MAPKAP kinase 2 (MK2) is one of several kinases that are regulated through direct phosphorylation by p38 MAP kinase. By introducing a targeted mutation into the mouse MK2 gene, we have determined the physiological function of MK2 in vivo. Mice that lack MK2 show increased stress resistance and survive LPS-induced endotoxic shock. This is due to a reduction of approximately 90% in the production of tumor necrosis factor-alpha (TNF-alpha) and not to a change in signalling from the TNF receptor. The level and stability of TNF-alpha mRNA is not reduced and TNF-alpha secretion is not affected. We conclude that MK2 is an essential component in the inflammatory response which regulates biosynthesis of TNF-alpha at a post-transcriptional level.

We demonstrate that lipopolysaccharide-induced tumor necrosis factor (TNF) biosynthesis becomes independent of MAPKAP kinase 2 (MK2) when the AU-rich element (ARE) of the TNF gene is deleted. In spleen cells and macrophages where TNF biosynthesis is restored as a result of this deletion, interleukin (IL)-6 biosynthesis is still dependent on MK2. In MK2-deficient macrophages the half-life of IL-6 mRNA is reduced more than 10-fold, whereas the half-life of TNF mRNA is only weakly decreased. It is shown that the stability of a reporter mRNA carrying the AU-rich 3-untranslated region (3-UTR) of IL-6 is increased by MK2. The data provide in vivo evidence that the AU-rich 3-UTRs of TNF and IL-6 are downstream to MK2 signaling and make MK2 an essential component of mechanisms that regulate biosynthesis of IL-6 at the levels of mRNA stability, and of TNF mainly through TNF-ARE-dependent translational control.

STAT1 is an essential transcription factor for macrophage activation by IFN-gamma and requires phosphorylation of the C-terminal Ser727 for transcriptional activity. In macrophages, Ser727 phosphorylation in response to bacterial lipopolysaccharide (LPS), UV irradiation, or TNF-alpha occurred through a signaling path sensitive to the p38 mitogen-activated protein kinase (p38 MAPK) inhibitor SB203580 whereas IFN-gamma-mediated Ser727 phosphorylation was not inhibited by the drug. Consistently, SB203580 did not affect IFN-gamma-mediated, Stat1-dependent transcription but inhibited its enhancement by LPS. Furthermore, LPS, UV irradiation, and TNF-alpha caused activation of p38 MAPK whereas IFN-gamma did not. An essential role for p38 MAPK activity in STAT1 Ser727 phosphorylation was confirmed by using cells expressing an SB203580-resistant p38 MAPK. In such cells, STAT1 Ser727 phosphorylation in response to UV irradiation was found to be SB203580 insensitive. Targeted disruption of the mapkap-k2 gene, encoding a kinase downstream of p38 MAPK with a key role in LPS-stimulated TNF-alpha production and stress-induced heat shock protein 25 phosphorylation, was without a significant effect on UV-mediated Ser727 phosphorylation. The recombinant Stat1 C terminus was phosphorylated in vitro by p38MAPKalpha and beta but not by MAPK-activated protein kinase 2. Janus kinase 2 activity, previously reported to be required for IFN-gamma-mediated Ser727 phosphorylation, was not needed for LPS-mediated Ser727 phosphorylation, and activation of Janus kinase 2 did not cause the appearance of STAT1 Ser727 kinase activity. Our data suggest that STAT1 is phosphorylated at Ser727 by a stress-activated signaling pathway either through p38 MAPK directly or through an unidentified kinase downstream of p38MAPK.

Several growth factor-and calcium-regulated kinases such as pp90rsk or CaM kinase IV can phosphorylate the transcription factor serum response factor (SRF) at serine 103 (Ser-103). However, it is unknown whether stress-regulated kinases can also phosphorylate SRF. We show that treatment of cells with anisomycin, arsenite, sodium fluoride, or tetrafluoroaluminate induces phosphorylation of SRF at Ser-103 in both HeLa and NIH3T3 cells. This phosphorylation is dependent on the kinase p38/SAPK2 and correlates with the activation of MAPKAP kinase 2 (MK2). MK2 phosphorylates SRF in vitro at Ser-103 with similar efficiency as the small heat shock protein Hsp25 and significantly better than CREB. Comparison of wild type murine fibroblasts with those derived from MK2-deficient mice (Mk(؊/؊)) reveals MK2 as the major SRF kinase induced by arsenite. These results demonstrate that SRF is targeted by several signal transduction pathways within cells and establishes SRF as a nuclear target for MAPKAP kinase 2.

