Subject(s)
Apoptosis/physiology , JNK Mitogen-Activated Protein Kinases , Mitogen-Activated Protein Kinase Kinases/physiology , NF-kappa B/physiology , Tumor Necrosis Factor-alpha/physiology , Antigens, Differentiation/physiology , Bone Morphogenetic Proteins/physiology , Cell Survival/physiology , Gene Expression Regulation , Intracellular Signaling Peptides and Proteins , MAP Kinase Kinase 4 , MAP Kinase Signaling System , Mitogen-Activated Protein Kinase Kinases/antagonists & inhibitors , Models, Biological , NF-kappa B/antagonists & inhibitors , Proteins/physiology , X-Linked Inhibitor of Apoptosis Protein , GADD45 ProteinsABSTRACT
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.
Subject(s)
Cell Differentiation , Erythroid Precursor Cells/metabolism , Heat-Shock Proteins/metabolism , MAP Kinase Kinase 4 , Mitogen-Activated Protein Kinase Kinases/metabolism , Mitogen-Activated Protein Kinases/metabolism , Transcription Factors/metabolism , Anisomycin/pharmacology , Enzyme Activation/drug effects , Enzyme Inhibitors/pharmacology , Heat-Shock Proteins/genetics , Hemin/pharmacology , Humans , JNK Mitogen-Activated Protein Kinases , K562 Cells , MAP Kinase Kinase 7 , Mitogen-Activated Protein Kinases/antagonists & inhibitors , Protein Isoforms , Proto-Oncogene Proteins c-jun/genetics , Proto-Oncogene Proteins c-jun/metabolism , Staurosporine/pharmacology , Transcription Factors/geneticsABSTRACT
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.
Subject(s)
Adaptor Proteins, Signal Transducing , Alzheimer Disease/metabolism , Amyloid beta-Protein Precursor/metabolism , Carrier Proteins/metabolism , Mitogen-Activated Protein Kinases/metabolism , Nuclear Proteins/metabolism , Trans-Activators/metabolism , Amino Acid Motifs/physiology , Amino Acid Substitution , Animals , Brain/metabolism , Carrier Proteins/genetics , Gene Library , Humans , JNK Mitogen-Activated Protein Kinases , Mice , Mice, Inbred ICR , Mutagenesis, Site-Directed , Nerve Tissue Proteins/metabolism , Nuclear Proteins/genetics , Phosphotyrosine/metabolism , Protein Binding/physiology , Protein Isoforms/genetics , Protein Isoforms/metabolism , Protein Structure, Tertiary/genetics , Protein Structure, Tertiary/physiology , Reverse Transcriptase Polymerase Chain Reaction , Structure-Activity Relationship , Trans-Activators/genetics , Two-Hybrid System TechniquesABSTRACT
A continuing focus of our work has been an effort to understand the signal transduction pathways through which insulin achieves its cellular actions. In the mid-1970s, we and others observed that insulin promoted an increase in Ser/Thr phosphorylation of a subset of cellular proteins. This finding was unanticipated, inasmuch as nearly all of the actions of insulin then known appeared to result from protein dephosphorylation. In fact, nearly 15 years elapsed before any physiologic response to insulin attributable to stimulated (Ser/Thr) phosphorylation was established. Nevertheless, based on the hypothesis that insulin-stimulated Ser/Thr phosphorylation reflected the activation of protein (Ser/Thr) kinases downstream of the insulin receptor, we sought to detect and purify these putative, insulin-responsive protein (Ser/Thr) kinases. Our effort was based on the presumption that an understanding of the mechanism for their activation would provide an entry into the biochemical reactions through which the insulin receptor activated its downstream effectors. To a degree that, in retrospect, is surprising, this goal was accomplished, much in the way originally envisioned. It is now well known that receptor tyrosine kinases (RTKs) recruit a large network of protein (Ser/Thr) kinases to execute their cellular programs. The first of these insulin-activated protein kinase networks to be fully elucidated was the Ras-Raf-mitogen-activated protein kinase (MAPK) cascade. This pathway is a central effector of cellular differentiation in development; moreover, its inappropriate and continuous activation provides a potent promitogenic force and is a very common occurrence in human cancers. Conversely, this pathway contributes minimally, if at all, to insulin's program of metabolic regulation. Nevertheless, the importance of the Ras-MAPK pathway in metazoan biology and human malignancies has impelled us to an ongoing analysis of the functions and regulation of Ras and Raf. This chapter will summarize briefly the way in which work from this and other laboratories on insulin signaling led to the discovery of the mammalian MAP kinase cascade and, in turn, to the identification of unique role of the Raf kinases in RTK activation of this protein (Ser/Thr) kinase cascade. We will then review in more detail current understanding of the biochemical mechanism through which the Ras proto-oncogene, in collaboration with the 14-3-3 protein and other protein kinases, initiates activation of the Raf kinase.
