Signal transduction pathways regulated by mitogen-activated/extracellular response kinase kinase kinase induce cell death.

Mitogen-activated/extracellular response kinase kinase (MEK) kinase (MEKK) is a serine-threonine kinase that regulates sequential protein phosphorylation pathways, leading to the activation of mitogen-activated protein kinases (MAPK), including members of the Jun kinase (JNK)/stress-activated protein kinase (SAPK) family. In Swiss 3T3 and REF52 fibroblasts, activated MEKK induces cell death involving cytoplasmic shrinkage, nuclear condensation, and DNA fragmentation characteristic of apoptosis. Expression of activated MEKK enhanced the apoptotic response to ultraviolet irradiation, indicating that MEKK-regulated pathways sensitize cells to apoptotic stimuli. Inducible expression of activated MEKK stimulated the transactivation of c-Myc and Elk-1. Activated Raf, the serine-threonine protein kinase that activates the ERK members of the MAPK family, stimulated Elk-1 transactivation but not c-Myc; expression of activated Raf does not induce any of the cellular changes associated with MEKK-mediated cell death. Thus, MEKK selectively regulates signal transduction pathways that contribute to the apoptotic response.

MEKK1 phosphorylates MEK1 and MEK2 but does not cause activation of mitogen-activated protein kinase.

A constitutively active fragment of rat MEK kinase 1 (MEKK1) consisting of only its catalytic domain (MEKK-C) expressed in bacteria quantitatively activates recombinant mitogen-activated protein (MAP) kinase/extracellular signal-regulated protein kinase (ERK) kinases 1 and 2 (MEK1 and MEK2) in vitro. Activation of MEK1 by MEKK-C is accompanied by phosphorylation of S218 and S222, which are also phosphorylated by the protein kinases c-Mos and Raf-1. MEKK1 has been implicated in regulation of a parallel but distinct cascade that leads to phosphorylation of N-terminal sites on c-Jun; thus, its role in the MAP kinase pathway has been questioned. However, in addition to its capacity to phosphorylate MEK1 in vitro, MEKK-C interacts with MEK1 in the two-hybrid system, and expression of mouse MEKK1 or MEKK-C in mammalian cells causes constitutive activation of both MEK1 and MEK2. Neither cotransfected nor endogenous ERK2 is highly activated by MEKK1 compared to its stimulation by epidermal growth factor in spite of significant activation of endogenous MEK. Thus, other as yet undefined mechanisms may be involved in determining information flow through the MAP kinase and related pathways.

Regulation of recombinant MEK1 and MEK2b expressed in Escherichia coli.

Mitogen-activated protein kinase (MAPK) activation is an important signal involved in regulating cellular proliferation and/or differentiation. The immediate upstream kinase MAPK kinase, referred to as MEK, activates MAPK by phosphorylation on specific tyrosine and threonine residues. To date, two MEK's have been cloned and characterized, MEK1 and MEK2. Here we report the cloning of MEK2b from mouse pituitary cDNA. Rat and mouse MEK2 amino acid sequences vary by only three amino acids; the three changes are conserved in the MEK1 sequence. Analysis of recombinant MEK2b and MEK1 demonstrated similar activation by upstream kinases and phosphotransferase activity toward MAPK, while they differed in autophosphorylation and the ability to serve as a substrate for MAPK. The findings demonstrate significant differences in potential regulatory mechanisms of MEK1 and MEK2/2b but not in their activation by upstream regulators.

Direct interaction between Ras and the kinase domain of mitogen-activated protein kinase kinase kinase (MEKK1).

