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Recent evidence for cross-talk between protein kinase B (PKB) and the Raf-1 and NF-kappaB signalling pathways has provided new hints to the complex roles that PKB may play in regulating gene transcription and also raised questions about where and when these targets are relevant.

The importance of apoptosis as a natural means to eliminate unwanted or damaged cells has been realized over the past decade. Many components required to exercise programmed cell death have been identified and shown to pre-exist in most, if not all, cells. Such ubiquity requires that apoptosis be tightly controlled and suggests the propensity of cells to trigger the cellular death machinery can be regulated. Recently, several signaling pathways have been demonstrated to impact the apoptotic potential of cells, most notably the phosphatidylinositol 3' kinase (PI3'K) pathway. The 3' phosphorylated lipid products generated by this enzyme promote activation of a protein-serine kinase, PKB/AKT, which is necessary and sufficient to confer cell PI3'K-dependent survival signals. The relevance of this pathway to human cancer was revealed by the recent finding that the product of the PTEN tumor suppressor gene acts to antagonize PI3'K. This review focuses on the regulation and mechanisms by which PKB activation protects cells and the oncologic consequences of dysregulation of the pathway.

Tumor necrosis factor-alpha (TNF), a major inflammatory cytokine, generates a wide variety of cellular responses via key cytoplasmic adaptor molecules named TNF receptor-associated factors (TRAFs). We report that TRAF2, TRAF5 and TRAF6 associate with apoptosis signal-regulating kinase 1 (ASK1), and a catalytically-inactive ASK1 mutant blocks stress-activated protein kinase (SAPK)/Jun NH2-terminal kinase (JNK) activation by these TRAFs. A truncated derivative of TRAF2, which inhibits SAPK activation by TNF, blocks TNF-induced ASK1 activation. Furthermore, protection from TNF-induced cell death conferred by an ASK1 mutant is dependent upon TRAF2. Hence, ASK1 is a common mediator of TRAF-regulated SAPK and apoptosis signaling, and the TRAF2 - ASK1 connection completes the signaling cascade from TNF to SAPK/JNK activation.

4-1BB is a member of the TNFR superfamily expressed on activated CD4+ and CD8+ T cells. 4-1BB can costimulate IL-2 production by resting primary T cells independently of CD28 ligation. In this study, we report signaling events following 4-1BB receptor aggregation using an Ak-restricted costimulation-dependent T cell hybridoma, C8.A3. Aggregation of 4-1BB on the surface of C8.A3 cells induces TNFR-associated factor 2 recruitment, which in turn recruits and activates apoptosis signal-regulating kinase-1, leading to downstream activation of c-Jun N-terminal/stress-activated protein kinases (JNK/SAPK). 4-1BB ligation also enhances anti-CD3-induced JNK/SAPK activation in primary T cells. Overexpression of a catalytically inactive form of apoptosis signal-regulating kinase-1 in C8.A3 T cells interferes with activation of the SAPK cascade and with IL-2 secretion, consistent with a critical role for JNK/SAPK activation in 4-1BB-dependent IL-2 production. Given the ability of both CD28 and 4-1BB to induce JNK/SAPK activation, we asked whether hyperosmotic shock, another inducer of this cascade, could function to provide a costimulatory signal to T cells. Osmotic shock of resting primary T cells in conjunction with anti-CD3 treatment was found to costimulate IL-2 production by the T cells, consistent with a pivotal role for JNK/SAPK in T cell costimulation.

Part of the cellular response to toxins, physical stresses and inflammatory cytokines occurs by signalling via the stress-activated protein kinase (SAPK) and p38 reactivating kinase pathways. This results in modification of cellular gene expression. These stress-responsive kinase pathways are structurally similar, but functionally distinct, from the archetypal mitogen-activated protein kinases (MAPKs or ERKs). The ERK pathway is a hierarchical cascade originating at the cell membrane with receptors for mitogens or growth factors, which recruit, via adapter proteins and exchange factors, the small guanosine triphosphatase (GTPase) Ras (see fig. 1). Ras activates raf, a serine threonine kinase, which activates MEK (MAPK/ERK kinase). MEK, in turn, phosphorylates and activates ERK1 and ERK2, which translocate to the nucleus and transactivate transcription factors, changing gene expression to promote growth, differentiation or mitosis. By transducing signals through a cascade of kinases, several options for control are introduced for amplifying and/or modifying the output signal. The SAPK and p38 pathways are also hierarchically arranged, but less is known about the upstream components and the downstream effects of stimulation of these pathways. Among the processes modulated by stress-responsive pathways are apoptosis, transformation, development, immune activation, inflammation and adaptation to environmental changes. This review outlines the upstream componentry of these pathways that interact with a variety of agonists to modify the activity of SAPK and p38, and explores the downstream functions of this activation.

The protein-serine kinase Shaggy(Zeste-white3) (Sgg(Zw3)) is the Drosophila homolog of mammalian glycogen synthase kinase-3 and has been genetically implicated in signal transduction pathways necessary for the establishment of patterning. Sgg(Zw3) is a putative component of the Wingless (Wg) pathway, and epistasis analyses suggest that Sgg(Zw3) function is repressed by Wg signaling. Here, we have investigated the biochemical consequences of Wg signaling with respect to the Sgg(Zw3) protein kinase in two types of Drosophila cell lines and in embryos. Our results demonstrate that Sgg(Zw3) activity is inhibited following exposure of cells to Wg protein and by expression of downstream components of Wg signaling, Drosophila frizzled 2 and dishevelled. Wg-dependent inactivation of Sgg(Zw3) is accompanied by serine phosphorylation. We also show that the level of Sgg(Zw3) activity regulates the stability of Armadillo protein and modulates the level of phosphorylation of D-Axin and Armadillo. Together, these results provide direct biochemical evidence in support of the genetic model of Wg signaling and provide a model for dissecting the molecular interactions between the signaling proteins.

Transient expression of I2PP2A, a potent inhibitor of protein phosphatase 2A (PP2A), in HEK-293 cells increased the concentration and DNA binding of the proto-oncogene c-Jun. In contrast, expression of the catalytic subunit of PP2A (PP2AC) markedly decreased the concentration and DNA binding of c-Jun. Expression of I2PP2A also increased the transcriptional activity of activator protein-1, and this effect was diminished in a dose-dependent manner by expression of PP2AC. Densitometric analysis following Western blotting of extracts with antibodies specific for phospho-Ser63 and Ser73 suggests that the effects of I2PP2A and PP2AC expression might be mediated, in part, by changes in the phosphorylation of c-Jun at Ser63. The results indicate that I2PP2A elicits effects that are consistent with it acting as an inhibitor of PP2A in intact cells, and suggest that PP2A might exhibit site selectivity with respect to c-Jun phosphorylation.