Subject(s)
MAP Kinase Signaling System , Protein-Tyrosine Kinases/metabolism , Proto-Oncogene Proteins c-raf/metabolism , ras Proteins/metabolism , 14-3-3 Proteins , Animals , Cell Membrane/metabolism , Enzyme Activation , Humans , Mice , Models, Biological , Phosphorylation , Protein Binding , Proto-Oncogene Mas , Receptor Protein-Tyrosine Kinases/metabolism , Receptor, Insulin/metabolism , Serine/metabolism , Signal Transduction , Threonine/metabolism , Tyrosine 3-Monooxygenase/metabolismABSTRACT
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).
Subject(s)
Inflammation/physiopathology , MAP Kinase Signaling System/physiology , Mitogen-Activated Protein Kinases/metabolism , Stress, Physiological/physiopathology , Animals , Cytokines/physiology , Humans , Mammals , p38 Mitogen-Activated Protein KinasesABSTRACT
Chronic stresses, including the mechanical strain caused by hypertension or excess pulmonary ventilation pressure, lead to important clinical consequences, including hypertrophy and acute respiratory distress syndrome. Pathologic hypertrophy contributes to decreased organ function and, ultimately, organ failure; and cardiac and diabetic renal hypertrophy are major causes of morbidity and morality in the developed world. Likewise, acute respiratory distress syndrome is a serious potential side effect of mechanical pulmonary ventilation. Whereas the deleterious effects of chronic stress are well established, the molecular mechanisms by which these stresses affect cell function are still poorly characterized. gene 33 (also called mitogen-inducible gene-6, mig-6) is an immediate early gene that is transcriptionally induced by a divergent array of extracellular stimuli. The physiologic function of Gene 33 is unknown. Here we show that gene 33 mRNA levels increase sharply in response to a set of commonly occurring chronic stress stimuli: mechanical strain, vasoactive peptides, and diabetic nephropathy. Induction of gene 33 requires the stress-activated protein kinases (SAPKs)/c-Jun NH(2)-terminal kinases. This expression pattern suggests that gene 33 is a potential marker for diabetic nephropathy and other pathologic responses to persistent sublethal stress. The structure of Gene 33 indicates an adapter protein capable of binding monomeric GTPases of the Rho subfamily. Consistent with this, Gene 33 interacts in vivo and, in a GTP-dependent manner, in vitro with Cdc42Hs; and transient expression of Gene 33 results in the selective activation of the SAPKs. These results imply a reciprocal, positive feedback relationship between Gene 33 expression and SAPK activation. Expression of Gene 33 at sufficient levels may enable a compensatory reprogramming of cellular function in response to chronic stress, which may have pathophysiological consequences.
Subject(s)
Adaptor Proteins, Signal Transducing , Carrier Proteins , Mitogen-Activated Protein Kinases/metabolism , Proteins/genetics , Proteins/metabolism , Transcription, Genetic , cdc42 GTP-Binding Protein/metabolism , 3T3 Cells , Amino Acid Sequence , Animals , Cell Line , Diabetic Nephropathies/genetics , Enzyme Activation , Guanosine Triphosphate/metabolism , Humans , Intracellular Signaling Peptides and Proteins , JNK Mitogen-Activated Protein Kinases , Kidney , Mice , Models, Biological , Molecular Sequence Data , Protein-Tyrosine Kinases/chemistry , Protein-Tyrosine Kinases/genetics , Proteins/chemistry , Rats , Recombinant Fusion Proteins/biosynthesis , Sequence Alignment , Sequence Homology, Amino Acid , Transfection , Tumor Suppressor ProteinsABSTRACT
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.
Subject(s)
MAP Kinase Kinase Kinases/metabolism , Proteins/metabolism , Receptors, Tumor Necrosis Factor/metabolism , Signal Transduction , Thioredoxins/metabolism , Tumor Necrosis Factor-alpha/metabolism , Cell Line , Enzyme Activation , Gene Expression Regulation , Humans , MAP Kinase Kinase Kinase 5 , MAP Kinase Kinase Kinases/genetics , Proteins/genetics , Receptors, Tumor Necrosis Factor/genetics , Signal Transduction/genetics , TNF Receptor-Associated Factor 2 , Transfection , Tumor Necrosis Factor-alpha/geneticsABSTRACT
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.