Mitogen-activated protein kinase kinase kinase (MEKK1) is a serine-threonine kinase that regulates sequential protein kinase pathways involving stress-activated protein kinases and mitogen-activated protein kinases. MEKK1 is activated in response to growth factor stimulation of cells and by expression of activated Ras. We demonstrate that the kinase domain of MEKK1 (MEKKCOOH) binds to GST-RasV12 in a GTP-dependent manner. Purified bacterially expressed MEKKCOOH binds to GST-RasV12(GTP gamma S) (GTP gamma S is guanosine 5'-3-O-(thio)triphosphate), demonstrating a direct interaction of the two proteins. A Ras effector domain peptide blocks the binding of MEKKCOOH to GST-RasV12(GTP gamma S). MEKKCOOH complexed with GST-RasV12(GTP gamma S) is capable of phosphorylating MEK1. These findings indicate that MEKK1 directly binds Ras.GTP. Thus, Ras interacts with protein kinases of both the Raf and MEKK families.

Identification of a dual specificity kinase that activates the Jun kinases and p38-Mpk2.

One Ras-dependent protein kinase cascade leading from growth factor receptors to the ERK (extracellular signal-regulated kinases) subgroup of mitogen-activated protein kinases (MAPKs) is dependent on the protein kinase Raf-1, which activates the MEK (MAPK or ERK kinase) dual specificity kinases. A second protein kinase cascade leading to activation of the Jun kinases (JNKs) is dependent on MEKK (MEK kinase). A dual-specificity kinase that activates JNK, named JNKK, was identified that functions between MEKK and JNK. JNKK activated the JNKs but did not activate the ERKs and was unresponsive to Raf-1 in transfected HeLa cells. JNKK also activated another MAPK, p38 (Mpk2; the mammalian homolog of HOG1 from yeast), whose activity is regulated similarly to that of the JNKs.

Tumor necrosis factor alpha rapidly activates the mitogen-activated protein kinase (MAPK) cascade in a MAPK kinase kinase-dependent, c-Raf-1-independent fashion in mouse macrophages.

Tumor necrosis factor alpha (TNF alpha) is bound by two cell surface receptors, CD120a (p55) and CD120b (p75), that belong to the TNF/nerve growth factor receptor family and whose signaling is initiated by receptor multimerization in the plane of the plasma membrane. The initial signaling events activated by receptor crosslinking are unknown, although activation of the mitogen-activated protein kinase (MAPK) cascade occurs shortly after ligand binding to CD120a. In this study, we investigated the upstream kinases that mediate the activation of the 42-kDa MAPK p42mapk/erk2 following crosslinking of CD120a in mouse macrophages. Exposure of mouse macrophages to TNF alpha stimulated a time-dependent increase in the activity of MAPK/ERK kinase (MEK) that temporally preceded peak activation of p42mapk/erk2. MEKs, dual-specificity threonine/tyrosine kinases, act as a convergence point for several signaling pathways including Ras/Raf, MEK kinase (MEKK), and Mos. Incubation of macrophages with TNF alpha was found to transiently stimulate a MEKK that peaked in activity within 30 sec of exposure and progressively declined toward basal levels by 5 min. By contrast, under these conditions, activation of either c-Raf-1 or Raf-B was not detected. These data suggest that the activation of the MAPK cascade in response to TNF alpha is mediated by the sequential activation of a MEKK and a MEK in a c-Raf-1- and Raf-B-independent fashion.

Differential activation of ERK and JNK mitogen-activated protein kinases by Raf-1 and MEKK.

Growth factors activate mitogen-activated protein kinases (MAPKs), including extracellular signal-regulated kinases (ERKs) and Jun kinases (JNKs). Although the signaling cascade from growth factor receptors to ERKs is relatively well understood, the pathway leading to JNK activation is more obscure. Activation of JNK by epidermal growth factor (EGF) or nerve growth factor (NGF) was dependent on H-Ras activation, whereas JNK activation by tumor necrosis factor alpha (TNF-alpha) was Ras-independent. Ras activates two protein kinases, Raf-1 and MEK (MAPK, or ERK, kinase) kinase (MEKK). Raf-1 contributes directly to ERK activation but not to JNK activation, whereas MEKK participated in JNK activation but caused ERK activation only after overexpression. These results demonstrate the existence of two distinct Ras-dependent MAPK cascades--one initiated by Raf-1 leading to ERK activation, and the other initiated by MEKK leading to JNK activation.