Bcl-2 is a key inhibitor of a broad range of apoptotic pathways, yet neither the mechanism of action nor the role of Bcl-2 subcellular localization are well understood. The subcellular localization of Bcl-2 includes the mitochondrial membrane as well as the contiguous membrane of the endoplasmic reticulum and nuclear envelope. Most studies suggest that the ability of Bcl-2 to confer cell survival is dependent upon its localization to the mitochondria. In this manuscript, we show that Bcl-2 targeted to the endoplasmic reticulum can inhibit Myc-, but not etoposide-induced apoptosis in the Rat-1 fibroblast cell line. By contrast, wild type Bcl-2 can inhibit apoptosis triggered by either death agonist. We further show both Myc and etoposide trigger disruption of mitochondrial membrane potential (MMP) and induce poly-ADP ribose polymerase (PARP) cleavage, but release of calcium was not evident. Bcl-2 abrogates apoptosis at or upstream of MMP depletion showing that Bcl-2 does not have to reside at the mitochondria to prevent apoptosis. These results further elucidate the biochemical events associated with Myc- and etoposide-induced apoptosis and significantly advance our understanding of Bcl-2 function.

L6 myoblasts stably transfected with a GLUT4 cDNA harboring an exofacial myc epitope tag (L6-GLUT4myc myoblasts) were used to study the role of protein kinase B alpha (PKBalpha)/Akt1 in the insulin-induced translocation of GLUT4 to the cell surface. Surface GLUT4myc was detected by immunofluorescent labeling of the myc epitope in nonpermeabilized cells. Insulin induced a marked translocation of GLUT4myc to the plasma membrane within 20 min. This was prevented by transient transfection of a dominant inhibitory construct of phosphatidylinositol (PI) 3-kinase (Deltap85alpha). Transiently transfected cells were identified by cotransfection of green fluorescent protein. A constitutively active PKBalpha, created by fusion of a viral Gag protein at its N terminus (GagPKB), increased the cell surface density of GLUT4myc compared to that of neighboring nontransfected cells. A kinase-inactive, phosphorylation-deficient PKBalpha/Akt1 construct with the mutations K179A (substitution of alanine for the lysine at position 179), T308A, and S473A (AAA-PKB) behaved as a dominant-negative inhibitor of insulin-dependent activation of cotransfected wild-type hemagglutinin (HA)-tagged PKB. Furthermore, AAA-PKB markedly inhibited the insulin-induced phosphorylation of cotransfected BAD, demonstrating inhibition of the endogenous PKB/Akt. Under the same conditions, AAA-PKB almost entirely blocked the insulin-dependent increase in surface GLUT4myc. PKBalpha with alanine substitutions T308A and S473A (AA-PKB) or K179A (A-PKB) alone was a less potent inhibitor of insulin-dependent activation of wild-type HA-PKB or GLUT4myc translocation than was AAA-PKB. Cotransfection of AAA-PKB with a fourfold DNA excess of HA-PKB rescued insulin-stimulated GLUT4myc translocation. AAA-PKB did not prevent actin bundling (membrane ruffling), though this response was PI 3-kinase dependent. Therefore, it is unlikely that AAA-PKB acted by inhibiting PI 3-kinase signaling. These results outline an important role for PKBalpha/Akt1 in the stimulation of glucose transport by insulin in muscle cells in culture.

The stress signaling kinase SEK1/MKK4 is a direct activator of stress-activated protein kinases (SAPKs; also called Jun-N-terminal kinases, JNKs) in response to a variety of cellular stresses, such as changes in osmolarity, metabolic poisons, DNA damage, heat shock or inflammatory cytokines. We have disrupted the sek1 gene in mice using homologous recombination. Sek1(-/- )embryos display severe anemia and die between embryonic day 10.5 (E10.5) and E12.5. Haematopoiesis from yolk sac precursors and vasculogenesis are normal in sek1(-/- )embryos. However, hepatogenesis and liver formation were severely impaired in the mutant embryos and E11.5 and E12.5 sek1(-/- )embryos had greatly reduced numbers of parenchymal hepatocytes. Whereas formation of the primordial liver from the visceral endoderm appeared normal, sek1(-/-) liver cells underwent massive apoptosis. These results provide the first genetic link between stress-responsive kinases and organogenesis in mammals and indicate that SEK1 provides a crucial and specific survival signal for hepatocytes.

Glycogen synthase kinase-3 (GSK-3) is required during metazoan development to mediate the effects of the extracellular signal wingless/Wnt-1 and hence is necessary for correct cell type specification. GSK-3 also regulates cell fate during Dictyostelium development, but in this case it appears to mediate the effects of extracellular cAMP. By direct measurement of GSK-3 kinase activity during Dictyostelium development, we find that there is a rise in activity at the initiation of multicellular development which can be induced by cAMP. The timing of the rise correlates with the requirement for the Dictyostelium homologue of GSK-3, GSKA, to specify cell fate. We show that loss of the cAMP receptor cAR3 almost completely abolishes the rise in kinase activity and causes a mis-specification of cell fate that is equivalent to that seen in a gskA- mutant. The phenotype of a cAR3(-) mutant however is less severe than loss of gskA and ultimately gives rise to an apparently wild-type fruiting body. These results indicate that in Dictyostelium extracellular cAMP acts via cAR3 to cause a rise in GSKA kinase activity which regulates cell type patterning during the initial stages of multicellularity.

While a plethora of extracellular molecules exist that modulate cellular functions via binding to membrane receptors inside the cell, their actions are mediated by relatively few signalling mechanisms. One of these is activation of phosphatidylinositol 3-kinase (PI-3K), which results in the generation of a membrane-restricted second messenger, polyphosphatidylinositides containing a 3'-phosphate. How these molecules transduced the effects of agonists of PI-3K was unclear until the recent discovery that several protein kinases become activated upon exposure to 3'-phosphorylated inositol lipids. These enzymes include protein kinase B (PKB)/AKT and PtdIns(3,4, 5)P3-dependent kinases 1 and 2, the first two of which interact with 3'-phosphorylated phosphoinositides via pleckstrin homology domains. Once targeted to the membrane by this motif, PKB becomes phosphorylated at two residues, which relieves intermolecular inhibition, allowing the activated complex to dissociate and modify its targets. Identification of these substrates is the subject of intensive research, since at least one must play a key role in suppressing apoptosis, as demonstrated by expression of activated alleles of PKB. The generation of effective transdominant mutants, coupled with genetic analysis of the protein kinase in simpler organisms, should help in elucidating outstanding questions in the functions, targets and regulation of this important mediator of PI-3K signalling.

