Phosphoinositide 3-Kinase γ Is a Mediator of Gβγ-dependent Jun Kinase Activation (original) (raw)
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Phosphoinositide 3-Kinase Is a Mediator of G-dependent Jun Kinase Activation
Jun kinases (JNK) are involved in the stress response of mammalian cells. Stimulation of JNK can be induced by stress factors and by agonists of tyrosine kinase and G protein-coupled receptors. G protein-dependent receptors stimulate JNK via G subunits of heterotrim-eric G proteins, but the subsequent signaling reaction has been undefined. Here we demonstrate JNK activation in COS-7 cells by G-stimulated phosphoinositide 3-kinase (PI3K). Signal transduction from PI3K to JNK can be suppressed by dominant negative mutants of Ras, Rac, and the protein kinase PAK. These results identify PI3K as a mediator of G-dependent regulation of JNK activity. Interaction of cells with a wide variety of agonists results in a stimulation of intracellular mitogen-activated protein kinase (MAPK) 1 cascades. MAPKs control the expression of genes that are important for the regulation of many cell functions including proliferation and differentiation. In mammalian cells three parallel MAPK pathways have been characterized so far (1). The canonical MAPK cascade composed of Raf, MEK, and ERK is regulated by the Ras GTPase in response to agonists of tyrosine kinase and G protein-coupled receptors. ERK species catalyze the phosphorylation of transcription factors including Elk-1, thus controlling the expression of several genes (2). A second MAPK cascade that is stimulated by osmotic stress regulates the activity of the protein kinase p38. Signal trans-duction to p38 and the function of this pathway are still unclear. Like p38 the elements of the Jun kinase (JNK) cascade as a third MAPK pathway are involved in the stress response of mammalian cells. JNK can be stimulated by stress factors like interleukin 1 and tumor necrosis factor but also by agonists of tyrosine kinase and G protein-coupled receptors (3, 4). Available evidences point to an involvement of the small GTPase Rac and several protein kinases in the regulation of JNK activity (5). In some cellular systems the STE 20 homologue PAK was found to act as a JNK kinase kinase kinase (6). PAK is able to activate JNK via sequential stimulation of the protein kinases MEKK and SEK. The mechanism of signal transduction from G protein-coupled receptors to the JNK cascade is only partially understood. Using COS-7 cells as a transient expression system a recent report showed the involvement of G subunits of heterotrim-eric G proteins in the stimulation of JNK by agonists of the G protein-coupled muscarinergic receptor m2 (7). Additionally the small GTPases Ras and Rac have been demonstrated to mediate signal transduction from G protein-coupled receptor to JNK, but the topology of the signaling path from G to Ras and Rac remains unknown (7-9). One candidate for the link of G protein-coupled receptor and JNK cascade is phosphoinositide 3-kinase (PI3K). This sub-species of the PI3K family is stimulated in vitro by G and recently has been shown to be involved in signal transduction from G protein-coupled receptor to the ERK path of MAPK cascades (10). We now present evidences that JNK stimulation by an agonist of the m2 muscarinergic receptor and by G also implies a PI3K. Overexpression of PI3K in COS-7 cells induces a significant increase of JNK activity. Stimulation of JNK by G can be suppressed by the PI3K inhibitor wortman-nin and a lipid kinase negative mutant of PI3K. Dominant negative mutants of Ras, Rac, and PAK significantly reduce the stimulatory effect of PI3K on JNK. Thus PI3K seems to act as an intermediate connecting G protein-coupled receptors to the JNK cascade. EXPERIMENTAL PROCEDURES Cell Culture and Transfection-COS-7 cells (ATCC) were cultured in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum. Cells were split the day before transfection. Subconfluent cells were transfected with pcDNAIII-HA-JNK or pcDNAIII-HA-MAPK and additional DNAs, following the DEAE-dextran technique, adjusting the total amount of DNA to 5 g per plate with vector DNA when necessary. Assays were performed 48 h after transfection (9). For JNK assays the cells were serum-starved for 2 h, whereas for MAPK the cells were starved overnight in serum-free medium.
