Docking interactions in the c-Jun N-terminal kinase pathway

The c-Jun N-terminal kinase (JNK) signaling pathway is a major mediator of stress responses in cells. Similar to other mitogen-activated protein kinases (MAPKs), JNK activity is controlled by a cascade of protein kinases and by protein phosphatases, including dual-specificity MAPK phosphatases. Components of the JNK pathway associate with scaffold proteins that modulate their activities and cellular localization. The JNK-interacting protein-1 (JIP-1) scaffold protein specifically binds JNK, MAPK kinase 7 (MKK7), and members of the mixed lineage kinase (MLK) family, and regulates JNK activation in neurons. In this study we demonstrate that distinct regions within the N termini of MKK7 and the MLK family member dual leucine zipper kinase (DLK) mediate their binding to JIP-1. We have also identified amino acids in JNK required for: (a) binding to JIP-1 and for JIP-1-mediated JNK activation, (b) docking to MAPK kinase 4 (MKK4) and efficient phosphorylation by MKK4, and (c) docking to its substrate c-Jun and efficient c-Jun phosphorylation. None of the amino acids identified were essential for JNK docking to MKK7 or the dual-specificity phosphatase MAPK phosphatase 7 (MKP7). These findings uncover molecular determinants of JIP-1 scaffold complex assembly and demonstrate that there are overlapping, but also distinct, binding determinants within JNK that mediate interactions with scaffold proteins, activators, phosphatases, and substrates.     

JSAP1, a Novel Jun N-Terminal Protein Kinase (JNK)-Binding Protein That Functions as a Scaffold Factor in the JNK Signaling Pathway

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 were distinct from one another. JNK and MEKK1 also bound JSAP1 in vitro, suggesting that these interactions are direct. In contrast, only the activated form of SEK1 associated with JSAP1 in cotransfected COS-7 cells. The unstimulated SEK1 bound to MEKK1; thus, SEK1 might indirectly associate with JSAP1 through MEKK1. Although JSAP1 coprecipitated with MEK1 MAPKK and Raf-1 MAPKKK, and not MKK6 or MKK7 MAPKK, in cotransfected COS-7 cells, MEK1 and Raf-1 do not interfere with the binding of SEK1 and MEKK1 to JSAP1, respectively. Overexpression of full-length JSAP1 in COS-7 cells led to a considerable enhancement of JNK3 activation, and modest enhancement of JNK1 and JNK2 activation, by the MEKK1-SEK1 pathway. Deletion of the JNK- or MEKK1-binding regions resulted in a significant reduction in the enhancement of the JNK3 activation in COS-7 cells. These results suggest that JSAP1 functions as a scaffold protein in the JNK3 cascade. We also discuss a scaffolding role for JSAP1 in the JNK1 and JNK2 cascades.     

MEKK4 stimulation of p38 and JNK activity is negatively regulated by GSK3beta

The MAPK kinase kinase MEKK4 is required for neurulation and skeletal patterning during mouse development. MEKK4 phosphorylates and activates MKK4/MKK7 and MKK3/MKK6 leading to the activation of JNK and p38, respectively. MEKK4 is believed to be auto-inhibited, and its interaction with other proteins controls its dimerization and activation. TRAF4, GADD45, and Axin each bind and activate MEKK4, with TRAF4 and Axin binding to the kinase domain and GADD45 binding within the N-terminal regulatory domain. Here we show that similar to the interaction with TRAF4 and Axin, the kinase domain of MEKK4 interacts with the multifunctional serine/threonine kinase GSK3beta. GSK3beta binding to MEKK4 blocks MEKK4 dimerization that is required for MEKK4 activation, effectively inhibiting MEKK4 stimulation of the JNK and p38 MAPK pathways. Inhibition of GSK3beta kinase activity with SB216763 results in enhanced MEKK4 kinase activity and increased JNK and p38 activation, indicating that an active state of GSK3beta is required for binding and inhibition of MEKK4 dimerization. Furthermore, GSK3beta phosphorylates specific serines and threonines in the N terminus of MEKK4. Together, these findings demonstrate that GSK3beta binds to the kinase domain of MEKK4 and regulates MEKK4 dimerization. However, unlike TRAF4, Axin, and GADD45, GSK3beta inhibits MEKK4 activity and prevents its activation of JNK and p38. Thus, control of MEKK4 dimerization is regulated both positively and negatively by its interaction with specific proteins.     