Subject(s)
Mitogen-Activated Protein Kinase Kinases/physiology , Receptors, Cytoplasmic and Nuclear/physiology , Animals , Cell Nucleus/enzymology , Humans , MAP Kinase Signaling System/physiology , Receptor Cross-Talk/physiologyABSTRACT
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.
Subject(s)
Mitogen-Activated Protein Kinases , Signal Transduction , Transcription Factor AP-1/metabolism , Tumor Necrosis Factor-alpha/metabolism , Amino Acid Sequence , Animals , Calcium-Calmodulin-Dependent Protein Kinases/metabolism , Gene Expression Regulation , Humans , Molecular Sequence Data , Proteins/metabolism , Receptors, Tumor Necrosis Factor/metabolism , TNF Receptor-Associated Factor 1 , p38 Mitogen-Activated Protein KinasesABSTRACT
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.
Subject(s)
Calcium-Calmodulin-Dependent Protein Kinases/metabolism , Signal Transduction , Animals , Apoptosis , Cell Cycle , Enzyme Activation , GTP-Binding Proteins/metabolism , Mammals , Oxidative Stress , Receptors, Tumor Necrosis Factor/metabolismSubject(s)
Germinal Center/enzymology , Protein Serine-Threonine Kinases/metabolism , Signal Transduction , rab GTP-Binding Proteins , Animals , Binding Sites , Enzyme Activation , GTP-Binding Proteins/metabolism , Germinal Center Kinases , Protein Binding , Protein Conformation , Protein Serine-Threonine Kinases/chemistry , src Homology DomainsABSTRACT
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.
Subject(s)
Calcium-Calmodulin-Dependent Protein Kinases/metabolism , Cardiomegaly/physiopathology , Endothelin-1/physiology , Heart/physiology , Mitogen-Activated Protein Kinase Kinases , Mitogen-Activated Protein Kinases , Myocardium/enzymology , Protein Serine-Threonine Kinases/metabolism , Protein-Tyrosine Kinases/metabolism , Adenoviridae , Animals , Animals, Newborn , Calcium-Calmodulin-Dependent Protein Kinases/antagonists & inhibitors , Cardiomegaly/enzymology , Cells, Cultured , Endothelin-1/pharmacology , Enzyme Activation , Enzyme Inhibitors/pharmacology , Flavonoids/pharmacology , Genetic Vectors , Imidazoles/pharmacology , Mitogen-Activated Protein Kinase 3 , Models, Cardiovascular , Myocardium/cytology , Protein Serine-Threonine Kinases/genetics , Protein-Tyrosine Kinases/genetics , Pyridines/pharmacology , Rats , Recombinant Proteins/metabolism , Signal Transduction/drug effects , Transfection , p38 Mitogen-Activated Protein KinasesABSTRACT
Tumor necrosis factor (TNF) elicits a diverse array of inflammatory responses through engagement of its type-1 receptor (TNFR1). Many of these responses require de novo gene expression mediated by the activator protein-1 (AP-1) transcription factor. We investigated the mechanism by which TNFR1 recruits the stress-activated protein kinases (SAPKs) and the p38s, two mitogen-activated protein kinase (MAPK) families that together regulate AP-1. We show that the human SPS1 homologue germinal center kinase (GCK) can interact in vivo with the TNFR1 signal transducer TNFR-associated factor-2 (TRAF2) and with MAPK/ERK kinase kinase 1 (MEKK1), a MAPK kinase kinase (MAPKKK) upstream of the SAPKs, thereby coupling TRAF2 to the SAPKs. Receptor interacting protein (RIP) is a second TNFR signal transducer which can bind TRAF2. We show that RIP activates both p38 and SAPK; and that TRAF2 activation of p38 requires RIP. We also demonstrate that the RIP noncatalytic intermediate domain associates in vivo with an endogenous MAPKKK that can activate the p38 pathway in vitro. Thus, TRAF2 initiates SAPK and p38 activation by binding two proximal protein kinases: GCK and RIP. GCK and RIP, in turn, signal by binding MAPKKKs upstream of the SAPKs and p38s.
Subject(s)
Calcium-Calmodulin-Dependent Protein Kinases/metabolism , Signal Transduction , Tumor Necrosis Factor-alpha/metabolism , Binding Sites , Enzyme Activation , Humans , Protein Binding , Proteins/metabolism , Receptors, Tumor Necrosis Factor/metabolism , Recombinant Proteins/metabolism , TNF Receptor-Associated Factor 2ABSTRACT
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.
