Dual specificity of the interleukin 1- and tumor necrosis factor-activated beta casein kinase.

Tumor necrosis factor (TNF) and interleukin 1 (IL1) activate a protein kinase, TIP kinase, which phosphorylates beta casein in vitro. We have now identified its main phosphorylation site on beta casein, Ser124 (Km approximately 28 mu M), and a minor phosphorylation site, Ser142 (Km approximately 0.7 mM). The sequence motif that determined the phosphorylation of Ser124 by the kinase was studied with synthetic peptides bearing deletions or substitutions of the neighboring residues. This allowed synthesis of improved substrates (Km approximately 6 mu M) and showed that efficient phosphorylation of Ser124 was favored by the presence of large hydrophobic residues at positions +1, +9, +11, and +13 (counted relative to the position of the phosphoacceptor amino acid) and of a cysteine at position -2. Peptides in which Ser124 was replaced by tyrosine were also phosphorylated by TIP kinase, showing it to have dual specificity. It is unable to phosphorylate the MAP kinases in vitro and is therefore not directly involved in their activation. Its biochemical characteristics indicate that TIP kinase is a novel dual specificity kinase, perhaps related to the mixed lineage kinases. It copurified with a phosphoprotein of about 95 kDa, which could correspond either to the autophosphorylated kinase or to an associated substrate.

T cell proliferation in response to interleukins 2 and 7 requires p38MAP kinase activation.

Interleukin-2 (IL-2) is a potent T cell mitogen. However, the signaling pathways by which IL-2 mediates its mitogenic effect are not fully understood. One of the members of the mitogen-activated protein kinase (MAPK) family, p42/44MAPK (ERK2/1), is known to be activated by IL-2. We have now investigated the response to IL-2 of two other members of the MAP kinase family, p54MAP kinase (stress-activated protein kinase (SAPK)/Jun-N-terminal kinase (JNK)) and p38MAP kinase (p38/Mpk2/CSBP/RK), which respond primarily to stressful and inflammatory stimuli (e.g. tumor necrosis factor-alpha, IL-1, and lipopolysaccharide). Here we show that IL-2, and another T cell growth factor, IL-7, activate both SAPK/JNK and p38MAP kinase. Furthermore, inhibition of p38MAP kinase activity with a specific pyrinidyl imidazole inhibitor SB203580 that prevents activation of its downstream effector, MAPK-activating protein kinase-2, correlated with suppression of IL-2- and IL-7-driven T cell proliferation. These data indicate that in T cells p38MAP kinase has a role in transducing the mitogenic signal.

Role for p38 mitogen-activated protein kinase in platelet aggregation caused by collagen or a thromboxane analogue.

p38 mitogen-activated protein kinase (MAPK) was identified in platelets on the basis of (a) its reactivity with antibodies to C-terminal and N-terminal peptides, and (b) its ability to activate MAPK-activated protein kinase-2, which phosphorylates the small heat shock protein, hsp27. p38 MAPK was activated in platelets by collagen fibers, a collagen-related cross-linked peptide, thrombin, or the thromboxane analogue U46619. A highly specific inhibitor of p38 MAPK, a pyridinyl imidazole known as SB203580, inhibited the platelet enzyme in vitro (IC50 approximately 0.5 microM). At similar concentrations it also inhibited agonist-stimulated phosphorylation of hsp27 in platelets, and platelet aggregation and secretion induced by minimal aggregatory concentrations of collagen or U46619, but not thrombin. Inhibition of aggregation was overcome by increasing agonist dose. SB203580 might act by inhibiting thromboxane generation, but this was only inhibited by 10-20% at low agonist concentrations. p38 MAPK provides a crucial signal, which is necessary for aggregation caused by minimal concentrations of collagen fibers or U46619. Thrombin or high doses of these agonists generate signals that bypass the enzyme, or render the enzyme no longer rate-limiting.

Interleukin-1 activates a novel protein kinase cascade that results in the phosphorylation of Hsp27.

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.

Interleukin 1 and tumour necrosis factor activate the mitogen-activated protein (MAP) kinase kinase in cultured cells.

Interleukin 1 (IL1) activated mitogen-activated protein (MAP) kinase kinase in human gingival and foreskin fibroblasts and KB cells. Maximal activity was found in cytosolic extracts made after stimulating cells for 15 min. On anion-exchange chromatography two differently charged forms of MAP kinase kinase were identified, both phosphorylated a kinase-defective mutant MAP kinase, and activated recombinant wild type MAP kinase to phosphorylate MBP. Both were inhibited by an antiserum to recombinant MAP kinase kinase and the less acidic form was identified on Western blotting as an antigen of approximately 43 kDa. Indistinguishable forms were very much more strongly induced by phorbol myristate acetate (PMA). TNF had a similar effect to that of IL1.