Mitogen-Activated Protein Kinase Pathways Mediated by ERK, JNK, and p38 Protein Kinases
Science, 2002
Multicellular organisms have three well-characterized subfamilies of mitogenactivated protein kinases (MAPKs) that control a vast array of physiological processes. These enzymes are regulated by a characteristic phosphorelay system in which a series of three protein kinases phosphorylate and activate one another. The extracellular signal-regulated kinases (ERKs) function in the control of cell division, and inhibitors of these enzymes are being explored as anticancer agents. The c-Jun amino-terminal kinases (JNKs) are critical regulators of transcription, and JNK inhibitors may be effective in control of rheumatoid arthritis. The p38 MAPKs are activated by inflammatory cytokines and environmental stresses and may contribute to diseases like asthma and autoimmunity. Protein kinases are enzymes that covalently attach phosphate to the side chain of either serine, threonine, or tyrosine of specific proteins inside cells. Such phosphorylation of proteins can control their enzymatic activity, their interaction with other proteins and molecules, their location in the cell, and their propensity for degradation by proteases. Mitogen-activated protein kinases (MAPKs) compose a family of protein kinases whose function and regulation have been conserved during evolution from unicellular organisms such as brewers' yeast to complex organisms including humans (1). MAPKs phosphorylate specific serines and threonines of target protein substrates and regulate cellular activities ranging from gene expression, mitosis, movement, metabolism, and programmed death. Because of the many important cellular functions controlled by MAPKs, they have been studied extensively to define their roles in physiology and human disease. MAPK-catalyzed phosphorylation of substrate proteins functions as a switch to turn on or off the activity of the substrate protein. Substrates include other protein kinases, phospholipases, transcription factors, and cytoskeletal proteins. Protein phosphatases remove the phosphates that were transferred to the protein substrate by the MAPK. In this manner, the action of MAPKs and protein phosphatases reciprocally and rapidly alter the behavior of cells as they respond to changes in their environment. MAPKs are part of a phosphorelay system composed of three sequentially activated kinases, and, like their substrates, MAPKs are regulated by phosphorylation (Fig. 1) (2). MAPKs serve as phosphorylation substrates for
Protein kinase cascades activated by stress and inflammatory cytokines
BioEssays, 1996
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-a and IL-1-p, 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-a actions. These two cascades signal cell cycle delay, cellular repair or apoptosis in most cells, as well as activation of immune and Accepted reticuloendothelial cells.
Current Biology, 1996
The ERK, JNK/SAPK and p38/RK MAP kinase subtypes (reviewed in [1]) are differentially activated in mammalian cells by various stimuli, which elicit induction of immediate-early (IE) genes, such as c-fos and c-jun (reviewed in [1-3]), as well as phosphorylation of histone H3 [4] and HMG-14 [5]. Anisomycin and UV radiation have been suggested to induce c-fos and c-jun transcription via JNK/SAPKmediated phosphorylation of TCF (ternary complex factor), for c-fos induction [6-8], and c-Jun and/or ATF-2 for c-jun induction [9-13]. We report here that anisomycin and ultraviolet radiation (UV) activate MAP kinase kinase-6 (MKK6) [14,15], p38/RK [16-18] and MAPKAP kinase-2 (MAPKAP K-2) [17-19]. By using the p38/RK inhibitor SB 203580 [20,21], we show that activation of p38/RK and/or its downstream effectors are essential for anisomycin-and UV-stimulated cfos/c-jun induction and histone H3/HMG-14 phosphorylation, whereas JNK/SAPK activation and phosphorylation of c-Jun and ATF-2 are insufficient for these responses.