The decision between survival and death is an important aspect of cellular regulation during development and malignancy. Central to this regulation is the process of apoptosis, which is conserved in multicellular organisms [1]. A variety of signalling cascades have been implicated in modulation of apoptosis, including the phosphatidylinositol (Pl) 3-kinase pathway. Activation of Pl 3-kinase is protective, and inhibition of this lipid kinase enhances cell death under several conditions including deregulated expression of c-Myc, neurotrophin withdrawal and anoikis [2-7]. Recently, the protective effects of Pl 3-kinase have been linked to its activation of the pleckstrin homology (PH)-domain-containing protein kinase B (PKB or AKT) [8]. PKB/AKT was identified from an oncogene, v-akt, found in a rodent T-cell lymphoma [9]. To initiate a genetic analysis of PKB, we have isolated and characterized a Drosophila PKB/AKT mutant (termed Dakt1) that exhibits ectopic apoptosis during embryogenesis as judged by induction of membrane blebbing, DNA fragmentation and macrophage infiltration. Apoptosis caused by loss of Dakt function is rescued by caspase suppression but is distinct from the previously described reaper/grim/hid functions. These data implicate Dakt1 as a cell survival gene in Drosophila, consistent with cell protection studies in mammals.

Stimulation of c-Jun transcriptional activity via phosphorylation mediated by the stress-activated or c-Jun amino-terminal (SAPK/JNK) subgroup of mitogen-activated protein kinases (MAP kinases) is thought to depend on a kinase-docking site (the delta region) within the amino-terminal activation domain, which is deleted from the oncogenic derivative, v-Jun [1] [2] [3]. This mutation markedly enhances v-Jun oncogenicity [4] [5]; however, its transcriptional consequences have not been resolved. In part, this reflects uncertainty as to whether binding of SAPK/JNK inhibits c-Jun function directly [6] [7] or, alternatively, serves to facilitate and maintain the specificity of positive regulatory phosphorylation [8]. Using a two-hybrid approach, we show that SAPK/JNK stimulates c-Jun transactivation in yeast and that this depends on both catalytic activity and physical interaction between the kinase and its substrate. Furthermore, c-Jun is active when tethered to DNA via SAPK/JNK, demonstrating that kinase binding does not preclude transactivation. Taken together, these results suggest that SAPK/JNK acts primarily as a positive regulator of c-Jun transactivation in situ, and that loss of the docking site physically uncouples v-Jun from this control. This loss-of-function model accounts for the deficit of v-Jun regulatory phosphorylation and repression of TPA response element (TRE)-dependent transcription observed in v-Jun-transformed cells and predicts that an important property of the oncoprotein is to antagonise SAPK/JNK-dependent gene expression.

Background: The molecular mechanisms underlying both beneficial and undesirable opioid actions are poorly understood. Recently, the three currently known mammalian mitogen-activated protein kinase (MAPK) signaling cascades (extracellular signal-related kinase [ERK], stress-activated protein kinase, and p38 kinase) were shown to play important roles in transducing receptor-mediated signaling processes., Methods: To determine whether any of these kinase cascades were activated by opioids, mu, delta, or kappa opioid receptors were transiently introduced into COS-7 cells together with MAPKs tagged to allow recognition by specific antibodies, and then exposed to opioids. Mitogen-activated protein kinase activation was determined by an in vitro MAPK activation assay. In addition, C6 glioma cells with either mu, delta, or kappa receptors stably introduced were exposed to opioids and MAPK activation determined by in vitro activation assay or antibody detection of activated forms., Results: Transient experiments in COS cells revealed potent stimulation of ERK by mu and delta receptor activation, weak stimulation of stress-activated protein kinase by all receptor types, and no activation of p38. In stably transfected C6 glioma cells, only ERK activation was observed. Extracellular signal-related kinase induction was rapid, peaking 5 min after stimulation, and its activation was receptor-type specific. Mu and delta receptor stimulation activated ERK, but kappa stimulation did not., Conclusions: These results show that acute opioid signaling is not only inhibitory, but can strongly activate an important signaling cascade. Extracellular signal-related kinase activation may contribute to desirable responses to opioids, such as analgesia and sedation, and also to undesirable adaptive responses, such as tolerance, physical dependence, and possibly addiction. Further study of this system could provide greater insight into the molecular mechanisms underlying these clinical problems.

The dual specific kinase SAPK/ERK1 kinase (SEK1; mitogen-activated protein kinase kinase 4/Jun NH2 terminal kinase [ JNK] kinase) is a direct activator of stress-activated protein kinases ([SAPKs]/JNKs) in response to CD28 costimulation, CD40 signaling, or activation of the germinal center kinase. Here we show that SEK1(-/-) recombination-activating gene (RAG)2(-/-) chimeric mice have a partial block in B cell maturation. However, peripheral B cells displayed normal responses to IL-4, IgM, and CD40 cross-linking. SEK1(-/-) peripheral T cells showed decreased proliferation and IL-2 production after CD28 costimulation and PMA/Ca2+ ionophore activation. Although CD28 expression was absolutely crucial to generate vesicular stomatitis virus (VSV)-specific germinal centers, SEK1(-/-)RAG2(-/-) chimeras mounted a protective antiviral B cell response, exhibited normal IgG class switching, and made germinal centers in response to VSV. Interestingly, PMA/Ca2+ ionophore stimulation, which mimics TCR-CD3 and CD28-mediated signal transduction, induced SAPK/JNK activation in peripheral T cells, but not in thymocytes, from SEK1(-/-) mice. These results show that signaling pathways for SAPK activation are developmentally regulated in T cells. Although SEK1(-/-) thymocytes failed to induce SAPK/JNK in response to PMA/Ca2+ ionophore, SEK1(-/-)RAG2(-/-) thymocytes proliferated and made IL-2 after PMA/Ca2+ ionophore and CD3/CD28 stimulation, albeit at significantly lower levels compared to SEK1(+/+)RAG2(-/-) thymocytes, implying that CD28 costimulation and PMA/Ca2+ ionophore-triggered signaling pathways exist that can mediate proliferation and IL-2 production independently of SAPK activation. Our data provide the first genetic evidence that SEK1 is an important effector molecule that relays CD28 signaling to IL-2 production and T cell proliferation.