Ras-dependent growth factor regulation of MEK kinase in PC12 cells.

Mitogen-activated protein kinases (MAPKs) are rapidly activated in response to stimulation of diverse receptor types. MAPKs are positively regulated by phosphorylation on threonine and tyrosine by MAP kinase or extracellular signal-regulated kinase (ERK) kinases (MEKs). MEK kinase (MEKK) is part of a family of serine-threonine protein kinases that phosphorylate and activate MEKs independently of Raf. MEKK was rapidly and persistently activated in response to stimulation of resting PC12 cells with epidermal growth factor (EGF). Nerve growth factor (NGF) and 12-O-tetradecanoylphorbol-13-acetate (TPA) also activated MEKK, although to a lesser degree than did EGF. Activation of MEKK and B-Raf in response to EGF was inhibited by expression of dominant negative N17Ras. Expression of oncogenic Ras resulted in activation of MEKK. Stimulation of synthesis of cyclic adenosine 3',5'-monophosphate abolished activation of MEKK and B-Raf by EGF, NGF, and TPA. Thus, Ras simultaneously controls the activation of members of the Raf and MEKK families of protein kinases.

Mammalian mitogen-activated protein kinase kinase kinase (MEKK) can function in a yeast mitogen-activated protein kinase pathway downstream of protein kinase C.

Mitogen-activated protein kinase cascades are conserved in fungal, plant, and metazoan species. We expressed murine MAP kinase kinase kinase (MEKK) in the yeast Saccharomyces cerevisiae to determine whether this kinase functions as a general or specific activator of genetically and physiologically distinct MAP-kinase-dependent signaling pathways and to investigate how MEKK is regulated. Expression of MEKK failed to correct the mating deficiency of a ste11 delta mutant that lacks an MEKK homolog required for mating. MEKK expression also failed to induce expression of a reporter gene controlled by the HOG1 gene product (Hog1p), a yeast MAP kinase homolog involved in response to osmotic stress. Expression of MEKK did correct the cell lysis defect of a bck1 delta mutant that lacks an MEKK homolog required for cell-wall assembly. MEKK required the downstream MAP kinase homolog in the BCK1-dependent pathway, demonstrating that it functionally replaces the BCK1 gene product (Bck1p) rather than bypassing the pathway. MEKK therefore selectively activates one of three distinct MAP-kinase-dependent pathways. Possible explanations for this selectivity are discussed. Expression of the MEKK catalytic domain, but not the full-length molecule, corrected the cell-lysis defect of a pkc1 delta mutant that lacks a protein kinase C homolog that functions upstream of Bck1p. MEKK therefore functions downstream of the PKC1 gene product (Pkc1p). The N-terminal noncatalytic domain of MEKK, which contains several consensus protein kinase C phosphorylation sites, may, therefore, function as a negative regulatory domain. Protein kinase C phosphorylation may provide one mechanism for activating MEKK.

How does the G protein, Gi2, transduce mitogenic signals?

