate post-receptor events are not well understood, both have the potential to activate a range of protein kinases. These include the three types of mitogen activated protein (MAP) kinase (ERK, JNK/p54 and p38) and a beta-casein kinase. The mechanisms by which these kinases are activated is discussed and the significance of their activation for particular biological responses is assessed.

The regulation of the inhibitor of nuclear factor kappa B (I kappa B) by interleukin 1 (IL1) was investigated in HeLa cells. Two forms of I kappa B were resolved by ion-exchange chromatography. The major form (75%) was identified as MAD3 by specific antisera. IL1 generated rapidly (6 min) an electrophoretically retarded form of MAD3 that was stable in acid and was converted into the unmodified form by phosphatase 2A. It thus corresponded to a phosphorylation of the protein on serine or threonine. IL1 also caused the disappearance of MAD3 from the cells, which was complete 15 min after stimulation and coincided with a 46% reduction of cellular I kappa B activity. Newly-synthesized MAD3 accumulated to pre-stimulation levels between 60 and 90 min after stimulation and this coincided with the down-regulation of the phosphorylating activity. The serine proteinase inhibitors 3,4-dichloroisocoumarin (DCI) and tosylphenylalanyl chloromethylketone (TPCK) prevented phosphorylation and disappearance of MAD3. At the same concentrations (10-100 microM), they also increased basal phosphorylation of the small heat shock protein (hsp27) and prevented the IL1- and phorbol 12-myristate 13-acetate-induced increases of its phosphorylation. The inhibitors were thus interfering with protein kinases when blocking degradation of MAD3. Recombinant MAD3 phosphorylated in vitro by protein kinase C was not electrophoretically retarded, suggesting that MAD3 was phosphorylated by another kinase in IL1-stimulated cells. Our results suggest that the IL1-induced phosphorylation of MAD3 on serine or threonine leads to its degradation. DCI and TPCK blocked phosphorylation mechanisms and it could not be concluded that serine proteinases were involved in the breakdown of MAD3.

Increases (5-fold) in the rate of phosphorylation of beta-casein were observed in extracts of human gingival fibroblasts that had been stimulated by interleukin 1 (IL-1) or tumour necrosis factor (TNF). The induced kinase was cytosolic and had little activity on alpha-casein. Its chromatographic behaviour on anion-exchange and gel-filtration columns was similar to that of beta-casein kinase, an enzyme detected originally in MRC-5 cells stimulated by IL-1 and TNF. Phosphopeptide maps of beta-casein confirmed that the kinase activated in gingival fibroblasts had the same substrate specificity as beta-casein kinase. In gingival fibroblasts, beta-casein kinase activity was maximum after 15 min of stimulation by IL-1 or TNF, and remained activated for several hours. Activations of small heat-shock protein (hsp27) kinase and mitogen-activated protein (MAP) kinase were also maximum 15 min after stimulation, but decreased to background levels within the next 30 min. Study of the effects of 21 agents other than IL-1 or TNF showed that none activated beta-casein kinase, whereas several activated MAP kinase or hsp27 kinase. beta-Casein kinase was also detected in extracts of bovine articular chondrocytes and human endothelial cells stimulated by IL-1 or TNF. Semi-purified preparations of fibroblast beta-casein kinase were not inactivated by phosphatases in vitro. Our results suggest that it may be involved in responses specific to IL-1 and TNF in a wide range of cell types and that its activation probably involves mechanisms other than its phosphorylation.

An IL-1-stimulated protein kinase cascade resulting in phosphorylation of the small heat shock protein hsp27 has been identified in KB cells. It is distinct from the p42 MAP kinase cascade. An upstream activator kinase phosphorylated a 40 kDa kinase (p40) upon threonine and tyrosine residues, which in turn phosphorylated a 50 kDa kinase (p50) upon threonine (and some serine) residues. p50 phosphorylated hsp27 upon serine. p40 and p50 were purified to near homogeneity. All three components were inactivated by protein phosphatase 2A, and p40 was inactivated by protein tyrosine phosphatase 1B. The substrate specificity of p40 differed from that of p42 and p54 MAP kinases. The upstream activator was not a MAP kinase kinase. p50 resembled MAPKAPK-2 and may be identical.