Stress-activated Protein Kinases: Activation, Regulation and Function
Cellular Signalling, 1997
The response of cells to extracellular stimuli is mediated in part by a number of intracellular kinase and phosphatase enzymes. Within this area of research the activation of the p42 and p44 isoforms of mitogen-activated protein (MAP) kinases have been extensively described and characterised as central components of the signal transduction pathways stimulated by both growth factors and G-protein-coupled receptor agonists. Signaling events mediated by these kinases are fundamental to cellular functions such as proliferation and differentiation. More recently, homologues of the p42 and p44 isoforms of MAP kinase have been described, namely the stress-activated protein kinases (SAPKs) or alternatively the c-jun N-terminal kinases (JNKs) and p38 MAP kinase (the mammalian homologue of yeast HOG1). These MAP kinase homologues are integral components of parallel MAP kinase cascades activated in response to a number of cellular stresses including inflammatory cytokines (e.g., Interleukin-1 (Il-1) and tumour necrosis factor-␣ (TNF-␣), heat and chemical shock, bacterial endotoxin and ischaemia/cellular ATP depletion. Activation of these MAP kinase homologues mediates the transduction of extracellular signals to the nucleus and are pivotal events in the regulation of the transcription events that determine functional outcome in response to such stresses. In this review we highlight the identification and characterisation of the stress-activated MAP kinase homologues, their role as components of parallel MAP kinase pathways and the regulation of cellular responses following exposure to cellular stress. cell signal 9;6:403-410, 1997. © 1997 Elsevier Science Inc.
Biochemical and Biophysical Research Communications, 2001
Rho family small GTPases, in mammalian cells. Here we show that JNK activation by the prototypic Gqcoupled ␣1B-adrenergic receptor is mediated by the ␣ subunit of Gq (G␣q), not by G␥, using a transient transfection system in human embryonic kidney cells. JNK activation by the ␣1B-adrenergic receptor/G␣q was selectively mediated by mitogen-activated protein kinase kinase 4 (MKK4), but not MKK7. Also, MKK4 activation by the ␣1B-adrenergic receptor/G␣q required c-Src and Rho family small GTPases. Furthermore, activation of the ␣1B-adrenergic receptor stimulated JNK activity through Src family tyrosine kinases and Rho family small GTPases in hamster smooth muscle cells that natively express the ␣1Badrenergic receptor. Together, these results suggest that the ␣1B-adrenergic receptor/G␣q may up-regulate JNK activity through a MKK4 pathway dependent on c-Src and Rho family small GTPases in mammalian cells.
Sounding the Alarm: Protein Kinase Cascades Activated by Stress and Inflammation
Journal of Biological Chemistry, 1996
Eukaryotic cells respond to extracellular stimuli by recruiting signal transduction pathways, many of which employ protein Ser/ Thr kinases of the ERK 1 family. The ubiquity of ERKs and their upstream activators, the MEKs, in signal transduction was first appreciated from studies of yeast (1, 2). Although a 54-kDa rat liver c-Jun kinase (SAPK-p54␣1) with properties similar to the Rasregulated MAPKs had been characterized (3-5), the physiologic roles and regulation of this and related mammalian enzymes have emerged only recently. Molecular cloning of the SAPKs and p38s, together with the paradigms derived from the "classical" MAPKs and work in lower eukaryotes has enabled rapid elucidation of the regulation and cellular functions of these newer mammalian ERK pathways. Although architecturally homologous to the Ras/MAPK pathway, the SAPK and p38 pathways are not activated primarily by mitogens but by cellular stresses and inflammatory cytokines, which stimuli result in growth arrest, apoptosis, or activation of immune and reticuloendothelial cells. p38-mpk2/Reactivating Kinase/Upstream Activator/CSAIDbinding Proteins/Mxi2: the Mammalian HOG1 Homologues Efforts to uncover signaling mechanisms activated by inflammation and environmental stress identified mammalian homologues of the