Subject(s)
Calcium/metabolism , Cell Death , Cytosol/metabolism , Hypoxia/enzymology , Protein Serine-Threonine Kinases/metabolism , Saccharomyces cerevisiae Proteins , Animals , Dogs , Enzyme Activation , Intracellular Signaling Peptides and Proteins , LLC-PK1 Cells , MAP Kinase Kinase Kinases , Oxidative Stress , Reactive Oxygen Species , Signal Transduction , SwineABSTRACT
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.
Subject(s)
Genes, Tumor Suppressor , MAP Kinase Kinase 4 , Mitogen-Activated Protein Kinase Kinases , Neoplasm Proteins/deficiency , Neoplasms/genetics , Protein Serine-Threonine Kinases/physiology , Protein-Tyrosine Kinases/physiology , DNA, Neoplasm/genetics , Genotype , Heat-Shock Proteins/metabolism , Humans , Lung Neoplasms/enzymology , Lung Neoplasms/genetics , Lung Neoplasms/pathology , Neoplasm Proteins/genetics , Neoplasm Proteins/metabolism , Neoplasms/enzymology , Neoplasms/pathology , Pancreatic Neoplasms/enzymology , Pancreatic Neoplasms/genetics , Pancreatic Neoplasms/pathology , Phosphorylation , Protein Processing, Post-Translational , Protein Serine-Threonine Kinases/deficiency , Protein Serine-Threonine Kinases/genetics , Protein-Tyrosine Kinases/deficiency , Protein-Tyrosine Kinases/genetics , Sequence Deletion , Signal Transduction , Tumor Cells, CulturedABSTRACT
c-Jun N-terminal kinases/stress-activated protein kinases (JNKs/SAPKs) are mitogen-activated protein kinase (MAPK)-related protein kinases that are involved in several cellular events, including growth, differentiation, and apoptosis. Mixed lineage kinases (MLKs) form a family of protein kinases sharing two leucine zipper-like motifs and a kinase domain whose primary structure is similar to both the tyrosine-specific and the serine/threonine-specific kinase classes. We have reported that a member of the MLK family, MUK/DLK/ZPK, can activate JNK/SAPK in vivo, and here we show that another member of the MLK family, MST/MLK2, activates JNK/SAPK. Both MUK/DLK/ZPK and MST/MLK2 cause a slight activation of p38/Mpk2 when overexpressed in COS-1 cells, whereas MST/MLK2, but not MUK/DLK/ZPK, activates extracellular response kinase (ERK) to a certain degree. The activity of SEK1/MKK4/JNKK, a MAPK kinase class protein kinase designated as a direct activator of JNK/SAPK, is also induced by MUK/DLK/ZPK or MST/MLK2 overexpression. Furthermore, recombinant MST/MLK2 produced in bacteria directly phosphorylates and activates SEK1/MKK4/JNKK in vitro, showing that MST/MLK2 acts like a MAPK kinase kinase. Taken together, these results suggest that MLK family members are MAPK kinase kinases preferentially acting on the JNK/SAPK pathway.
Subject(s)
Calcium-Calmodulin-Dependent Protein Kinases/metabolism , MAP Kinase Kinase 4 , Mitogen-Activated Protein Kinase Kinases , Mitogen-Activated Protein Kinases , Protein Kinases/metabolism , Protein Serine-Threonine Kinases/metabolism , Animals , COS Cells , Enzyme Activation , JNK Mitogen-Activated Protein Kinases , Phosphorylation , Recombinant Proteins/metabolismABSTRACT
Antimitogenic stimuli such as environmental or genotoxic stress, transforming growth factor-beta, and the inflammatory cytokines tumor necrosis factor and interleukin-1 activate two extracellular signal-regulated kinase (ERK)-based signaling pathways: the stress-activated protein kinase (SAPK/JNK) pathway and the p38 pathway. Activated p38 phosphorylates transcription factors important in the regulation of cell growth and apoptosis, including activating transcription factor 2 (ATF2), Max, cAMP response element-binding protein-homologous protein/growth arrest DNA damage 153 (CHDP/GADD153). In turn, p38 lies downstream of the Rho family GTPases Cdc42Hs and Rac1, as well as at least three mitogen-activated protein kinase (MAPK)/ERK-kinases (MEKs): MAPK kinases-3, -6, and SAPK/ERK-kinase-1. Although many of the stimuli that activate p38 can also inhibit cell cycle progression, a clear-cut role for the p38 pathway in cell cycle regulation has not been established. Using a quantitative microinjection approach, we show here that Cdc42Hs, but not Rac1 or RhoA, can inhibit cell cycle progression at G1/S through a mechanism requiring activation of p38. These results suggest a novel role for Cdc42Hs in cell cycle inhibition. Furthermore, these results suggest that although both Cdc42Hs and Rac1 can activate p38 in situ, the effects of Cdc42Hs and Rac1 on cell cycle progression are, in fact, quite distinct.
