Raf meets Ras: completing the framework of a signal transduction pathway.

The Ras oncoprotein, a GTP-activated molecular switch, interacts directly with the Raf oncoprotein to recruit the MAP kinases and their subordinates. In this way, a mitogenic signal initiated by tyrosine kinases is converted by Ras into a wave of regulatory phosphorylation on serine and threonine residues that, depending on its intensity and duration, and the variety of substrates available, results in cell differentiation or cell division.

Role of the stress-activated protein kinases in endothelin-induced cardiomyocyte hypertrophy.

The signal transduction pathways governing the hypertrophic response of cardiomyocytes are not well defined. Constitutive activation of the stress-activated protein kinase (SAPK) family of mitogen-activated protein (MAP) kinases or another stress-response MAP kinase, p38, by overexpression of activated mutants of various components of the pathways is sufficient to induce a hypertrophic response in cardiomyocytes, but it is not clear what role these pathways play in the response to physiologically relevant hypertrophic stimuli. To determine the role of the SAPKs in the hypertrophic response, we used adenovirus-mediated gene transfer of SAPK/ERK kinase-1 (KR) [SEK-1(KR)], a dominant inhibitory mutant of SEK-1, the immediate upstream activator of the SAPKs, to block signal transmission down the SAPK pathway in response to the potent hypertrophic agent, endothelin-1 (ET-1). SEK-1(KR) completely inhibited ET-1-induced SAPK activation without affecting activation of the other MAP kinases implicated in the hypertrophic response, p38 and extracellular signal-regulated protein kinases (ERK)-1/ERK-2. Expression of SEK-1(KR) markedly inhibited the ET-1-induced increase in protein synthesis. In contrast, the MAPK/ERK kinase inhibitor, PD98059, which blocks ERK activation, and the p38 inhibitor, SB203580, had no effect on ET-1-induced protein synthesis. ET-1 also induced a significant increase in atrial natriuretic factor mRNA expression as well as in the percentage of cells with highly organized sarcomeres, responses which were also blocked by expression of SEK-1(KR). In summary, inhibiting activation of the SAPK pathway abrogated the hypertrophic response to ET-1. These data are the first demonstration that the SAPKs are necessary for the development of agonist-induced cardiomyocyte hypertrophy, and suggest that in response to ET-1, they transduce critical signals governing the hypertrophic response.

Activation of apoptosis signal-regulating kinase 1 (ASK1) by tumor necrosis factor receptor-associated factor 2 requires prior dissociation of the ASK1 inhibitor thioredoxin.

The stress-activated protein kinases (SAPKs, also called c-Jun NH(2)-terminal kinases) and the p38s, two mitogen-activated protein kinase (MAPK) subgroups activated by cytokines of the tumor necrosis factor (TNF) family, are pivotal to the de novo gene expression elicited as part of the inflammatory response. Apoptosis signal-regulating kinase 1 (ASK1) is a MAPK kinase kinase (MAP3K) that activates both the SAPKs and p38s in vivo. Here we show that TNF receptor (TNFR) associated factor 2 (TRAF2), an adapter protein that couples TNFRs to the SAPKs and p38s, can activate ASK1 in vivo and can interact in vivo with the amino- and carboxyl-terminal noncatalytic domains of the ASK1 polypeptide. Expression of the amino-terminal noncatalytic domain of ASK1 can inhibit TNF and TRAF2 activation of SAPK. TNF can stimulate the production of reactive oxygen species (ROS), and the redox-sensing enzyme thioredoxin (Trx) is an endogenous inhibitor of ASK1. We also show that expression of TRAF2 fosters the production of ROS in transfected cells. We demonstrate that Trx significantly inhibits TRAF2 activation of SAPK and blocks the ASK1-TRAF2 interaction in a reaction reversed by oxidants. Finally, the mechanism of ASK1 activation involves, in part, homo-oligomerization. We show that expression of ASK1 with TRAF2 enhances in vivo ASK1 homo-oligomerization in a manner dependent, in part, upon the TRAF2 RING effector domain and the generation of ROS. Thus, activation of ASK1 by TNF requires the ROS-mediated dissociation of Trx possibly followed by the binding of TRAF2 and consequent ASK1 homo-oligomerization.

Human mitogen-activated protein kinase kinase 4 as a candidate tumor suppressor.