Serpentine receptors coupled to the heterotrimeric G protein, Gi2, are capable of stimulating DNA synthesis in a variety of cell types. A common feature of the Gi2-coupled stimulation of DNA synthesis is the activation of the mitogen-activated protein kinases (MAPKs). The regulation of MAPK activation by the Gi2-coupled thrombin and acetylcholine muscarinic M2 receptors occurs by a sequential activation of a network of protein kinases. The MAPK kinase (MEK) which phosphorylates and activates MAPK is also activated by phosphorylation. MEK is phosphorylated and activated by either Raf or MEK kinase (MEKK). Thus, Raf and MEKK converge at MEK to regulate MAPK. Gi2-coupled receptors are capable of activating MEK and MAPK by Raf-dependent and Raf-independent mechanisms. Pertussis toxin catalyzed ADP-ribosylation of alpha i2 inhibits both the Raf-dependent and -independent pathways activated by Gi2-coupled receptors. The Raf-dependent pathway involves Ras activation, while the Raf-independent activation of MEK and MAPK does not involve Ras. The Raf-independent activation of MEK and MAPK most likely involves the activation of MEKK. The vertebrate MEKK is homologous to the Ste11 and Byr2 protein kinases in the yeast Saccharomyces cerevisiae and Schizosaccharomyces pombe, respectively. The yeast Ste11 and Byr2 protein kinases are involved in signal transduction cascades initiated by pheromone receptors having a 7 membrane spanning serpentine structure coupled to G proteins. MEKK appears to be conserved in the regulation of G protein-coupled signal pathways in yeast and vertebrates. Raf represents a divergence in vertebrates from the yeast pheromone-responsive protein kinase system.(ABSTRACT TRUNCATED AT 250 WORDS)

MEK-1 phosphorylation by MEK kinase, Raf, and mitogen-activated protein kinase: analysis of phosphopeptides and regulation of activity.

MEK-1 is a dual threonine and tyrosine recognition kinase that phosphorylates and activates mitogen-activated protein kinase (MAPK). MEK-1 is in turn activated by phosphorylation. Raf and MAPK/extracellular signal-regulated kinase kinase (MEKK) independently phosphorylate and activate MEK-1. Recombinant MEK-1 is also capable of autoactivation. Purified recombinant wild type MEK-1 and a mutant kinase inactive MEK-1 were used as substrates for MEKK, Raf, and autophosphorylation. MEK-1 phosphorylation catalyzed by Raf, MEKK, or autophosphorylation resulted in activation of MEK-1 kinase activity measured by phosphorylation of a mutant kinase inactive MAPK. Phosphoamino acid analysis and peptide mapping identified similar MEK-1 tryptic phosphopeptides after phosphorylation by MEK kinase, Raf, or MEK-1 autophosphorylation. MEK-1 is phosphorylated by MAPK at sites different from that for Raf and MEKK. Phosphorylation of MEK-1 by MAPK does not affect MEK-1 kinase activity. Several phosphorylation sites present in MEK-1 immunoprecipitated from 32P-labeled cells after stimulation with epidermal growth factor were common to the in vitro phosphorylated enzyme. The major site of MAPK phosphorylation in MEK-1 is threonine 292. Mutation of threonine 292 to alanine eliminates 90% of MAPK catalyzed phosphorylation of MEK-1 but does not influence MEK-1 activity. The results demonstrate that MEKK and Raf regulate MEK-1 activity by phosphorylation of common residues and thus, two independent protein kinases converge at MEK-1 to regulate the activity of MAPK.

A divergence in the MAP kinase regulatory network defined by MEK kinase and Raf.

Mitogen-activated protein kinases (MAPKs) are rapidly phosphorylated and activated in response to various extracellular stimuli in many different cell types. Such regulation of MAPK results from sequential activation of a series of protein kinases. The kinases that phosphorylate MAPKs, the MAP kinase kinases (MEKs) are also activated by phosphorylation. MEKs are related in sequence to the yeast protein kinases Byr1 (from Schizosaccharomyces pombe) and Ste7 (from Saccharomyces cerevisiae), which function in the pheromone-induced signaling pathway that results in mating. Byr1 and Ste7 are in turn regulated by the protein kinases Byr2 and Ste11. The amino acid sequence of the mouse homolog of Byr2 and Ste11, denoted MEKK (MEK kinase), was elucidated from a complementary DNA sequence encoding a protein of 672 amino acid residues (73 kilodaltons). MEKK was expressed in all mouse tissues tested, and it phosphorylated and activated MEK. Phosphorylation and activation of MEK by MEKK was independent of Raf, a growth factor-regulated protein kinase that also phosphorylates MEK. Thus, MEKK and Raf converge at MEK in the protein kinase network mediating the activation of MAPKs by hormones, growth factors, and neurotransmitters.