The trophoblast of the developing ruminant conceptus secretes large quantities (up to 100 micrograms, or 10(7) IU, per 24 h) of a number of characteristic Type I interferons. Secretion (gene expression) commences at Day 8 or 9 (in the sheep; Day 10 in cattle) and ends at about Day 22 (Day 25). The function of this material is in the inhibition of uterine prostaglandin secretion, and hence in the maintenance of the corpus luteum (the 'maternal recognition of pregnancy'). Such rapid onset and cessation of gene transcription begs questions about the function of trophoblast interferon gene promoters, and a limited number of studies have now been carried out with reporter gene constructs, with inconclusive results. It seems unlikely, however, that viral responsiveness accounts for the phenomenon, as in adult interferon genes, although the trophoblast interferons are highly active antiviral agents.

A type I interferon (IFN) secreted by the trophoblast of early sheep and cow embryos is thought to be responsible for the maternal recognition of pregnancy. The expression of trophoblast IFN is tissue specific and temporally controlled. However, the isolated bovine trophoblast IFN promoter did not confer tissue specificity on the expression of a bacterial chloramphenicol acetyl transferase (CAT) reporter gene, and could not be induced by virus, unlike other type I IFNs. Trophoblast IFN acts locally within the uterus to prevent luteolysis and prolong progesterone secretion. Endometrial IFN receptors are present, and trophoblast IFN decreases expression of endometrial oxytocin receptor and increases expression of endometrial beta 2-microglobulin, MHC class I antigens and Mx (a mediator of IFN antiviral activity) only in the pregnant horn of pregnant ewes with a transected uterus. The primary effect of trophoblast IFN during early pregnancy appears to be an inhibition of oxytocin receptor expression, although studies in ovariectomized ewes suggest that luteal oxytocin may be required to facilitate the inhibition of prostaglandin F secretion by trophoblast IFN. An investigation of the isolated oxytocin receptor promoter should confirm its critical role in the maternal recognition of pregnancy.

Interleukin 1 and tumor necrosis factor activate serine/threonine kinase activity in cells within minutes of receptor interaction. Kinases increasing phosphorylation of the small heat shock protein (HSP 27) and epidermal growth factor receptor have been identified and shown to be activated independent of protein kinase A or C. Such kinases are also implicated in activation of the transcription factor nuclear factor kappa B. The cytokines have novel signal transduction pathways that could offer potential therapeutic targets.

Interleukin 1 signals through an 80kDa receptor to change protein kinase activity in fibroblasts. Two increases in protein phosphorylation have been identified: the EGF receptor and the small heat shock protein (hsp 27). Both involve serine and are similarly transient (maximal 5-15 minutes). The kinase activity differs from PKA or C. Hsp 27 can be used for its assay in extracts of stimulated cells.

Pig heart tissue have been shown to contain 3 different 60,000 Da phosphoproteins. Different purification procedures were used in order to separate them, suggesting that the 3 phosphoproteins differ in their environmental parameters. The 2 major ones appear essentially as peripheral phosphoproteins that are associated with cellular membranes through ionic forces, whereas the third minor phosphoprotein behaves as an integral plasma membrane protein. The three phosphoproteins also differ in their relative amount of phosphorylated serine, threonine and tyrosine residues after in vitro protein kinase assay. Evidence that the 3 phosphoproteins are related arises from the similarity between their respective phosphopeptide maps after partial hydrolysis with proteases, an experiment that also points out relatedness in primary structure between them and the transforming protein of Rous sarcoma virus, pp60v-src. The 3 phosphoproteins, however, do not appear to be immunologically related to pp60v-src since none of them is immunoprecipitated by sera that precipitate pp60v-src. The possibility that the three 60,000 Da phosphoproteins under study represent 3 differentially localized and phosphorylated products of c-src and/or c-src related genes is discussed.

Preincubation of purified plasma membranes from rat liver with EGF stimulates the level of phosphorylation on serine and tyrosine residues in a 59-kD protein. Such an increased phosphoserine and phosphotyrosine content of the 59-kD protein occurs at the expense of the phosphorylation on threonine residues. The effect is observed under conditions where the plasma membranes have been extracted at pH 10. It is not observed when the membranes are simply washed at pH 7.5 before further purification. A number of experiments, including TBR-IgG phosphorylation in immunoprecipitates and partial hydrolysis with varying concentrations of the V8 protease, suggest that the 59-kD protein modified upon EGF treatment could be a representative of the c-src gene product from hepatocytes.