Functional binding between Gbeta and the LIM domain of Ste5 is required to activate the MEKK Ste11

BACKGROUND: In the budding yeast Saccharomyces cerevisiae, the pheromones that induce haploid cells of opposite cell types to mate activate the Gbeta and Ggamma subunits of a heterotrimeric G protein. These subunits signal through the PAK kinase Ste20 to activate a mitogen-activated protein (MAP) kinase cascade comprising the MEKK Ste11, the MEK Ste7 and two MAP kinases, Fus3 and Kss1. The pathway requires Ste5, a scaffold protein that tethers the MAP kinase cascade enzymes into a high molecular weight complex. Ste5 is thought to associate with Gbeta in a pheromone-independent manner, but it is not known if this interaction affects signaling. RESULTS: A ste5C180A mutant - which expresses Ste5 disrupted in the LIM domain, a putative metal-binding motif that has been proposed to be essential for Ste5 oligomerization - could not transmit the pheromone signal from Gbeta through Ste20 to Ste11. The Ste5C180A protein was impaired in binding Gbeta, although it could oligomerize, bind Ste11, Ste7 and Fus3, facilitate the basal activation of Ste11, and relay the Ste11 signal to MAP kinases. Ste5 bound to Gbeta in a pheromone-dependent manner and preferentially associated with a phosphorylated form of Gbeta in wild-type and ste20Delta, but not in ste5C180A, strains. CONCLUSIONS: Pheromone induces binding of Gbeta to Ste5 through its LIM domain. This binding is essential for activation of Ste11 and is distinct from the ability of Ste5 to oligomerize or to serve as a scaffold and relay the signal from Ste11 to the MAP kinases. Pheromone also induces Ste5-dependent phosphorylation of Gbeta.     

Ste5 RING-H2 domain: role in Ste4-promoted oligomerization for yeast pheromone signaling

Ste5 is a scaffold for the mitogen-activated protein kinase (MAPK) cascade components in a yeast pheromone response pathway. Ste5 also associates with Ste4, the beta subunit of a heterotrimeric guanine nucleotide-binding protein, potentially linking receptor activation to stimulation of the MAPK cascade. A RING-H2 motif at the Ste5 amino terminus is apparently essential for function because Ste5(C177S) and Ste5(C177A C180A) mutants did not rescue the mating defect of a ste5Delta cell. In vitro Ste5(C177A C180A) bound each component of the MAPK cascade, but not Ste4. Unlike wild-type Ste5, the mutant did not appear to oligomerize; however, when fused to a heterologous dimerization domain (glutathione S-transferase), the chimeric protein restored mating in an ste5Delta cell and an ste4Delta ste5Delta double mutant. Thus, the RING-H2 domain mediates Ste4-Ste5 interaction, which is a prerequisite for Ste5-Ste5 self-association and signaling.     

Role of MEKK1 in cell survival and activation of JNK and ERK pathways defined by targeted gene disruption

Targeted disruption of the gene encoding MEK kinase 1 (MEKK1), a mitogen-activated protein kinase (MAPK) kinase kinase, defined its function in the regulation of MAPK pathways and cell survival. MEKK1(-/-) embryonic stem cells from mice had lost or altered responses of the c-Jun amino-terminal kinase (JNK) to microtubule disruption and cold stress but activated JNK normally in response to heat shock, anisomycin, and ultraviolet irradiation. Activation of JNK was lost and that of extracellular signal-regulated protein kinase (ERK) was diminished in response to hyperosmolarity and serum factors in MEKK1(-/-) cells. Loss of MEKK1 expression resulted in a greater apoptotic response of cells to hyperosmolarity and microtubule disruption. When activated by specific stresses that alter cell shape and the cytoskeleton, MEKK1 signals to protect cells from apoptosis.     

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.     

MEKK1 binds directly to the c-Jun N-terminal kinases/stress-activated protein kinases

Mitogen-activated protein (MAP) kinases mediate responses to a wide array of cellular stimuli. These cascades consist of a MAP kinase or extracellular signal-regulated kinase (ERK), activated by a MAP/ERK kinase (MEK), in turn activated by a MEK kinase (MEKK). MEKK1 has been shown to be a strong activator of the c-Jun N-terminal kinase/stress-actived protein kinase (JNK/SAPK) pathway. We report here that JNK/SAPK binds directly to the N-terminal, noncatalytic domain of MEKK1 in vitro and in transfected cells. Immobilized MEKK1-derived peptides extract JNK/SAPK selectively from cell lysates. MEKK1 coimmunoprecipitates with multiple JNK/SAPK isoforms in transfected cells. Expression of the N terminus of MEKK1 lacking the kinase domain increases activation of endogenous JNK/SAPK by MEKK1. The data are consistent with a model in which MEKK1-JNK/SAPK binding facilitates the receipt of signals from upstream inputs and localizes JNK/SAPK to intracellular targets of the pathway.     