yeast-osmosensing ERK HOG1 (6). p38 was first purified as a macrophage polypeptide that became Tyr-phosphorylated in situ in response to bacterial lipopolysaccharide (7). Molecular cloning revealed similarities between p38 and HOG1 (6, 7). Lipopolysaccharide induces shock in part by causing release of TNF-␣ and IL-1. Novel anti-inflammatory drugs, CSAIDs, can inhibit lipopolysaccharide-stimulated TNF-␣ and IL-1 production. Two major intracellular CSAID-binding proteins were identified as isoforms of p38. CSAIDs directly inhibit p38 kinase activity, pointing to a role for p38 in cytokine release (8). The p38 kinase was also identified as part of a protein kinase cascade activated by IL-1 or physiologic stress, which culminates in MAPKAP kinase-2 activation and Hsp25/Hsp27 phosphorylation; p38 phosphorylates and activates MAPKAP kinase-2 in vitro (9, 11). MAPKAP kinase-2 phosphorylates the heat shock protein Hsp25/Hsp27 in vitro at the sites phosphorylated in situ in response to stress (10). Although p42
Differential regulation of Jun N-terminal kinase and p38MAP kinase by Gα12
Oncogene, 2004
Based on the findings that the overexpression of the wildtype Ga 12 (Ga 12 WT) result in the oncogenic transformation of NIH3T3 cells in a serum-dependent manner, a model system has been established in which the mitogenic and subsequent cell transformation pathways activated by Ga 12 can be turned on or off by the addition or removal of serum. Using this model system, our previous studies have shown that the stimulation of Ga 12 WT or the expression of an activated mutant of Ga 12 (Ga 12 QL) leads to increased cell proliferation and subsequent oncogenic transformation of NIH3T3 cells, as well as persistent activation of Jun N-terminal kinases (JNKs). In the present studies, we show that the stimulation of Ga 12 WT or the expression of Ga 12 QL results in a potent inhibition of p38MAPK, and that the mechanism by which Ga 12 inhibits p38MAPK activity involves the dual specificity kinases upstream of p38MAPK. The results indicate that Ga 12 attenuates the activation of MKK3 and MKK4, which are known to stimulate only p38MAPK or p38MAPK and JNK, respectively. The results also suggest that Ga 12 activates JNKs specifically through the stimulation of the JNK-specific upstream kinase MKK7. These findings demonstrate for the first time that Ga 12 differentially regulates JNK and p38MAPK by specifically activating MKK7, while inhibiting MKK3 and MKK4 in NIH3T3 cells. Since the stimulation of p38MAPK is often associated with apoptotic responses, our findings suggest that Ga 12 stimulates cell proliferation and neoplastic transformation of NIH3T3 cells by attenuating p38MAPK-associated apoptotic responses, while activating the mitogenic responses through the stimulation of ERK-and JNK-mediated signaling pathways.
Molecular and cellular biology, 1999
The major components of the mitogen-activated protein kinase (MAPK) cascades are MAPK, MAPK kinase (MAPKK), and MAPKK kinase (MAPKKK). Recent rapid progress in identifying members of MAPK cascades suggests that a number of such signaling pathways exist in cells. To date, however, how the specificity and efficiency of the MAPK cascades is maintained is poorly understood. Here, we have identified a novel mouse protein, termed Jun N-terminal protein kinase (JNK)/stress-activated protein kinase-associated protein 1 (JSAP1), by a yeast two-hybrid screen, using JNK3 MAPK as the bait. Of the mammalian MAPKs tested (JNK1, JNK2, JNK3, ERK2, and p38alpha), JSAP1 preferentially coprecipitated with the JNKs in cotransfected COS-7 cells. JNK3 showed a higher binding affinity for JSAP1, compared with JNK1 and JNK2. In similar cotransfection studies, JSAP1 also interacted with SEK1 MAPKK and MEKK1 MAPKKK, which are involved in the JNK cascades. The regions of JSAP1 that bound JNK, SEK1, and MEKK1 ...
The stress-activated protein kinase subfamily of c-Jun kinases
Nature, 1994
Diabetes Research Laboratory, Medical Services, Massachusetts General Hospital East, 149 13th Street, Charlestown, Massachusetts 02129, USA † The Ontario Cancer Institute, Princess Margaret Hospital, 500 Sherbourne Street, ...