Subject(s)
Calcium-Calmodulin-Dependent Protein Kinases/metabolism , Cell Cycle Proteins/metabolism , Cell Cycle , GTP Phosphohydrolases/metabolism , GTP-Binding Proteins/metabolism , Mitogen-Activated Protein Kinases , Signal Transduction , 3T3 Cells , Animals , G1 Phase , Gene Expression Regulation, Enzymologic , Mice , S Phase , cdc42 GTP-Binding Protein , p38 Mitogen-Activated Protein Kinases , rac GTP-Binding ProteinsABSTRACT
SEK-1, a dual specificity protein kinase that serves as one of the immediate upstream activators of the stress-activated protein kinases (SAPKs), associates specifically with the actin-binding protein, ABP-280, in vitro and in situ. SEK-1 binds to the carboxyl-terminal rod segment of ABP-280, upstream of the ABP carboxyl-terminal dimerization domain. Activation of SEK-1 in situ increases the SEK-1 activity bound to ABP-280 without changing the amount of SEK-1 polypeptide bound. The influence of ABP-280 on SAPK regulation was evaluated in human melanoma cells that lack ABP-280 expression, and in stable transformants of these cells expressing wild type ABP, or an actin-binding but dimerization-deficient mutant ABP (ABPDeltaCT109). ABP-280-deficient cells show an activation of SAPK in response to most stimuli that is comparable to that seen in ABP-280-replete cells; ABP-280-deficient cells, however, fail to show the brisk tumor necrosis factor-alpha (TNF-alpha) activation of SAPK seen in ABP-replete cells and have an 80% reduction in SAPK activation by lysophosphatidic acid. Expression of the dimerization-deficient mutant ABP-280 fails to correct the defective SAPK response to lysophosphatidic acid, but essentially normalizes the TNF-alpha activation of SAPK. Thus, a lack of ABP-280 in melanoma cells causes a defect in the regulation of SAPK that is selective for TNF-alpha and is attributable to the lack of ABP-280 polypeptide itself rather than to the disordered actin cytoskeleton that results therefrom. ABP-280 participates in TNF-alpha signal transduction to SAPKs, in part through the binding of SEK-1.
Subject(s)
Contractile Proteins/metabolism , MAP Kinase Kinase 4 , Microfilament Proteins/metabolism , Mitogen-Activated Protein Kinase Kinases , Protein Kinases/metabolism , Tumor Necrosis Factor-alpha/pharmacology , Actins/metabolism , Animals , Anisomycin/pharmacology , B-Lymphocytes/immunology , B-Lymphocytes/metabolism , Cell Line , Contractile Proteins/isolation & purification , Dimerization , Enzyme Activation , Epidermal Growth Factor/pharmacology , Filamins , Glutathione Transferase/metabolism , Humans , Melanoma , Mice , Microfilament Proteins/isolation & purification , Mutagenesis, Site-Directed , Phosphorylation , Recombinant Fusion Proteins/metabolism , T-Lymphocytes/immunology , T-Lymphocytes/metabolism , Transfection , Tumor Cells, CulturedABSTRACT
SPRK (also called PTK-1 and MLK-3), a member of the mixed lineage kinase subfamily of (Ser/Thr) protein kinases, encodes an amino-terminal SH3 domain followed by a kinase catalytic domain, two leucine zippers interrupted by a short spacer, a Rac/Cdc42 binding domain, and a long carboxyl-terminal proline-rich region. We report herein that SPRK activates the stress-activated protein kinases (SAPKs) but not ERK-1 during transient expression in COS cells; the p38 kinase is activated modestly (1.3-2 fold) but consistently. SPRK also activates cotransfected SEK-1/MKK-4, a dual specificity kinase which phosphorylates and activates SAPK. Reciprocally, expression of mutant, inactive SEK-1 inhibits completely the basal and SPRK-activated SAPK activity. Immunoprecipitated recombinant SPRK is able to phosphorylate and activate recombinant SEK-1 in vitro to an extent comparable to that achieved by MEK kinase-1. These results identify SPRK as a candidate upstream activator of the stress-activated protein kinases, acting through the phosphorylation and activation of SEK-1.