Mammalian mitogen-activated protein kinase pathways are regulated through formation of specific kinase-activator complexes

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.     

Stimulation of Jun N-terminal kinase (JNK) by gonadotropin-releasing hormone in pituitary alpha T3-1 cell line is mediated by protein kinase C, c-Src, and CDC42

The signaling of ligands operating via heterotrimeric G proteins is mediated by a complex network that involves sequential phosphorylation events. Signaling by the G protein-coupled receptor GnRH was shown to include elevation of Ca2+ and activation of phospholipases, protein kinase C (PKC) and extra-cellular signal-regulated kinase (ERK). In this study, GnRH was shown to activate Jun N-Terminal Kinase (JNK)/SAPK in alpha T3-1 cells in a PKC- and tyrosine kinase-dependent manner. GnRH as well as tumor-promoting agent (TPA) also increased c-Src activity, which peaked at 2 min after GnRH stimulation and was sensitive both to PKC and to tyrosine kinase inhibitors. Coexpression of Csk, which serves as a Src-dominant interfering kinase, and constitutively active forms of Src, together with JNK, confirmed the involvement of c-Src downstream of PKC in the GnRH-JNK pathway. Coexpression of dominant negative and constitutively active forms of CDC42, Rac1, Ras, MEKK1, and MEK1 with JNK indicated that JNK activation by GnRH and TPA is mediated by CDC42 and MEKK1. Ras and MEK1, which are involved in a related mitogen-activated protein kinase (MAPK) pathway, did not affect JNK activation in alpha T3-1 cells. Taken together, our results suggest that GnRH stimulation of JNK activity is mediated by a unique pathway that includes sequential activation of PKC, c-Src, CDC42, and probably also MEKK1.     

A pilot study of a short cognitive-behavioral group treatment for female recurrent suicide attempters

The SLT2(MPK1) mitogen-activated protein kinase signal transduction pa thway has been implicated in several biological processes in Saccharomyces cerevisiae, including the regulation of cytoskeletal and cell wall structure, polarized cell growth, and response to nutrient availability, hypo-osmotic shock and heat shock. We examined the conditions under which the SLT2 pathway is activated. We found that the SLT2 kinase is tyrosine phosphorylated and activated during periods in which yeast cells are undergoing polarized cell growth, namely during bud formation of vegetative cell division and during projection formation upon treatment with mating pheromone. BCK1(SLK1), a MEK kinase, is required for SLT2 activation in both of these situations. Upstream of BCK1(SLK1), we found that the STE20 kinase was required for SLT2 activation by mating pheromone, but was unnecessary for its activation during the vegetative cell cycle. Finally, SLT2 activation during vegetative growth was partially dependent on CDC28 in that the stimulation of SLT2 tyrosine phosphorylation was significantly reduced directly after a temperature shift in cdc28 ts mutants. Our data are consistent with a role for SLT2 in promoting polarized cell growth.     

Control of thrombopoietin-induced megakaryocytic differentiation by the mitogen-activated protein kinase pathway

Thrombopoietin (TPO) is the major regulator of both growth and differentiation of megakaryocytes. We previously showed that both functions can be generated by TPO in the megakaryoblastic cell line UT7, in which murine Mpl was introduced, and are independently controlled by distinct regions of the cytoplasmic domain of Mpl. Particularly, residues 71 to 94 of this domain (deleted in the mutant mpl delta3) were found to be required for megakaryocytic maturation but dispensable for proliferation. We show here that TPO-induced differentiation in UT7 cells is tightly dependent on a strong, long-lasting activation of the mitogen-activated protein kinase (MAPK) pathway. Indeed, (i) in UT7-mpl cells, TPO induced a strong activation of extracellular signal-regulated kinases (ERK) which was persistent until at least 4 days in TPO-containing medium; (ii) a specific MAPK kinase (MEK) inhibitor inhibited TPO-induced megakaryocytic gene expression; (iii) the Mpl mutant mpl delta3, which displayed no maturation activity, transduced only a weak and transient ERK activation in UT7 cells; and (iv) TPO-induced megakaryocytic differentiation in UT7-mpl delta3 cells was partially restored by expression of a constitutively activated mutant of MEK. The capacity of TPO to trigger a strong and prolonged MAPK signal depended on the cell in which Mpl was introduced. In BAF3-mpl cells, TPO triggered a weak and transient ERK activation, similar to that induced in UT7-mpl delta3 cells. In these cells, no difference in MAPK activation was found between normal Mpl and mpl delta3. Thus, depending on the cellular context, several distinct regions of the cytoplasmic domain of Mpl and signaling pathways may contribute to generate quantitative variations in MAPK activation.     