Interleukin 1 and tumor necrosis factor stimulate two novel protein kinases that phosphorylate the heat shock protein hsp27 and beta-casein.

We have partially purified and characterized two protein kinases that were strongly activated by interleukin-1 (IL-1) or tumor necrosis factor (TNF) in MRC-5 fibroblasts. The kinases were separated by anion exchange chromatography of cytosolic fractions. They phosphorylated in vitro the small heat shock protein (hsp27) or beta-casein and were stimulated 3- and 4.5-fold, respectively, in cells that had been exposed to IL-1 or TNF for 10 min. They were distinct from the mitogen-activated protein kinases, whose activation by IL-1 or TNF has been reported recently. The hsp27 kinase phosphorylated its substrate on serine residues. Its molecular mass was estimated to be 45-kDa by gel filtration. It is probably involved in the increase in hsp27 phosphorylation seen in intact cells. The beta-casein kinase behaved as a 65-kDa protein. It phosphorylated its substrate on serine and threonine residues and had little activity on alpha-casein. The hsp27 and beta-casein kinases were not activated after stimulation of the cells with phorbol myristate acetate (PMA). In contrast, the MAP kinases were activated to a similar extent (2-3-fold) by the cytokines and by PMA. The hsp27- and beta-casein kinases probably correspond to novel enzymes whose mechanisms of activation may be independent of protein kinase C or MAP kinases.

Interleukin 1 induces NF-kappa B through its type I but not its type II receptor in lymphocytes.

It is not known whether one or both of the interleukin 1 (IL1) receptors mediates the induction of the DNA-binding protein NF-kappa B. Nuclear extracts of the murine lines EL4.NOB.1 and 70Z/3, which bear the type I (80 kDa) and type II (67 kDa) IL1 receptor, respectively, were analyzed by an electrophoretic mobility shift assay. A 265-base pair sequence of the human serum amyloid A gene or a synthetic oligonucleotide each containing the NF-kappa B site were used as the DNA probes. IL1 induction of NF-kappa B was rapid (optimal at 15-30 min) and transient in both cell types. The IL1 receptor antagonist (IL1ra), which binds strongly to the type I receptor, inhibited the NF-kappa B response in both cell lines. IL1ra did not bind to the type II receptor on 70Z/3 cells as judged by competition for binding with 125I-IL1 alpha. When 125I-IL1ra binding to 70Z/3 cells was measured, a small number (10/cell) of high affinity sites (Kd = 5 x 10(-12) M) were detected. These were likely to have been type I receptor because an antibody to this inhibited the NF-kappa B induction in 70Z/3 cells (as well as EL4). Potential signal transduction mechanisms involving protein kinase C or oxygen radicals were studied. Phorbol 12-myristate 13-acetate induced NF-kappa B with a similar time course to IL1 in 70Z/3 but only after 4 h in EL4.IL1 was unaffected by a protein kinase C inhibitor (staurosporine). H2O2 did not mimic IL1, and IL1 was not inhibited by an antioxidant. The type I receptor mediates the induction of NF-kappa B in response to IL1 via a signaling mechanism that still remains to be identified.

The influence of interferon alpha and gamma, singly or in combination on human natural cell mediated cytotoxicity.

The influence of interferon alpha and gamma alone or in combination on the augmentation of human natural cytotoxicity was studied. Treatment of peripheral blood lymphocytes with IFN- alpha led to a rapid augmentation of NK activity, in contrast to IFN-gamma where target cell killing was observed only following 18 hrs exposure of lymphocytes to IFN-gamma. The results of the single cell assay paralleled those obtained using the Chromium release test, but neither interferon type caused an increase in the number of target binding lymphocytes. The combined effect of IFN-alpha and IFN-gamma in stimulating human natural cytotoxicity demonstrated individual lymphocyte responses to be variable. Exposure of lymphocytes to IFN-alpha and IFN-gamma for 18 hrs prior to assay for cytotoxicity usually decreased the level of cytotoxicity compared with control values, whereas other treatment regimes gave an additive and sometimes synergistic effect. Only treatment with IFN-alpha for 18 hrs and IFN-gamma for one hr produced a synergistic response in the majority of individuals tested. We conclude from this study that individual responses to IFN-alpha and IFN-gamma alone or in combination are variable and dependent upon timing of exposure of lymphocytes to individual interferon types, and possibly reflects the donor status at the time of sampling.