Mitogen-activated protein kinases function in signal transduction pathways that are involved in controlling key cellular processes in many organisms. A mammalian member of this kinase family, MKK4/JNKK1/SEK1, has been reported to link upstream MEKK1 to downstream stress-activated protein kinase/JNK1 and p38 mitogen-activated protein kinase. This mitogen-activated protein kinase pathway has been implicated in the signal transduction of cytokine- and stress-induced apoptosis in a variety of cell types. Here, we report that two human tumor cell lines, derived from pancreatic carcinoma and lung carcinoma, harbor homozygous deletions that eliminate coding portions of the MKK4 locus at 17p, located approximately 10 cM centromeric of p53. In addition, in a set of 88 human cancer cell lines prescreened for loss of heterozygosity, we detected two nonsense and three missense sequence variants of MKK4 in cancer cell lines derived from human pancreatic, breast, colon, and testis cells. In vitro biochemical assays revealed that, when stimulated by MEKK1, four of the five altered MKK4 proteins lacked the ability to phosphorylate stress-activated protein kinase. Thus, the incidence of coding mutations of MKK4 in the set of cell lines is 6 of 213 (approximately 3%). These findings suggest that MKK4 may function as a suppressor of tumorigenesis or metastasis in certain types of cells.

Stress-activated protein kinases bind directly to the delta domain of c-Jun in resting cells: implications for repression of c-Jun function.

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.

c-Jun N-terminal kinase (JNK)-interacting protein-1b/islet-brain-1 scaffolds Alzheimer's amyloid precursor protein with JNK.

Using a yeast two-hybrid method, we searched for amyloid precursor protein (APP)-interacting molecules by screening mouse and human brain libraries. In addition to known interacting proteins containing a phosphotyrosine-interaction-domain (PID)-Fe65, Fe65L, Fe65L2, X11, and mDab1, we identified, as a novel APP-interacting molecule, a PID-containing isoform of mouse JNK-interacting protein-1 (JIP-1b) and its human homolog IB1, the established scaffold proteins for JNK. The APP amino acids Tyr(682), Asn(684), and Tyr(687) in the G(681)YENPTY(687) region were all essential for APP/JIP-1b interaction, but neither Tyr(653) nor Thr(668) was necessary. APP-interacting ability was specific for this additional isoform containing PID and was shared by both human and mouse homologs. JIP-1b expressed by mammalian cells was efficiently precipitated by the cytoplasmic domain of APP in the extreme Gly(681)-Asn(695) domain-dependent manner. Reciprocally, both full-length wild-type and familial Alzheimer's disease mutant APPs were precipitated by PID-containing JIP constructs. Antibodies raised against the N and C termini of JIP-1b coprecipitated JIP-1b and wild-type or mutant APP in non-neuronal and neuronal cells. Moreover, human JNK1beta1 formed a complex with APP in a JIP-1b-dependent manner. Confocal microscopic examination demonstrated that APP and JIP-1b share similar subcellular localization in transfected cells. These data indicate that JIP-1b/IB1 scaffolds APP with JNK, providing a novel insight into the role of the JNK scaffold protein as an interface of APP with intracellular functional molecules.

Insulin, epidermal growth factor and fibroblast growth factor elicit distinct patterns of protein tyrosine phosphorylation in BC3H1 cells.

The polypeptides which are phosphorylated at tyrosine residues in the murine muscle-like cell line, BC3H1, in response to insulin, epidermal growth factor (EGF) and fibroblast growth factor (FGF) were detected by immunoblotting with antiphosphotyrosine antibodies. Each ligand elicited the tyrosine phosphorylation of a characteristic, largely nonoverlapping set of polypeptide substrates, as classified by subunit Mr, pI, behavior on subcellular fractionation and adsorption to lectin (what germ agglutinin-Sepharose) columns. The dose-response curves for all stimulated tyrosine phosphorylations elicited by a single ligand were superimposable. By contrast, the temporal pattern of the responses elicited by each ligand differed in regard to speed of onset and persistence of the stimulation. Phosphorylation in response to insulin was maximal in a virtually instantaneous fashion and was fully maintained for at least 30 min. The response to EGF increased steadily over the initial 15-60 s to peak values, and fell progressively thereafter. FGF-stimulated phosphorylation was not detectable until 4 min after FGF addition, abruptly rose to maximal within the next 30 s, and declined subsequently. Exposure of BC3H1 cells to active phorbol esters prior to hormone addition altered the response to hormones in a differential fashion. FGF responses were abolished, EGF responses were partially inhibited, whereas the response to insulin was unaffected. Thus, acting on a single cell, insulin, EGF and FGF each mediate the tyrosine phosphorylation of a characteristic, largely nonoverlapping array of polypeptide substrates, indicating that each of these receptor tyrosine kinases exhibits a fundamentally distinct substrate specificity. Differences in the kinetic and regulatory properties of the response to each ligand are also apparent, and reflect the differing regulatory properties of each receptor tyrosine kinase acting in situ.