Docking interactions of the JNK scaffold protein WDR62

JNK (c-Jun N-terminal kinase) is part of a MAPK (mitogen-activated protein kinase) signalling cascade. Scaffold proteins simultaneously associate with various components of the MAPK signalling pathway and play a crucial role in signal transmission and MAPK regulation. WDR62 (WD repeat domain 62) is a JNK scaffold protein. Recessive mutations within WDR62 result in severe cerebral cortical malformation. In the present study we demonstrate the association of WDR62 with endogenous and overexpressed proteins of both JNK2 and the JNK2-activating kinase MKK7 (MAPK kinase 7). Association of WDR62 with JNK2 and MKK7 occurs via direct protein-protein interactions. We mapped the docking domain of WDR62 responsible for the association with JNK. WDR62 interacts with all JNK isoforms through a D domain motif located at the C-terminus. A WDR62 mutant lacking the putative JNK-binding domain fails to activate and recruit JNK to cellular granules. Furthermore, a synthetic peptide composed of the WDR62 docking domain inhibits JNK2 activity in vitro. WDR62 association with JNK2 requires both the JNK CD and ED domains, and the binding requisite is distinct from that of the previously described JNK2 association with JIP1 (JNK-interacting protein 1). Next, we characterized the association between WDR62 and MKK7. WDR62 associates directly with the MKK7β1 isoform independently of JNK binding, but fails to interact with MKK7α1. Furthermore, MKK7β1 recruits a protein phosphatase that dephosphorylates WDR62. Interestingly, a premature termination mutation in WDR62 that results in severe brain developmental defects does not abrogate WDR62 association with either JNK or MKK7. Therefore such mutations represent a loss of WDR62 function independent of JNK signalling.     

Synergistic interaction of MEK kinase 2, c-Jun N-terminal kinase (JNK) kinase 2, and JNK1 results in efficient and specific JNK1 activation

Mitogen-activated protein kinases (MAPKs) are activated through cascades or modules consisting of a MAPK, a MAPK kinase (MAPKK), and a MAPKK kinase (MAPKKK). Investigating the molecular basis of activation of the c-Jun N-terminal kinase (JNK) subgroup of MAPK by the MAPKKK MEKK2, we found that strong and specific JNK1 activation by MEKK2 was mediated by the MAPKK JNK kinase 2 (JNKK2) rather than by JNKK1 through formation of a tripartite complex consisting of MEKK2, JNKK2, and JNK1. No scaffold protein was required for the MEKK2-JNKK2-JNK1 tripartite-complex formation. Expression of JNK1, JNKK2, and MEKK2 significantly augmented the coprecipitation of, respectively, MEKK2-JNKK2, MEKK2-JNK1, and JNKK2-JNK1, indicating that the interaction of MEKK2, JNKK2, and JNK1 is synergistic. Finally, the JNK1 was activated more efficiently in the MEKK2-JNKK2-JNK1 complex than was the JNK1 excluded from the complex. Thus, formation of a signaling complex through synergistic interaction of a MAPKKK, a MAPKK, and a MAPK molecule like MEKK2-JNKK2-JNK1 is likely to be responsible for the efficient, specific flow of information via MAPK cascades.     

ASK1 regulates influenza virus infection-induced apoptotic cell death

Apoptosis occurs in influenza virus (IV)-infected cells. There are a number of mechanisms for the regulation of apoptosis. However, the molecular mechanism of IV infection-induced apoptosis is still controversial. Apoptosis signal-regulating kinase1 (ASK1) is a ubiquitously expressed mitogen-activated protein kinase kinase kinase (MAPKKK) that activates the SEK1-c-Jun N-terminal kinase (JNK) and MKK3/MKK6-p38 MAPK signaling cascades. ASK1 has been implicated in cytokine- and stress-induced apoptosis. Here, we show the following: (1) IV infection activated ASK1 and concomitantly phosphorylated JNK and p38 MAPK in human bronchial epithelial cells; (2) the activation of JNK and p38 MAPK but not extracellular-regulated kinase (ERK) in embryonic fibroblasts (MEFs) derived from ASK1 knockout mice (ASK1(-/-) MEFs) was depressed compared to MEFs derived from wild type mice (ASK1(+/+) MEFs); and (3) ASK1(-/-) MEFs were defective in IV infection-induced caspase-3 activation and cell death. These results indicate that apoptosis in IV-infected BEC is mediated through ASK1-dependent cascades.     