MEK kinase 1, a substrate for DEVD-directed caspases, is involved in genotoxin-induced apoptosis

MEK kinase 1 (MEKK1) is a 196-kDa protein that, in response to genotoxic agents, was found to undergo phosphorylation-dependent activation. The expression of kinase-inactive MEKK1 inhibited genotoxin-induced apoptosis. Following activation by genotoxins, MEKK1 was cleaved in a caspase-dependent manner into an active 91-kDa kinase fragment. Expression of MEKK1 stimulated DEVD-directed caspase activity and induced apoptosis. MEKK1 is itself a substrate for CPP32 (caspase-3). A mutant MEKK1 that is resistant to caspase cleavage was impaired in its ability to induce apoptosis. These findings demonstrate that MEKK1 contributes to the apoptotic response to genotoxins. The regulation of MEKK1 by genotoxins involves its activation, which may be part of survival pathways, followed by its cleavage, which generates a proapoptotic kinase fragment able to activate caspases. MEKK1 and caspases are predicted to be part of an amplification loop to increase caspase activity during apoptosis.     

Activation of the p42 mitogen-activated protein kinase pathway inhibits Cdc2 activation and entry into M-phase in cycling Xenopus egg extracts

We have added constitutively active MAP kinase/ERK kinase (MEK), an activator of the mitogen-activated protein kinase (MAPK) signaling pathway, to cycling Xenopus egg extracts at various times during the cell cycle. p42MAPK activation during entry into M-phase arrested the cell cycle in metaphase, as has been shown previously. Unexpectedly, p42MAPK activation during interphase inhibited entry into M-phase. In these interphase-arrested extracts, H1 kinase activity remained low, Cdc2 was tyrosine phosphorylated, and nuclei continued to enlarge. The interphase arrest was overcome by recombinant cyclin B. In other experiments, p42MAPK activation by MEK or by Mos inhibited Cdc2 activation by cyclin B. PD098059, a specific inhibitor of MEK, blocked the effects of MEK(QP) and Mos. Mos-induced activation of p42MAPK did not inhibit DNA replication. These results indicate that, in addition to the established role of p42MAPK activation in M-phase arrest, the inappropriate activation of p42MAPK during interphase prevents normal entry into M-phase.     

T cell proliferation in response to interleukins 2 and 7 requires p38MAP kinase activation

Interleukin-2 (IL-2) is a potent T cell mitogen. However, the signaling pathways by which IL-2 mediates its mitogenic effect are not fully understood. One of the members of the mitogen-activated protein kinase (MAPK) family, p42/44MAPK (ERK2/1), is known to be activated by IL-2. We have now investigated the response to IL-2 of two other members of the MAP kinase family, p54MAP kinase (stress-activated protein kinase (SAPK)/Jun-N-terminal kinase (JNK)) and p38MAP kinase (p38/Mpk2/CSBP/RK), which respond primarily to stressful and inflammatory stimuli (e.g. tumor necrosis factor-alpha, IL-1, and lipopolysaccharide). Here we show that IL-2, and another T cell growth factor, IL-7, activate both SAPK/JNK and p38MAP kinase. Furthermore, inhibition of p38MAP kinase activity with a specific pyrinidyl imidazole inhibitor SB203580 that prevents activation of its downstream effector, MAPK-activating protein kinase-2, correlated with suppression of IL-2- and IL-7-driven T cell proliferation. These data indicate that in T cells p38MAP kinase has a role in transducing the mitogenic signal.     