Activation of glycogen synthesis in skeletal muscle in response to insulin results from the combined inactivation of glycogen synthase kinase-3 (GSK-3) and activation of the protein phosphatase-1, changing the ratio between the inactive phosphorylated state of the glycogen synthase to the active dephosphorylated state. In a search for genetic defects responsible for the decreased insulin stimulated glycogen synthesis seen in patients with non-insulin-dependent diabetes mellitus (NIDDM) and their glucose-tolerant first-degree relatives we have performed mutational analysis of the coding region of the 2 isoforms of GSK-3alpha and GSK-3beta in 72 NIDDM patients and 12 control subjects. No structural changes were detected apart from a few silent mutations. Mapping of the GSK-3alpha to chromosome 19q13.1-13.2 and the GSK-3beta to chromosome 3q13.3-q21 outside known genetic loci linked to NIDDM further makes it unlikely that these genes are involved in the pathogenesis of common forms of NIDDM.

Stress-activated protein kinases (SAPK) are stimulated by a variety of agents and conditions that also activate the Na+/H+ exchanger (NHE). Activation of the exchanger results in a rapid increase in intracellular pH (pHi), raising the possibility that cytosolic alkalinization may contribute to SAPK activation. This hypothesis was tested by manipulating the pHi of U937 cells using permeant weak bases. Three different bases increased pHi and caused a 4-12-fold increase in SAPK activity with a time course that paralleled intracellular alkalinization. p38, a related stress kinase, was also stimulated by the weak bases. Stimulation of the stress kinases was not accompanied by changes in cytosolic free calcium nor was the activation of SAPK achieved when calcium was elevated by thapsigargin or calcium ionophores. Weak bases not only alter the pH of the cytosol but also alkalinize endomembrane compartments such as endosomes and lysosomes. However, the latter do not appear to mediate the stimulation of SAPK, since neither bafilomycin A1 nor desipramine, agents that neutralize acidic endomembrane compartments, activated the kinase. Because hyperosmolarity acutely activates the NHE, we considered whether the resulting cytosolic alkalinization mediates the activation of SAPK upon cell shrinkage. The addition of amiloride or the omission of Na+, which were verified to inhibit NHE, did not prevent the osmotically induced activation of SAPK. We conclude that cytosolic alkalinization increases the activity of SAPK and p38 by a calcium-independent mechanism that does not involve acidic intracellular organelles. In addition, even though cell shrinkage is accompanied by alkalinization due to the activation of NHE, the increased pHi is not the main cause of the observed stimulation of SAPK upon hyperosmotic challenge.

Distinct and evolutionarily conserved signal transduction cascades mediate survival or death in response to developmental and environmental cues. The stress-activated protein kinases, or Jun N-terminal kinases (SAPKs/JNKs), are activated in response to a variety of cellular stresses such as changes in osmolarity and metabolism, DNA damage, heat shock, ischaemia, or inflammatory cytokines. Sek1 (JNKK/MKK4) is a direct activator of SAPKs/JNKs in response to environmental stresses or mitogenic factors. Here we investigate the role of Sek1 in development and apoptosis by deleting sek1 in embryonic stem (ES) cells by homologous recombination. We provide genetic evidence that different stresses utilize distinct signalling pathways for SAPK/JNK activation. sek1(-/-) rag2(-/-) chimaeric mice have normal numbers of mature T cells but fewer immature CD4+CD8+ thymocytes. The sek1 mutation did not affect the induction of apoptosis in response to environmental stresses in ES and T cells: instead, sek1 protected thymocytes from CD95 (Fas)- and CD3-mediated apoptosis. These data indicate that SEK1 mediates survival signals in T-cell development.

Interaction of the p55 tumor necrosis factor receptor 1 (TNF-R1)-associated signal transducer TRADD with FADD signals apoptosis, whereas the TNF receptor-associated factor 2 protein (TRAF2) is required for activation of the nuclear transcription factor nuclear factor kappa B. TNF-induced activation of the stress-activated protein kinase (SAPK) was shown to occur through a noncytotoxic TRAF2-dependent pathway. TRAF2 was both sufficient and necessary for activation of SAPK by TNF-R1; conversely, expression of a dominant-negative FADD mutant, which blocks apoptosis, did not interfere with SAPK activation. Therefore, SAPK activation occurs through a pathway that is not required for TNF-R1-induced apoptosis.

Mixed lineage kinase-3 (MLK-3) is a 97 kDa serine/threonine kinase with multiple interaction domains, including a Cdc42 binding motif, but unknown function. Cdc42 and the related small GTP binding protein Rac1 can activate the SAPK/JNK and p38/RK stress-responsive kinase cascades, suggesting that MLK-3 may have a role in upstream regulation of these pathways. In support of this role, we demonstrate that MLK-3 can specifically activate the SAPK/JNK and p38/RK pathways, but has no effect on the activation of ERKs. Immunoprecipitated MLK-3 catalyzed the phosphorylation of SEK1 in vitro, and co-transfected MLK-3 induced phosphorylation of SEK1 and MKK3 at sites required for activation, suggesting direct regulation of these protein kinases. Furthermore, interactions between MLK-3 and SEK and MLK-3 and MKK6 were observed in co-precipitation experiments. Finally, kinase-dead mutants of MLK-3 blocked activation of the SAPK pathway by a newly identified mammalian analog of Ste20, germinal center kinase, but not by MEKK, suggesting that MLK-3 functions to activate the SAPK/JNK and p38/RK cascades in response to stimuli transduced by Ste20-like kinases.