MAP kinases and the regulation of nuclear receptors.

The activity of nuclear receptors or ligand-activated transcription factors can be regulated both positively and negatively by signals transduced through mitogen-activated protein kinase (MAPK) signaling cascades. Kyriakis discusses how cross talk between MAPK signaling and nuclear receptor signaling occurs and the effect this cross talk has on nuclear receptor function.

Activating transcription factor-2 DNA-binding activity is stimulated by phosphorylation catalyzed by p42 and p54 microtubule-associated protein kinases.

Recent studies have detailed the ability of activating transcription factor-2 (ATF-2) to mediate adenoviral E1a stimulation of gene expression; however, an endogenous regulator for the transcriptional activity of this protein has not been described. To characterize the regulation of ATF-2 activity, we have expressed full-length and truncated peptides corresponding to various regions of the ATF-2 protein in bacteria and the baculovirus insect cell system. Bacterially expressed truncated (350-505) but not full-length ATF-2, was able to bind a consensus cAMP response element-containing oligonucleotide, suggesting the N-terminal moiety may serve as a negative regulator of DNA-binding activity. In contrast, the full-length ATF-2 protein expressed in Spodoptera frugiperda (Sf9) cells using a recombinant baculovirus was fully competent to bind DNA. Protein phosphatase 2A reversed the DNA-binding activity by dephosphorylating the ATF-2 polypeptide. Microtubule-associated protein kinase catalyzed the phosphorylation and stimulated the DNA-binding activity of bacterially expressed full-length ATF-2. Phosphopeptide mapping of phosphorylated ATF-2 proteins identified a single peptide in the N-terminal moiety of ATF-2 phosphorylated by p42 or p54 microtubule-associated protein kinase. Therefore, we propose that phosphorylation of this regulatory site is sufficient to induce an allosteric structural change in the ATF-2 protein, which allows dimerization and subsequent DNA binding.

Activation of the MKK4-JNK pathway during erythroid differentiation of K562 cells is inhibited by the heat shock factor 2-beta isoform.

In this study we report the activation of c-Jun N-terminal kinase (JNK) in human K562 erythroleukemia cells undergoing hemin-mediated erythroid differentiation, which occurs concomitantly with activation of heat shock factor 2 (HSF2) and leads to a simultaneous in vivo phosphorylation of c-Jun. The activation of JNK occurs through activation of mitogen-activated protein kinase kinase (MKK) 4 and not by activation of MKK7 or inhibition of JNK-directed phosphatases. We have previously shown that overexpression of the HSF2-beta isoform inhibits the activation of HSF2 upon hemin-induced erythroid differentiation. Here we demonstrate that HSF2-beta overexpression blocks the hemin-induced activation of the MKK4-JNK pathway, suggesting an erythroid lineage-specific JNK activation likely to be regulated by HSF2.

Making the connection: coupling of stress-activated ERK/MAPK (extracellular-signal-regulated kinase/mitogen-activated protein kinase) core signalling modules to extracellular stimuli and biological responses.

Signal-transduction pathways that employ members of the extracellular signal-regulated kinase (ERK)/mitogen-activated protein kinase (MAPK) family of protein Ser/Thr kinases are widely conserved among eukaryotes. The multiplicity of these pathways allows the cell to respond to divergent extracellular stimuli by initiating a broad array of responses ranging from cell growth to apoptosis. ERK/MAPK pathways are comprised of a three-tiered core-signalling module wherein ERK/MAPKs are regulated by MAPK/ERK kinases (MEKs) and MEKs, in turn, are regulated by MAPK kinase kinases (MAPKKKs). The regulation of MAPKKK-->MEK-->ERK/MAPK core-signalling modules by upstream components is poorly understood. Mammalian stress-activated ERK/MAPK pathways have been implicated in numerous important physiological functions, including inflammatory responses and apoptosis. In this review, I will discuss how mammalian stress-regulated ERK/MAPK core-signalling modules couple with members of the SPS1 family of protein kinases and to other upstream elements, and how these stress-regulated pathways influence cell function.

Activation of the AP-1 transcription factor by inflammatory cytokines of the TNF family.