8-Chloroadenosine mediates 8-chloro-cyclic AMP-induced down-regulation of cyclic AMP-dependent protein kinase in normal and neoplastic mouse lung epithelial cells by a cyclic AMP-independent mechanism.

The 8-chloro analogue of the regulatory molecule, cyclic AMP (cAMP), modulates the intracellular concentrations of cAMP-dependent protein kinases (PKA) and inhibits both in vitro and in vivo growth of several neoplastic cell types. Because 8-chloro-cyclic AMP (8-Cl-cAMP) can be converted to 8-chloroadenosine (8-Cl-adenosine) by serum enzymes contained in cell growth media, we tested whether 8-Cl-cAMP effects were mediated by its adenosine metabolite in normal and neoplastic cell lines of mouse lung epithelial origin. 8-Cl-adenosine, directly added to cells or derived from exogenously applied 8-Cl-cAMP, specifically decreased the intracellular concentration of the type I isozyme of cAMP-dependent protein kinase (PKA I). 8-Cl-adenosine and 8-Cl-cAMP were equipotent at inhibiting cell growth, and elicited similar changes in the proportion of cells in the G1, S, and G2-M phases of the cell cycle. The presence of adenosine deaminase, which converts 8-Cl-adenosine to 8-chloroinosine, completely prevented growth inhibition by 8-Cl-cAMP and the concomitant diminution of PKA I. 8-Cl-cAMP had no discernible effect on cells when its conversion into 8-Cl-adenosine was prevented by 3-isobutyl-1-methyl-xanthene, an inhibitor of phosphodiesterase. 6-(p-Nitrobenzyl)-thioinosine, an inhibitor of adenosine uptake, protected cells from cytostasis, indicating that 8-Cl-adenosine acts intracellularly. 8-Cl-adenosine greatly decreased RI (regulatory subunit of PKA I) and PKA catalytic (C) subunit protein concentrations without affecting RII (regulatory subunit of the PKA type II isozyme) or intracellular cAMP levels. Northern blot analysis of PKA subunit mRNAs following treatment of each cell line with 8-Cl-adenosine demonstrated decreased C alpha mRNA expression, increased RII alpha mRNA, and no change in RI alpha mRNA abundance. Our results indicate that 8-Cl-adenosine inhibits lung cell growth and induces PKA I down-regulation via a cAMP-independent mechanism.

Differential responsiveness to agents which stimulate cAMP production in normal versus neoplastic mouse lung epithelial cells.

We examined the responsiveness of normal and neoplastic lung cells to agents which stimulate cAMP production. While their basal cAMP levels were similar, spontaneous in vitro transformant E9 cells and tumor-derived PCC4 cells produced much less cAMP in response to 1 microM isoproterenol compared to non-tumorigenic C10 cells derived from normal mouse lung epithelium. Iodocyanopindolol binding studies indicated that both neoplastic lines contained fewer beta-adrenergic receptors than normal C10 cells. When receptors were bypassed via treatment with 10 pM cholera toxin, the pattern of cAMP-responsiveness was reversed; both neoplastic cell lines produced more cAMP than C10 cells. Direct stimulation of adenylate cyclase with 100 microM forskolin greatly increased cAMP concentrations in all three cell lines. These anomalies at both the receptor and G-protein levels in neoplastic lung epithelial cells may contribute to their deregulated growth.

Differential regulation of the stability of cyclic AMP-dependent protein kinase messenger RNA in normal versus neoplastic mouse lung epithelial cells.