The p38/stress-activated protein kinase 2 (p38/SAPK2) is activated by cellular stress and proinflammatory cytokines. Several transcription factors have been reported to be regulated by p38/SAPK2, and this kinase is involved in the control of expression of various genes. In human Jurkat T-cells, induction of the early growth response gene-1 (egr-1) by anisomycin is completely inhibited by SB203580, a specific inhibitor of p38/SAPK2a and -b. Northern blot and reporter gene experiments indicate that this block is at the level of mRNA biosynthesis. Using mutants of the egr-1 promoter, we demonstrate that a distal cAMP-responsive element (CRE; nucleotides ؊134 to ؊126) is necessary to control egr-1 induction by p38/SAPK2. Pull-down assays indicate that phospho-CRE binding protein (CREB) and phospho-activating transcription factor-1 (ATF1) bind to this element in a p38/SAPK2-dependent manner. In response to anisomycin, two known CREB kinases downstream to p38/SAPK2, MAPKAP kinase 2 (MK2) and mitogen-and stress-activated kinase 1 (MSK1), show increased activity. However, in MK2 ؊/؊ fibroblasts derived from mice carrying a disruption of the MK2 gene, the phosphorylation of CREB and ATF1 and the expression of egr-1 reach levels comparable with wild type cells. This finding excludes MK2 as an involved enzyme. We conclude that egr-1 induction by anisomycin is mediated by p38/ SAPK2 and probably by MSK1. Phosphorylated CREB and ATF1 then bind to the CRE of the egr-1 promoter and cause a stress-dependent transcriptional activation of this gene. Signal transduction via mitogen-activated protein (MAP)1 kinases plays a key role in a variety of cellular responses, including growth factor-induced proliferation, differentiation, and cell death (1-5). Several parallel MAP kinase signal transduction pathways have been defined in mammalian cells. These pathways include the extracellular signal regulated kinases (ERK), c-Jun N-terminal kinases (JNK, also known as SAPK1), and p38 MAP kinases (SAPK2).p38/SAPK2 is activated by bacterial lipopolysaccharide (6), physico-chemical changes in the extracellular milieu (heat, hyper-and hypo-osmolarity, UV irradiation, sodium arsenite, and anisomycin) (7-9), and proinflammatory cytokines (e.g. IL-1␤ and tumor necrosis factor-␣) (5,10,11).The activation of several transcription factors is regulated by the p38/SAPK2 pathway, and hence this pathway is involved in the control of expression of various genes including interferon-␥ (12), tumor necrosis factor-␣, IL-1, 14), IL-6 (10), inducible nitric-oxide synthase (15), and as shown more recently, E-selectin (16) and vascular cell adhesion molecule-1 (17). In vitro studies demonstrate that the transcription factor ATF2 is phosphorylated and activated by p38/SAPK2 (18,19). In addition, p38/SAPK2 activates the Elk-1 (19), CHOP (20), MEF2C (21), and SAP-1 (22) transcription factors. In addition to these factors, p38/SAPK2 also phosphorylates and thereby activates numerous downstream kinases (i.e. MNK1/2 (23, 24), MAPKAP kinase 2 (MK2) (25,26), PRAK (27),...