Arrestin-3 Binds c-Jun N-terminal Kinase 1 (JNK1) and JNK2 and Facilitates the Activation of These Ubiquitous JNK Isoforms in Cells via Scaffolding

Non-visual arrestins scaffold mitogen-activated protein kinase (MAPK) cascades. The c-Jun N-terminal kinases (JNKs) are members of MAPK family. Arrestin-3 has been shown to enhance the activation of JNK3, which is expressed mainly in neurons, heart, and testes, in contrast to ubiquitous JNK1 and JNK2. Although all JNKs are activated by MKK4 and MKK7, both of which bind arrestin-3, the ability of arrestin-3 to facilitate the activation of JNK1 and JNK2 has never been reported. Using purified proteins we found that arrestin-3 directly binds JNK1α1 and JNK2α2, interacting with the latter comparably to JNK3α2. Phosphorylation of purified JNK1α1 and JNK2α2 by MKK4 or MKK7 is increased by arrestin-3. Endogenous arrestin-3 interacted with endogenous JNK1/2 in different cell types. Arrestin-3 also enhanced phosphorylation of endogenous JNK1/2 in intact cells upon expression of upstream kinases ASK1, MKK4, or MKK7. We observed a biphasic effect of arrestin-3 concentrations on phosphorylation of JNK1α1 and JNK2α2 both in vitro and in vivo. Thus, arrestin-3 acts as a scaffold, facilitating JNK1α1 and JNK2α2 phosphorylation by MKK4 and MKK7 via bringing JNKs and their activators together. The data suggest that arrestin-3 modulates the activity of ubiquitous JNK1 and JNK2 in non-neuronal cells, impacting the signaling pathway that regulates their proliferation and survival.     

The JIP Group of Mitogen-Activated Protein Kinase Scaffold Proteins

Activation of the c-Jun NH(2)-terminal kinase (JNK) group of mitogen-activated protein (MAP) kinases is mediated by a protein kinase cascade. This signaling mechanism may be coordinated by the interaction of components of the protein kinase cascade with scaffold proteins. The JNK-interacting protein (JIP) group of scaffold proteins selectively mediates signaling by the mixed-lineage kinase (MLK)-->MAP kinase kinase 7 (MKK7)-->JNK pathway. The scaffold proteins JIP1 and JIP2 interact to form oligomeric complexes that accumulate in peripheral cytoplasmic projections extended at the cell surface. The JIP proteins function by aggregating components of a MAP kinase module (including MLK, MKK7, and JNK) and facilitate signal transmission by the protein kinase cascade.     

Identification of a motif in the carboxyl terminus of beta -arrestin2 responsible for activation of JNK3

Accumulating evidence indicates that the beta-arrestins act as scaffold molecules that couple G-protein-coupled receptors to mitogen-activated protein (MAP) kinase signaling pathways. Recently, we identified the c-Jun N-terminal kinase 3 (JNK3) as a beta-arrestin2-interacting protein in yeast-two hybrid and co-immunoprecipitation studies. Beta-arrestin2 acts as a scaffold to enhance signaling to JNK3 stimulated by overexpression of the MAP3 kinase ASK1 or by agonist activation of the angiotensin 1A receptor. Whereas beta-arrestin2 is a very strong activator of JNK3 signaling, beta-arrestin1 is very weak in this regard. The data also indicate that the specific step enhanced by beta-arrestin2 involves phosphorylation of JNK3 by the MAP2 kinase MKK4. We reasoned that defining the region (or domain) in beta-arrestin2 responsible for high level JNK3 activation would provide insight into the mechanism by which beta-arrestin2 enhances the activity of this signaling pathway. Using chimeric beta-arrestins, we have determined that sequences in the carboxyl-terminal region of beta-arrestin2 are important for the enhancement of JNK3 phosphorylation. More detailed analysis of the carboxyl-terminal domains of the beta-arrestins indicated that beta-arrestin2, but not beta-arrestin1, contains a sequence (RRSLHL) highly homologous to the conserved docking motif present in many MAP kinase-binding proteins. Replacement of the beta-arrestin2 RRS residues with the corresponding KP residues present in beta-arrestin1 dramatically reduced both JNK3 interaction and enhancement of JNK3 phosphorylation. Conversely, replacement of the KP residues in beta-arrestin1 with RRS significantly increased both JNK3 binding and enhancement of JNK3 phosphorylation. These results delineate a mechanism by which beta-arrestin2 functions as a scaffold protein in the JNK3 signaling pathway and implicate the conserved docking site in beta-arrestin2 as an important factor in binding JNK3 and stimulating the phosphorylation of JNK3 by MKK4.     

Antagonistic interactions between two MAP kinase cascades in plant development and immune signaling

Mitogen‐activated protein kinase (MAPK) signaling plays important roles in diverse biological processes. In Arabidopsis, MPK3/MPK6, MKK4/MKK5, and the MAPKKK YODA (YDA) form a MAPK pathway that negatively regulates stomatal development. Brassinosteroid (BR) stimulates this pathway to inhibit stomata production. In addition, MPK3/MPK6 and MKK4/MKK5 also serve as critical signaling components in plant immunity. Here, we report that MAPKKK3/MAPKKK5 form a kinase cascade with MKK4/MKK5 and MPK3/MPK6 to transduce defense signals downstream of multiple plant receptor kinases. Loss of MAPKKK3/MAPKKK5 leads to reduced activation of MPK3/MPK6 in response to different pathogen‐associated molecular patterns (PAMPs) and increased susceptibility to pathogens. Surprisingly, developmental defects caused by silencing of YDA are suppressed in the mapkkk3 mapkkk5 double mutant. On the other hand, loss of YDA or blocking BR signaling leads to increased PAMP‐induced activation of MPK3/MPK6. These results reveal antagonistic interactions between a developmental MAPK pathway and an immune signaling MAPK pathway.     