Neoplastic mouse lung epithelial cells contain greatly diminished activity, protein, and mRNA for the type I isozyme of cyclic AMP-dependent protein kinase (PKA I), while expression of the type II isozyme (PKA II) is similar to that of normal lung cells. A time course of PKA mRNA content in transcriptionally inhibited cells indicated that most PKA mRNAs are more stable in the neoplastic E9 cell line than in related nontumorigenic C10 cells. To address the basis of this differential stability, we treated both cell lines with cycloheximide, an inhibitor of protein synthesis, in the presence or absence of the transcriptional inhibitor, 5,6-dichloro-1-b-ribofuranosyl-benzimidazole (DRB). The rate of PKA II regulatory subunit alpha mRNA decay in the presence of DRB was unaffected by cycloheximide treatment in E9 cells but decreased upon the addition of cycloheximide to DRB-treated C10 cells. The combination of these two agents markedly destabilized PKA II mRNAs (PKA catalytic subunit alpha and PKA II regulatory subunit alpha) relative to DRB treatment alone in neoplastic E9 cells, causing them to decay at a rate equal to that in C10 cells. PKA II mRNA may be specifically stabilized by a protein with a relatively short half-life in neoplastic E9 cells. These results suggest the involvement of tumor-specific factor(s) in the regulation of PKA mRNA stability, a potential mechanism for conferring the observed differential responsiveness of normal and neoplastic lung cells to cyclic AMP.

Altered regulation of mRNA levels encoding the type I isozyme of cAMP-dependent protein kinase in neoplastic mouse lung epithelial cells.

Neoplastic mouse lung epithelial cells express greatly diminished activity, protein, and mRNA for the type I isozyme of cAMP-dependent protein kinase (PKA I). To address the mechanism of this decrease, the turnover rate of PKA subunit mRNA was examined. Northern blot analysis of PKA mRNAs from transcriptionally inhibited cells indicated that these messages exhibit different stabilities and that they are more stable in neoplastic E9 cells than in normal C10 cells. This suggests that the mechanism of decreased PKA I mRNA in E9 cells resides at the level of transcription. To examine whether this was due to an altered responsiveness to agents which regulate PKA transcription, PKA levels were experimentally manipulated in C10 and E9 cells by long-term treatment with forskolin or 8-chloro-cAMP. PKA activity and the concentration of RI (regulatory subunit of PKA I) and C (catalytic subunit) are coordinately regulated in both cell lines, but this does not reflect the changes in PKA I subunit mRNAs. RI alpha mRNA is specifically induced by forskolin in normal C10 cells, but not in E9 cells. C alpha mRNA is forskolin-inducible in E9 cells, but this enhanced level of expression does not approach that found constitutively in C10 cells. Thus, while C10 and E9 undergo similar changes in PKA I protein subunits following these treatments, a differential modulation of their PKA I mRNA occurs. These cell-specific mRNA responses to cAMP-mediated induction suggest that the mechanism of the decreased constitutive concentration of PKA I in E9 cells involves altered regulation of PKA I mRNAs.

Alterations in the cAMP signal transduction pathway in mouse lung tumorigenesis.

Alterations in the cAMP signal transduction pathway are associated with mouse lung neoplasia, cAMP effects are mediated by activating cAMP-dependent protein kinase isozymes, PKA I and PKA II. E9, a tumorigenic cell line, exhibited decreased PKA I levels compared to C10 cells, a nontumorigenic cell line of similar epithelial origin. Western immunoblots of PKA subunit proteins demonstrated low concentrations of both the catalytic (C) and regulatory (RI) PKA I subunits. Although RII (regulatory subunit of PKA II) concentrations were similar in both cell lines, RII from E9 cells was more highly phosphorylated than in C10 cells. RII phosphorylation status regulates cAMP activation of PKA II. Northern-blot analysis of mRNA content indicated diminished expression of both C and RI mRNA in E9 relative to C10 cells. Several endogenous PKA substrate proteins present in C10 cells were minimally phosphorylated by PKA in E9 cells. Forskolin, which raises cellular cAMP content, increased phosphorylation of a protein doublet in intact C10 cells, but not in E9 cells. Decreased PKA I expression and alterations in RII phosphorylation in lung neoplasia may contribute to anomalous regulation by cAMP, thereby diminishing cAMP-mediated growth inhibitory effects.