In mammalian cells, a specific stress-activated protein kinase (SAPK/JNK) pathway is activated in response to inflammatory cytokines, injury from heat, chemotherapeutic drugs and UV or ionizing radiation. The mechanisms that link these stimuli to activation of the SAPK/JNK pathway in different tissues remain to be identified. We have developed and applied a PCR-based subtraction strategy to identify novel genes that are differentially expressed at specific developmental points in hematopoiesis. We show that one such gene, hematopoietic progenitor kinase 1 (hpk1), encodes a serine/threonine kinase sharing similarity with the kinase domain of Ste20. HPK1 specifically activates the SAPK/JNK pathway after transfection into COS1 cells, but does not stimulate the p38/RK or mitogen-activated ERK signaling pathways. Activation of SAPK requires a functional HPK1 kinase domain and HPK1 signals via the SH3-containing mixed lineage kinase MLK-3 and the known SAPK activator SEK1. HPK1 therefore provides an example of a cell type-specific input into the SAPK/JNK pathway. The developmental specificity of its expression suggests a potential role in hematopoietic lineage decisions and growth regulation.

Background: Exposing eukaryotic cells to lithium ions (Li+) during development has marked effects on cell fate and organization. The phenotypic consequences of Li+ treatment on Xenopus embryos and sporulating Dictyostelium are similar to the effects of inhibition or disruption, respectively, of a highly conserved protein serine/threonine kinase, glycogen synthase kinase-3 (GSK-3). In Drosophila, the GSK-3 homologue is encoded by zw3sgg, a segment-polarity gene involved in embryogenesis that acts downstream of wg. In higher eukaryotes, GSK-3 has been implicated in signal transduction pathways downstream of phosphoinositide 3-kinase and mitogen-activated protein kinases., Results: We investigated the effect of Li+ on the activity of the GSK-3 family. At physiological doses, Li+ inhibits the activity of human GSK-3 beta and Drosophila Zw3Sgg, but has no effect on other protein kinases. The effect of Li+ on GSK-3 is reversible in vitro. Treatment of cells with Li+ inhibits GSK-3-dependent phosphorylation of the microtubule-associated protein Tau. Li+ treatment of Drosophila S2 cells and rat PC12 cells induces accumulation of cytoplasmic Armadillo/beta-catenin, demonstrating that Li+ can mimic Wingless signalling in intact cells, consistent with its inhibition of GSK-3., Conclusions: Li+ acts as a specific inhibitor of the GSK-3 family of protein kinases in vitro and in intact cells, and mimics Wingless signalling. This reveals a possible molecular mechanism of Li+ action on development and differentiation.

Mammalian cells contain at least three signaling systems which are structurally related to the mitogen-activated protein kinase (MAPK) pathway. Growth factors acting through Ras primarily stimulate the Raf/MEK/MAPK cascade of protein kinases. In contrast, many stress-related signals such as heat shock, inflammatory cytokines, and hyperosmolarity induce the MEKK/SEK(MKK4)/SAPK(JNK) and/or the MKK3 or MKK6/p38(hog) pathways. Physiological agonists of these pathway types are either qualitatively or quantitatively distinct, suggesting few common proximal signaling elements, although past studies performed in vitro, or in cells using transient over-expression, reveal interaction between the components of all three pathways. These studies suggest a high degree of cross-talk apparently not seen in vivo. We have examined the possible molecular basis of the differing agonist profiles of these three MAPK pathways. We report preferential association between MAP kinases and their activators in eukaryotic cells. Furthermore, using the yeast 2-hybrid system, we show that association between these components can occur independent of additional eukaryotic proteins. We show that SAPK(JNK) or p38(hog) activation is specifically impaired by co-expression of cognate dominant negative MAP kinase kinase mutants, demonstrating functional specificity at this level. Further divergence and insulation of the stress pathways occurs proximal to the MAPK kinases since activation of the MAPK kinase kinase MEKK results in SAPK(JNK) activation but does not cause p38(hog) phosphorylation. Therefore, in intact cells, the three MAPK pathways may be independently regulated and their components show specificity in their interaction with cognate cascade members. The degree of intermolecular specificity suggests that mammalian MAPK signaling pathways may remain distinct without the need for specific scaffolding proteins to sequester components of individual pathways.

The Drosophila gene product Wingless (Wg) is a secreted glycoprotein and a member of the Wnt gene family. Genetic analysis of Drosophila epidermal development has defined a putative paracrine Wg signalling pathway involving the zeste-white 3/shaggy (zw3/sgg) gene product. Although putative components of Wg- (and by inference Wnt-) mediated signalling pathways have been identified by genetic analysis, the biochemical significance of most factors remains unproven. Here we show that in mouse 10T1/2 fibroblasts the activity of glycogen synthase kinase-3 (GSK-3), the murine homologue of Zw3/Sgg, is inactivated by Wg. This occurs through a signalling pathway that is distinct from insulin-mediated regulation of GSK-3 in that Wg signalling to GSK-3 is insensitive to wortmannin. Additionally, Wg-induced inactivation of GSK-3 is sensitive to both the protein kinase C (PKC) inhibitor Ro31-8220 and prolonged pre-treatment of 10T1/2 fibroblasts with phorbol ester. These findings provide the first biochemical evidence in support of the genetically defined pathway from Wg to Zw3/Sgg, and suggest a previously uncharacterized role for a PKC upstream of GSK-3/Zw3 during Wnt/Wg signal transduction.

Background: Stimuli that stress cells, including inflammatory cytokines, ultra-violet irradiation, DNA-damaging chemotherapeutic drugs and heat shock, stimulate a recently identified cytoplasmic signaling system that is structurally related to the mitogen-activated protein kinase pathway. This pathway consists of a cascade of protein kinases including stress-activated protein kinase (SAPK), also termed Jun N-terminal kinase (JNK), and two kinases that activate it, MEKK and SEK/MKK4. Despite rapid progress in delineating the components of this pathway, the cellular consequence of its activation remains to be defined., Results: We have screened cells for defects in SAPK signaling and identified a cell line, previously characterized for its thermotolerance properties, as being more refractive to SAPK activation induced by heat stress than its thermosensitive parental line. Stable expression of dominant inhibiting SEK mutants in thermosensitive parental cells specifically and effectively blocked SAPK activation after heat shock. These lines also became markedly resistant to the cytocidal effects of thermal stress, confirming the phenotype of the thermotolerant line. These cell lines defective in SAPK activation were also resistant to the lethal effects of the DNA-damaging drug cis-platinum., Conclusions: Experimentally induced stable blockade of SAPK activation in cells with normal thermosensitivity is sufficient to confer resistance to cell death induced by diverse stimuli including heat and the chemotherapeutic agent cis-platinum. These results suggest that activation of the SAPK pathway by diverse cell stressors plays a critical part in mediating the toxicity of these treatments and inducing cell death. SAPK activation in this context could broadly influence cellular response to stress, modulate apoptosis during development or determine the clinical response of tumor cells to cytotoxic therapies.