Inflammatory cytokines of the tumor necrosis factor (TNF) family mediate a large variety of cellular and organismal inflammatory responses and are important to the pathogenesis of a number of important disease states including arthritis, septic shock, inflammatory bowel disease, and, possibly, type II diabetes. Many of the responses to these cytokines require de novo gene expression mediated by the activator protein-1 (AP-1) heterodimeric transcription factor. This review will discuss what is known of how cytokines of the TNF family, acting at the cell surface, recruit two mitogen-activated protein kinase (MAPK) subfamilies, the stress-activated protein kinases (SAPKs, also called JNKs) and the p38s, to transduce signals to AP-1.

Protein kinase cascades activated by stress and inflammatory cytokines.

Signal transduction pathways constructed around a core module of three consecutive protein kinases, the most distal being a member of the extracellular signal-regulated kinase (ERK) family, are ubiquitous among eukaryotes. Recent work has defined two cascades activated preferentially by the inflammatory cytokines TNF-alpha and IL-1-beta, as well as by a wide variety of cellular stresses such as UV and ionizing radiation, hyperosmolarity, heat stress, oxidative stress, etc. One pathway converges on the ERK subfamily known as the "stress activated' protein kinases (SAPKs, also termed Jun N-terminal kinases, JNKs), whereas the second pathway recruits the p38 kinases. Upstream inputs are diverse, and include small GTPases (primarily Rac and Cdc42; secondarily Ras) acting through mammalian homologs of the yeast Ste20 kinase, other kinase subfamilies (e.g. GC kinase) and ceramide, a putative second messenger for certain TNF-alpha actions. These two cascades signal cell cycle delay, cellular repair or apoptosis in most cells, as well as activation of immune and reticuloendothelial cells.

pp54 microtubule-associated protein 2 kinase. A novel serine/threonine protein kinase regulated by phosphorylation and stimulated by poly-L-lysine.

An hepatic protein kinase that phosphorylates microtubule-associated protein 2 (MAP-2) on Ser/Thr residues is markedly activated after intraperitoneal injection of cycloheximide in the rat. The enzyme has been purified greater than 10,000-fold to near homogeneity and corresponds to a 54-kDa polypeptide, based on auto-phosphorylation, renaturation of activity from sodium dodecyl sulfate gels, and gel filtration. The protein kinase activity is unaffected by prior autophosphorylation, Ca2+, diacylglycerol and phospholipids, cyclic nucleotides, staurosporine, and protein kinase inhibitor, but can be totally and specifically deactivated by the Ser/Thr protein phosphatase 2A. The enzyme is inhibited completely but reversible by transition metals and p-chloromercuribenzoate, and is strongly stimulated by poly-L-lysine toward most, but not all protein substrates. The activity of the cycloheximide-stimulated MAP-2 kinase (pp54 MAP-2 kinase) toward potential polypeptide substrates was compared to that of an insulin-stimulated MAP-2 kinase (pp42 MAP-2 kinase). Although both MAP-2 kinases exhibited little or no ability to phosphorylate histones and casein, the two kinases had a distinguishable substrate specificity. At comparable MAP-2 phosphorylating activities, pp42 MAP-2 kinase, but not pp54 MAP-2 kinase, phosphorylated and activated the Xenopus S6 protein kinase II. Moreover, pp42 MAP-2 kinase phosphorylated myelin basic protein at 10-12-fold higher rates than did pp54 MAP-2 kinase. Cycloheximide-activated pp54 MAP-2 protein kinase appears to be a previously uncharacterized protein kinase that is itself regulated through Ser/Thr phosphorylation and, perhaps, polypeptide regulators with basic domains. The identity of the upstream regulatory elements and the native substrates remain to be established.

Mammalian mitogen-activated protein kinase signal transduction pathways activated by stress and inflammation.

The molecular details of mammalian stress-activated signal transduction pathways have only begun to be dissected. This, despite the fact that the impact of these pathways on the pathology of chronic inflammation, heart disease, stroke, the debilitating effects of diabetes mellitus, and the side effects of cancer therapy, not to mention embryonic development, innate and acquired immunity, is profound. Cardiovascular disease and diabetes alone represent the most significant health care problems in the developed world. Thus it is not surprising that understanding these pathways has attracted wide interest, and in the past 10 years, dramatic progress has been made. Accordingly, it is now becoming possible to envisage the transition of these findings to the development of novel treatment strategies. This review focuses on the biochemical components and regulation of mammalian stress-regulated mitogen-activated protein kinase (MAPK) pathways. The nuclear factor-kappa B pathway, a second stress signaling paradigm, has been the subject of several excellent recent reviews (258, 260).