Mapping of a yeast G protein betagamma signaling interaction

The mating pathway of Saccharomyces cerevisiae is widely used as a model system for G protein-coupled receptor-mediated signal transduction. Following receptor activation by the binding of mating pheromones, G protein betagamma subunits transmit the signal to a MAP kinase cascade, which involves interaction of Gbeta (Ste4p) with the MAP kinase scaffold protein Ste5p. Here, we identify residues in Ste4p required for the interaction with Ste5p. These residues define a new signaling interface close to the Ste20p binding site within the Gbetagamma coiled-coil. Ste4p mutants defective in the Ste5p interaction interact efficiently with Gpa1p (Galpha) and Ste18p (Ggamma) but cannot function in signal transduction because cells expressing these mutants are sterile. Ste4 L65S is temperature-sensitive for its interaction with Ste5p, and also for signaling. We have identified a Ste5p mutant (L196A) that displays a synthetic interaction defect with Ste4 L65S, providing strong evidence that Ste4p and Ste5p interact directly in vivo through an interface that involves hydrophobic residues. The correlation between disruption of the Ste4p-Ste5p interaction and sterility confirms the importance of this interaction in signal transduction. Identification of the Gbetagamma coiled-coil in Ste5p binding may set a precedent for Gbetagamma-effector interactions in more complex organisms.     

Raf-1 independent stimulation of mitogen-activated protein kinase by leukemia inhibitory factor in 3T3-L1 cells

We show here that treatment of 3T3-L1 cells with leukemia inhibitory factor (LIF) stimulated Raf-1 activity in a time- and dose-dependent manner. Although phorbol ester failed to activate Raf-1 directly, a protein kinase C-stimulated signal was found to be necessary, but not sufficient, for LIF-mediated activation of Raf-1. Elevation of intracellular cAMP levels completely blocked Raf-1 activation by LIF, but was without effect on the magnitude of mitogen-activated protein kinase (MAPK) stimulation by the cytokine, suggesting the presence of a Raf-1-independent, cAMP-insensitive MAPK kinase kinase (MAPKKK) pathway in 3T3-L1 cells. Mono Q-fractionation of LIF-stimulated 3T3-L1 extracts identified a single peak of MAPKKK activity that was largely insensitive to elevated intracellular levels of cAMP, and that failed to correlate with stimulation of either Raf-1 or MEKK1 protein kinases. Our results demonstrate that LIF-mediated activation of the MAP kinase cascade in 3T3-L1 cells proceeds through both Raf-1-dependent and -independent pathways which differ in their sensitivity to inhibition by intracellular cAMP.     

Activation of the hematopoietic progenitor kinase-1 (HPK1)-dependent, stress-activated c-Jun N-terminal kinase (JNK) pathway by transforming growth factor beta (TGF-beta)-activated kinase (TAK1), a kinase mediator of TGF beta signal transduction

Transforming growth factor beta (TGF-beta)-activated kinase (TAK1) is known for its involvement in TGF-beta signaling and its ability to activate the p38-mitogen-activated protein kinase (MAPK) pathway. This report shows that TAK1 is also a strong activator of c-Jun N-terminal kinase (JNK). Both the wild-type and a constitutively active mutant of TAK1 stimulated JNK in transient transfection assays. Mitogen-activated protein kinase kinase 4 (MKK4)/stress-activated protein kinase/extracellular signal-regulated kinase (SEK1), a dual-specificity kinase that phosphorylates and activates JNK, synergized with TAK1 in activating JNK. Conversely, a dominant-negative (MKK4/SEK1 mutant inhibited TAK1-induced JNK activation. A kinasedefective mutant of TAK1 effectively suppressed hematopoietic progenitor kinase-1 (HPK1)-induced JNK activity but had little effect on germinal center kinase activation of JNK. There are two additional MAPK kinase kinases, MEKK1 and mixed lineage kinase 3 (MLK3), that are also downstream of HPK1 and upstream of MKK4/SEK mutant. However, because the dominant-negative mutants of MEKK1 and MLK3 did not inhibit TAK1-induced JNK activity, we conclude that activation of JNK1 by TAK1 is independent of MEKK1 and MLK3. In addition to TAK1, TGF-beta also stimulated JNK activity. Taken together, these results identify TAK1 as a regulator in the HPK1 --> TAK1 --> MKK4/SEK1 --> JNK kinase cascade and indicate the involvement of JNK in the TGF-beta signaling pathway. Our results also suggest the potential roles of TAK1 not only in the TGF-beta pathway but also in the other HPK1/JNK1-mediated pathways.