Mammalian cells respond to their immediate environment by inducing signal transduction cascades that regulate metabolism, secretion and gene expression. Several of these signalling pathways are structurally and organizationally related insofar as they require activation of a protein-serine kinase via it's phosphorylation on tyrosine and threonine; the archetype being mitogen-activated protein kinase (MAPK) which responds primarily to mitogenic stimuli via Ras. In contrast, two more recently identified cascades are responsive to cellular stresses such as heat, inflammatory cytokines, ischaemia and metabolic poisons. The recent identification of the components of these pathways has allowed manipulation of the stress-responsive pathways and evaluation of their physiological roles. These studies reveal a high degree of independence between the pathways not apparent from in vitro studies. Manipulation of the pathways in vivo will likely result in novel therapies for inflammatory disease and reperfusion injury.

The B cell-associated surface molecule CD40 plays a key role in T cell-dependent B cell maturation, as individuals with defects in either CD40 or its ligand are impaired in immunoglobulin isotype class switching and germinal center formation. CD40 signaling activates downstream effectors, including the tyrosine protein kinase, Lyn, the phosphatidylinositol-3-kinase (PI-3 kinase), and the transcription factor, NF-kappa B. In this study, we demonstrate that stress-activated protein kinases (SAPK) are activated after CD40 cross-linking on various B cell lines or human tonsillar B cells. The activation is rapid and transient and is mediated through a cyclosporin A-insensitive pathway. Furthermore, this signaling pathway appears not to rely on protein kinase C. While CD40 ligation strongly activates the SAPKs (up to 25-fold), it does not affect members of the mitogen-activated protein kinase family (MAPK; ERK1 and ERK2). Consistent with these data, CD40 signals up-regulate c-jun but not c-fos mRNA and alter the transcription factor ATF2 but not the Raf-1 protein. In summary, CD40 signaling preferentially induces SAPK but not MAPK.

The present study compares the mitogen-activated protein (MAP) kinase responses in T cells activated with the CD28 ligands B7-1 (CD80) and B7-2/B70 (CD86). Ligands B7-1 and B7-2 do not activate the Raf-1/ERK2 cascade, but share the ability to activate related Jun kinases. These natural ligands for CD28 had no stimulatory effect alone on Jun kinase activation, but the data show that B7-1 and B7-2 could both co-operate with intracellular Ca2+ increase and protein kinase C (PKC) activation to stimulate Jun kinases. The present study shows that the interaction of CD28 with its ligands B7-1 and B7-2 can induce identical signal transduction through the MAP kinase cascades.

The SAPKs represent novel conduits through which the effects of cellular insults or injury are transmitted to the nucleus to influence gene expression. The SAPK pathway consists of at least four levels of protein kinases that are activated by a wide range of agents that adversely affect cell growth. Unlike the structurally related MAPK pathway, the stress induced kinases are not required for mitogenesis and instead induce growth arrest. Given the modulation of the pathway by inflammatory cytokines, reperfusion injury and chemotherapeutics, determination of the physiological functions of the pathway may uncover new possibilities for diagnosis and therapeutic intervention.

We report the cloning of the skp1+ gene, a Schizosaccharomyces pombe homolog of the glycogen synthase kinase 3 (GSK-3) family whose members in higher eukaryotes are involved in cell fate determination, nuclear signalling, and hormonal regulation. skp1 is 67% identical to mammalian GSK-3 beta and displays similar biochemical properties in vitro. Like GSK-3 beta, skp1 is phosphorylated on a conserved tyrosine residue, and this phosphorylation is required for efficient activity. skp1 is also phosphorylated at a serine which has been identified as S-335. Phosphorylation at this site is likely to inhibit its function. Unlike the mammalian enzyme, skp1 both tyrosine autophosphorylates in yeast cells and can phosphorylate other proteins on tyrosine in bacteria. The skp1+ gene is not essential. However, cells with deletions in skp1+ are sensitive to heat shock and exhibit defects in sporulation. Overexpression of wild-type skp1+ specifically complements cdc14-118, one of several mutations causing a defect in cytokinesis. In addition, certain phosphorylation site mutants induce a delay or block in cytokinesis when overexpressed. Together, these data identify novel interactions of a fission yeast GSK-3 homolog with elements of the cytokinesis machinery.

Pyramidal neurons in affected regions of Alzheimer's disease (AD) brain contain neurofibrillary tangles (NFT), aggregates of paired helical filaments (PHF) composed mainly of phosphorylated microtubule-associated protein tau. To explore the role of tau phosphorylation in the aggregation of tau into PHF, we constructed mammalian cell culture systems producing high levels of intracellular phosphorylated tau. COS-1 fibroblast-like cells were transiently transfected to simultaneously express tau, MAP kinase (MAPK), and MAP kinase kinase (MAPKK), or alternatively to express tau and glycogen synthase kinase 3 (GSK3). B103 neuron-like cells (which contain MAPK but little tau or GSK3) were stably transfected to express tau or tau and GSK3. In both systems, GSK3-transfected cells contained tau AT8/M (defined by AT8 staining and tau PHF-like mobility), but MAPK-transfected cells required phosphatase inhibitors, such as okadaic acid (OKA) or calyculin (CAL), to produce tau AT8/M. In vitro, the same concentrations of CAL and OKA inhibit phosphatases 1 and 2A (PP1 and PP2A), except that 100-1000 times as much OKA is needed to inhibit PP1. Inducing tau phosphorylation at the AT8 site in MAPK-transfected cells required 2-10 times more OKA than CAL, suggesting both PP1 and PP2A helped block the phosphorylation. Though levels of tau AT8/M reached 2-8% of total cellular proteins in COS-1 cells, the ratio of particulate to supernatant tau levels did not increase, and no tangles were observed; perhaps post-translational modifications or co-aggregating proteins are needed to induce PHF.

Eukaryotic cells respond to different extracellular stimuli by recruiting homologous signalling pathways that use members of the MEKK, MEK and ERK families of protein kinases. The MEKK-->MEK-->ERK core pathways of Saccharomyces cerevisiae may themselves be regulated by members of the STE20 family of protein kinases. Here we report specific activation of the mammalian stress-activated protein kinase (SAPK) pathway by germinal centre kinase (GCK), a human STE20 homologue. SAPKs, members of the ERK family, are activated in situ by inflammatory stimuli, including tumour-necrosis factor (TNF) and interleukin-1, and phosphorylate and probably stimulate the transactivation function of c-Jun. Although GCK is found in many tissues, its expression in lymphoid follicles is restricted to the cells of the germinal centre, where it may participate in B-cell differentiation. Activation of the SAPK pathway by GCK illustrates further the striking conservation of eukaryotic signalling mechanisms and defines the first physiological function of a mammalian Ste20.