Regulation of nuclear transcription factors by stress signals.

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.

Changes in insulin binding to developing embryonic chick neural retina cells.

Specific cell surface insulin binding to embryonic chick neural retina cells has been demonstrated in vivo. Kinetics of insulin binding as well as hormonal specificity were similar to those reported for other vertebrate cells and tissues, both neural and nonneural. When surface insulin binding to retinal cells was studied as a function of embryonic age, a developmental relationship was observed. Scatchard analysis revealed that the number of cell surface insulin receptors decreased approximately 75% between days 10 and 16 of embryonic development. Receptor affinities remained fairly constant for this period.

Insulin stimulates choline acetyltransferase activity in cultured embryonic chicken retina neurons.

The effect of insulin on the appearance of the enzyme choline acetyltransferase (ChoAcT; acetyl-CoA:choline O-acetyltransferase, EC 2.3.1.6) in embryonic chicken retina neurons cultured in defined medium was studied. In the presence of a minimal level of insulin (1 ng/ml), ChoAcT activity increased with time in culture. A correspondence between the insulin concentration in the defined medium (1-100 ng/ml) and both the rate of increase and maximum attained level of ChoAcT activity was observed. Maximal ChoAcT activity was 2- to 3-fold greater in cells cultured in the presence of 100 ng of insulin per ml than in cells cultured in the presence of 1 ng of insulin per ml. To elicit maximum ChoAcT activity, insulin at 100 ng/ml was required in the medium for only the first 4 days of the culture period, at which time insulin could be reduced to maintenance levels (10 ng/ml) without affecting ChoAcT activity. Insulin binding assays performed during a 7-day culture period revealed that irrespective of the insulin concentration in the medium during culture, cell-surface insulin receptors decreased by approximately 90% between 4 and 7 days in culture. This decrease in insulin binding corresponded to the observed decrease in the sensitivity of ChoAcT activity to insulin. Our findings suggest that insulin plays a role in mediating cholinergic differentiation in the embryonic chicken retina.

Activation of the Ste20-like oxidant stress response kinase-1 during the initial stages of chemical anoxia-induced necrotic cell death. Requirement for dual inputs of oxidant stress and increased cytosolic [Ca2+].

Signal transduction mechanisms activated during the early stages of necrotic cell death are poorly characterized. We have recently identified the Sterile 20 (Ste20)-like oxidant stress response kinase-1, SOK-1, which is a member of the Ste20 kinase family. We report that SOK-1 is markedly activated as early as 20 min after chemical anoxia induced by exposure of Madin-Darby canine kidney or LLC-PK1 renal tubular epithelial cells to 2-deoxyglucose (2-DG) and any one of three inhibitors of the electron transport chain, cyanide (CN), rotenone, or antimycin A. Since oxidant stress activates SOK-1, we postulated that reactive oxygen species (ROS), which are produced by the electron transport chain during chemical anoxia, might be responsible for SOK-1 activation. The time course of CN/2-DG-induced SOK-1 activation and of production of ROS, measured in cells loaded with dichlorofluorescein, were compatible with a role for ROS in SOK-1 activation. Furthermore, preincubation of LLC-PK1 cells with three unrelated scavengers of ROS, pyrrolidine dithiocarbamate, pyruvate, or nordihydroguaiaretic acid, reduced both cellular oxidant stress and activation of SOK-1 by CN/2-DG. An increase in cytosolic free [Ca2+] ([Ca2+]i) was necessary but not sufficient for CN/2-DG-induced activation of SOK-1. Preincubation of cells with BAPTA-AM prevented activation of SOK-1. Incubation of cells with thapsigargin or the calcium ionophore, A23187, had no effect on SOK-1 activity, but preincubation of cells with either of these agents markedly enhanced CN/2-DG-induced activation of SOK-1 (20-fold versus 7-fold). In summary, chemical anoxia activates SOK-1 via an oxidant stress-dependent mechanism that is both critically dependent upon and markedly amplified by an increase in [Ca2+]i. This requirement for dual inputs of oxidant stress and an increase in [Ca2+]i may prevent inappropriate activation of the kinase by milder degrees of oxidant stress, which are insufficient to generate an increase in [Ca2+]i. The activation of SOK-1 may be one of the cell's earliest responses to inducers of necrotic cell death.