In the present review we consider regulation of the build‐up of the nitrate reducing apparatus (NO−3 → NH+4) in the course of light‐mediated plastidogenesis. In particular, the regulation of the appearance of nitrate reductase (NR, EC 1.6.6.1) and nitrite reductase (NIR, EC 1.7.7.1) will be treated. While both enzymes are nuclear‐encoded, NR is a cytosolic and NIR a plastidic protein. Substrate induction by nitrate, control by light (phytochrome) and dependence on a ‘plastidic factor’ are the main features of the control system. In mustard (Sinapis alba L.) seedling cotyledons the NR‐mRNA level was determined by a strong synergistic coaction of nitrate and light. Synthesis of NR (a cytosolic protein with two isoforms) correlated with the level of NR mRNA. A positive control of NR gene expression by the plastidic factor was observed. It appeared that the plant cell regulates NR gene expression as if NR would be a plastidic protein. Control of gene expression in the case of plastidic NIR in the same mustard cotyledons was conspicuously different. Whereas, in case of NR, the transcript level was determined by a synergism between light and nitrate, in the case of NIR the transcript level was controlled by light alone, with nitrate being ineffective. However, in both cases actual enzyme synthesis was controlled by nitrate, and the dependence on positive control by the plastidic factor was the same. Thus, the difference between NR and NIR gene expression appears to be a matter of transcriptional control. From a teleonomic viewpoint, the control system to establish the nitrate‐reducing apparatus appears (almost) perfect. In spinach (Spinacia oleracea L.) seedling cotyledons the NIR transcript level was unaffected by light but determined by nitrate. The strong action of light on NIR synthesis was multiplicatively superimposed on the action of nitrate, consistent with the conclusion that nitrate affects the transcript level while light controls enzyme synthesis. In tobacco (Nicotiana tabacum L., cv. Coker 176) seedlings the NIR transcript level was determined by a synergistic action between nitrate and light, i.e. both factors coact at the level of transcription. There was no effect of light on NIR synthesis in the absence of nitrate while a strong action of light on protein synthesis was observed in the presence of nitrate. The specific effect of blue light on enzyme appearance, i.e. an effect which cannot be attributed to phytochrome, was not seen at the transcript level. Thus, in tobacco a coaction of nitrate and light (phytochrome) is required to bring about a high NIR transcript level, while in mustard the NIR transcript level was determined by phytochrome alone and in spinach by nitrate alone. It is obvious that in different plants phytochrome and nitrate control NIR gene expression differently. Only the dependence on the plastidic factor appears to be the same in all cases studied so far. The conspicuous regulatory differences between spinach and tobacco offer a chance to address the crucial question of wh...

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UV irradiation of cells causes ligand-independent acChanges in gene expression are induced by extracellular stimuli most of which are perceived by cell surface receptors. One major class of surface receptors, receptor tyrosine kinases, are primed to react to specific growth factors. Upon ligand binding, the receptor subunits mutually phosphorylate each other. The phosphorylated tyrosine peptide motifs then become docking sites for numerous proteins of the signal transduction network. Most data are compatible with the idea that the ligand triggers this activity of receptor tyrosine kinase by stabilizing the association of subunits (1, 2).In addition to physiologic stimuli, many adverse agents such as carcinogens, metal toxins, oxidants, and radiation can induce signal transduction cascades and changes in gene expression. Ultraviolet (UV) 1 radiation of various wavelength has received most attention. The response to UV appears to depend on several primary radiation target molecules (3, 4). Interestingly, part of the immediate response to UV is mediated by the ligand-independent activation of growth factor receptors (5-7). For instance, the epidermal growth factor receptor (EGFR) and the platelet-derived growth factor receptor (PDGFR) are autophosphorylated within seconds of UV irradiation of cells in culture. The autophosphorylation appears to lead to fully functional receptor signaling, best shown for the EGFR (5, 8 -10) and the insulin receptor (6). Several components of signal transduction, Grb-2, phospholipase C-␥, and Shc that contain SH2 domains or phosphotyrosine-binding domains associate with the receptors in UV-irradiated cells. In consequence, Ras and the Ras-dependent signal transduction pathway to Erk and several other signaling pathways (11) are activated. From the successful activation by UV of receptor heterodimers the conclusion has been derived that a substantial fraction of the receptor tyrosine kinases are in dimer configuration in the absence of ligand and prior to the UV stimulus (Ref. 12, and references therein).How can an unspecific agent such as UV lead to a gain of function: up-regulation of enzymatic activity of the receptors? One suggestion has been, that UV causes cross-links of associated receptor monomers. The dose of UV that would be needed to substantially cross-link proteins, is, however, at least 1 order of magnitude higher than that efficiently activating the receptors. Clustering has, however, been observed for the tumor necrosis factor receptor after UV irradiation (7). It is not clear whether the clustering represents the primary event or whether it is the result rather than the cause of activation. An alternative suggestion has been derived from comparative studies of the kinetics of receptor dephosphorylation with or without UV irradiation (9). UV strongly retards dephosphorylation of both, EGFR and PDGFR. In vitro, irradiation of a plasma membrane vesicle preparation containing phosphatase inactivated the enzymatic activity, compatible with the idea that specific receptor-...