A proportion of the microtubule-associated protein, tau, is in an elevated state of phosphorylation in foetal and adult brain whereas all of the tau in paired helical filaments, which are characteristic of Alzheimer's disease is hyperphosphorylated; it is important therefore to elucidate the mechanisms that regulate tau phosphorylation. Here we describe results that show that although MAP kinase can hyperphosphorylate tau in vitro, activation of MAP kinase in transformed fibroblasts does not result in hyperphosphorylation of transfected tau, whereas glycogen synthase kinase-3 beta (GSK-3 beta) when co-transfected with tau does result in tau hyperphosphorylation. The findings imply that GSK-3 beta may be a stronger candidate than MAP kinase for inducing tau hyperphosphorylation in vivo.

Glycogen synthase kinase 3 (GSK-3) is homologous to the product of the Drosophila gene shaggy (zeste-white 3), which is required for signalling by wingless during Drosophila development. To test whether GSK-3 is also involved in vertebrate pattern formation, its role was investigated during early Xenopus development. It was found that dominant-negative GSK-3 mutants induced dorsal differentiation, whereas wild-type GSK-3 induced ventralization. These results indicate that GSK-3 is required for ventral differentiation, and suggest that dorsal differentiation may involve the suppression of GSK-3 activity by a wingless/wnt-related signal.

1. A diverse array of stressful stimuli induces changes in gene expression via post-translational modification of transcription factors. 2. Study of activator protein (AP-1) transcription factor regulation has revealed a novel family of protein-serine kinases. Molecular cloning and expression of these proteins have demonstrated their specific activation by stress stimuli such as UV irradiation, heat, ischaemia/reperfusion and metabolic toxins. 3. Dissection of the regulation of the stress-activated protein kinases has revealed a similar but distinct mechanism to mitogen-activated protein kinases suggesting that cellular responses to stress may partially overlap with normal growth responses and have common nuclear targets.

The transactivating function of the c-Jun proto-oncogene component of the AP-1 transcription factor is acutely regulated by a wide variety of cellular signals via modulation of phosphorylation of two serines (63 and 73). The viral oncoprotein, v-Jun, while containing homologous serines, is not phosphorylated in cells. A novel family of stress-activated protein kinases (SAPKs), also termed Jun N-terminal domain kinases (JNKs), are responsible for mediating S63/73 phosphorylation in response to a variety of cellular stimuli including tumor necrosis factor-alpha, heat stress and u.v. light. The p54 alpha 1, alpha 2, p54 beta and p46 beta SAPKs are shown to bind directly to c-Jun but not to v-Jun, with an absolute requirement for c-Jun amino acids 31-47, a region deleted in v-Jun. Inactive SAPKs tightly bind c-Jun in resting cells and may be a manifestation of the 'delta' inhibitor, a previously described repressor of c-Jun function.

The stress-activated protein kinases (SAPKs), which are distantly related to the MAP kinases, are the dominant c-Jun amino-terminal protein kinases activated in response to a variety of cellular stresses, including treatment with tumour-necrosis factor-alpha and interleukin-beta (refs 1, 2). SAPK phosphorylation of c-Jun probably activates the c-Jun transactivation function. SAPKs are part of a signal transduction cascade related to, but distinct from, the MAPK pathway. We have now identified a novel protein kinase, called SAPK/ERK kinase-1 (SEK1), which is structurally related to the MAP kinase kinases (MEKs). SEK1 is a potent activator of the SAPKs in vitro and in vivo. An inactive SEK1 mutant blocks SAPK activation by extracellular stimuli without interfering with the MAPK pathway. Although alternative mechanisms of SAPK activation may exist, as an immediate upstream activator of the SAPKs, SEK1 further defines a signalling cascade that couples cellular stress agonists to the c-Jun transcription factor.

A kinase distinct from the MEK activator Raf, termed MEK kinase-1 (MEKK), was originally identified by virtue of its homology to kinases involved in yeast mating signal cascades. Like Raf, MEKK is capable of activating MEK in vitro. High-level expression of MEKK in COS-7 cells or using vaccinia virus vectors also activates MEK and MAPK, indicating that MEKK and Raf provide alternative means of activating the MAPK signalling pathway. We have derived NIH3T3 cell sublines that can be induced to express active MEKK. Here we show that induction of MEKK does not result in the activation of MAPK, but instead stimulates the stress-activated protein kinases (SAPKs) which are identical to a Jun amino-terminal kinase. We find that MEKK regulates a new signalling cascade by phosphorylating an SAPK activator, SEK1 which in turn phosphorylates and activates SAPK.

Glycogen synthase kinase-3 (GSK-3), a protein-serine kinase implicated in cell-fate determination and differentiation, phosphorylates several regulatory proteins that are activated by dephosphorylation in response to hormones or growth factors. GSK-3 beta is phosphorylated in vitro at serine 9 by p70 S6 kinase and p90rsk-1, resulting in its inhibition [Sutherland, Leighton, and Cohen (1993) Biochem. J. 296, 15-19]. Using HeLa cells expressing GSK-3 beta or a mutant containing alanine at residue 9, we demonstrate that serine 9 is modified in intact cells and is targeted specifically by p90rsk-1, and that phosphorylation leads to loss of activity. Since p90rsk-1 is directly activated by mitogen-activated protein kinases, agonists of this pathway, such as insulin, repress GSK-3 function.

The signal transduction pathways that mediate activation of trans acting factors controlling an organ's response to ischemia are unknown. The stress-activated protein kinases (SAPKs), a subfamily of the extracellular signal-regulated kinases (ERKs), phosphorylate c-Jun within the amino-terminal transactivation domain and are activated in response to a variety of cellular stresses. We determined whether SAPKs are activated in response to ischemia, an extreme, albeit common, pathophysiologic stress. Rats underwent 40 min of renal ischemia followed by reperfusion for 0, 5, 20, or 90 min. SAPKs were immunoprecipitated from kidney lysates and kinase activity assayed with recombinant GST-c-Jun(1-135), containing the amino-terminal transactivation domain of c-Jun as substrate. SAPKs were not activated by ischemia alone, but reperfusion for as little as 5 min was associated with a 4.6-fold increase in kinase activity. Kinase activity was increased 7.6-fold at 20 min following reperfusion and remained elevated at 90 min of reperfusion (4.9-fold). In contrast, activity of the related ERK-1 and -2 was increased only 1.3-fold and only at the 5-min reperfusion time point. When SAPKs were immunodepleted from kidney extracts prior to incubation of the extracts with agarose-coupled GST-c-Jun(1-135), it was found that SAPKs accounted for the majority of the amino-terminal c-Jun kinase activity of kidney at 5 min following reperfusion. In Madin-Darby canine kidney epithelial cells, ATP repletion, following ATP depletion induced by chemical anoxia, was associated with a 9-15-fold activation of SAPKs with a similar time course of activation to that seen in the kidney after ischemia and reperfusion. In conclusion, the SAPKs are markedly activated very early after reperfusion of ischemic kidney and following ATP repletion of anoxic cells in culture. We propose that this activation of SAPKs may trigger part of the kidney's early genetic response to ischemia, possibly by enhancing trans acting activity of c-Jun.

Extensive in vitro phosphorylation of a purified preparation of control human brain tau consistently produces four rather than, as previously believed, three tau species on SDS-PAGE. The species thus generated are shifted on SDS-PAGE to positions that match those of PHF-tau isolated from Alzheimer's disease brain. A mixture of recombinant human tau isoforms phosphorylated by GSK-3 beta gave similar results to those obtained with control human brain tau. In vitro phosphorylation of the individual recombinant isoforms by GSK-3 beta showed that the four bands of PHF-tau are likely to consist of isoforms 3R,0 alone; 4R,0 with 3R,29; 4R,29 with 3R,58 and 4R,58 alone.

Glycogen synthase kinase-3 is a 'black sheep' among protein kinases. Although its name suggests a primary function in intermediary metabolism, its scope is far more diverse as indicated by both genetic and biochemical evidence. Over the past five years the enzyme has emerged as an important component of transduction pathways ultimately regulating cell fate and differentiation. The molecular mechanisms underlying these functions are only just being unraveled but point to a novel mode of cellular regulation conserved in all eukaryotes.

Growth factors, phorbol esters, and oncogenes such as ras, src, and sis are believed to stimulate c-Jun transcriptional activation by inducing increased phosphorylation at two serine residues (S63 and S73) within the N-terminal transactivation domain. Although S63 and S73 are conserved in the mutant v-Jun oncoprotein, they are not phosphorylated by two enzymes which target the corresponding residues in c-Jun in vitro; namely a partially purified c-Jun kinase from TPA-stimulated U937 cells and purified p54 mitogen activated protein (MAP) kinase. In addition, v-Jun activates transcription more strongly than c-Jun when fused to the Gal4 DNA-binding domain, and transcriptional activation by Gal4-v-Jun is unaffected when S63, S73, or both, are replaced with non-phosphorylatable alanine residues, amino acid substitutions which severely impair transcriptional activation by Gal4-c-Jun. The novel biochemical and transcriptional properties of v-Jun result from deletion of a 27 amino acid segment, termed delta, which is important for transforming activity. On the basis of these results we propose that unlike c-Jun, v-Jun transcriptional activation is independent of positive regulatory phosphorylation and that this may contribute to oncogenesis by v-Jun.

ATP citrate-lyase (CL), acetyl-CoA carboxylase (ACC) and glycogen synthase kinase-3 beta (GSK-3 beta) levels were measured in cytosol from 3T3-L1 cells during differentiation from fibroblasts into fat-cells. Protein levels were estimated from immunoblots using specific antisera. Cytosol from confluent cells contain significant amounts of GSK-3 beta, which fell during differentiation of these cells into adipocytes. CL from confluent cells was found to be mostly in the form of a single protein band of apparent mass 110 kDa. Levels of CL and ACC increased during cell differentiation into adipocytes. During the first 3 days of differentiation, CL migration changed, and it was expressed as a complex of protein bands of apparent mass 110 kDa, 113 kDa and 115 kDa. At later stages of differentiation, when these cells had assumed the phenotype of fat-cells, they expressed CL mainly as protein bands of 110 and 113 kDa. When samples containing these bands were treated with alkaline phosphatase, the 113 kDa protein band collapsed into the 110 kDa species. This suggests that the slower-migrating species of CL is a higher-order phosphorylation state of the same protein. Furthermore, when purified CL, mostly expressed as the 110 kDa species, was phosphorylated with cyclic AMP-dependent protein kinase alone or together with GSK-3 and resolved by SDS/PAGE, the phosphorylated CL now migrated more slowly as the 113 kDa and 115 kDa forms. CL phosphorylation was hormone-regulated, since, in samples from fat-cells that had the complex two-band pattern, when cultured in medium without serum or hormones, CL migration reverted to a single band of 110 kDa, similar to confluent cells. Treatment of these 'down-regulated' cells with insulin rapidly induced substantial amounts of the 113 kDa species, with a concomitant decrease in the 110 kDa species.

The mitogen-activated protein (MAP) kinases Erk-1 and Erk-2 are proline-directed kinases that are themselves activated through concomitant phosphorylation of tyrosine and threonine residues. The kinase p54 (M(r) 54,000), which was first isolated from cycloheximide-treated rats, is proline-directed like Erks-1/2, and requires both Tyr and Ser/Thr phosphorylation for activity. p54 is, however, distinct from Erks-1/2 in its substrate specificity, being unable to phosphorylate pp90rsk but more active in phosphorylating the c-Jun transactivation domain. Molecular cloning of p54 reveals a unique subfamily of extracellularly regulated kinases. Although they are 40-45% identical in sequence to Erks-1/2, unlike Erks-1/2 the p54s are only poorly activated in most cells by mitogens or phorbol esters. However, p54s are the principal c-Jun N-terminal kinases activated by cellular stress and tumour necrosis factor (TNF)-alpha, hence they are designated stress-activated protein kinases, or SAPKs. SAPKs are also activated by sphingomyelinase, which elicits a subset of cellular responses to TNF-alpha (ref. 9). SAPKs therefore define a new TNF-alpha and stress-activated signalling pathway, possibly initiated by sphingomyelin-based second messengers, which regulates the activity of c-Jun.