The Caenorhabditis elegans Ste20-Related Kinase and Rac-Type Small GTPase Regulate the c-Jun N-Terminal Kinase Signaling Pathway Mediating the Stress Response

Mitogen-activated protein kinases (MAPKs) are integral to the mechanisms by which cells respond to physiological stimuli and a wide variety of environmental stresses. In Caenorhabditis elegans, the stress response is controlled by a c-Jun N-terminal kinase (JNK)-like MAPK signaling pathway, which is regulated by MLK-1 MAPK kinase kinase (MAPKKK), MEK-1 MAPKK, and KGB-1 JNK-like MAPK. In this study, we identify the max-2 gene encoding a C. elegans Ste20-related protein kinase as a component functioning upstream of the MLK-1-MEK-1-KGB-1 pathway. The max-2 loss-of-function mutation is defective in activation of KGB-1, resulting in hypersensitivity to heavy metals. Biochemical analysis reveals that MAX-2 activates MLK-1 through direct phosphorylation of a specific residue in the activation loop of the MLK-1 kinase domain. Our genetic data presented here also show that MIG-2 small GTPase functions upstream of MAX-2 in the KGB-1 pathway. These results suggest that MAX-2 and MIG-2 play a crucial role in mediating the heavy metal stress response regulated by the KGB-1 pathway.Mitogen-activated protein kinase (MAPK) signal transduction pathways are evolutionarily conserved in eukaryotic cells and transduce signals in response to a variety of extracellular stimuli. Each pathway is composed of three classes of protein kinases: MAPK, MAPK kinase (MAPKK), and MAPK kinase kinase (MAPKKK) (4, 14). MAPKKK phosphorylates and activates MAPKK, which in turn activates MAPK by dual phosphorylation of threonine and tyrosine residues within a Thr-Xxx-Tyr motif. Three subgroups of MAPKs have been identified: the extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK), and p38 kinases (4, 14). JNK and p38 MAPKs function as key mediators of stress and immune signaling in mammals. The MKK4 and MKK7 MAPKKs have been shown to activate JNK, and the MKK3 and MKK6 MAPKKs serve as the major activators of p38 MAPK (4, 14). The specific MAPKKs are themselves phosphorylated and activated by specific MAPKKKs.Recent studies of Caenorhabditis elegans have revealed a high degree of conservation of JNK MAPK signaling components between C. elegans and mammals. The C. elegans JNK pathway, composed of an MKK7-type MAPKK JKK-1 and a JNK-type MAPK JNK-1, regulates coordinated movement via type D GABAergic (GABA stands for γ-aminobutyric acid) motor neurons (10) and has a role in synaptic vesicle transport (3). C. elegans also possesses another JNK-like MAPK pathway, composed of MLK-1 MAPKKK, MEK-1 MAPKK, and KGB-1 MAPK, which is homologous to the mammalian MLK-MKK7-JNK MAPK signaling cassette. KGB-1 has a novel activation site, consisting of Ser-Xxx-Tyr rather than Thr-Xxx-Tyr (19, 21). The KGB-1 pathway regulates the stress response to heavy metals (19). We have previously identified the vhp-1 and shc-1 genes as components functioning in the KGB-1 pathway. The vhp-1 and shc-1 genes encode a MAPK phosphatase (MKP) highly homologous to mammalian MKP-7 and a homolog of the mammalian Shc adaptor, respectively (19, 20). VHP-1 plays an important role in the heavy metal stress response in C. elegans by negatively regulating the KGB-1 pathway through dephosphorylation of KGB-1. SHC-1 mediates activation of the KGB-1 pathway by linking MEK-1 MAPKK with MLK-1 MAPKKK. However, it remains unknown what components function upstream of the MLK-1-MEK-1-KGB-1 pathway.In mammalian cells, the kinase activity of MLK family members is controlled by several different mechanisms, such as dimer formation, autoinhibition mediated by the Src homology 3 (SH3) domain of the MLKs itself, interaction with small GTPases, and phosphorylation by MAPKKK kinase (MAP4K) (6). In this study, we identified MAX-2, a member of the Ste20 group of protein kinases, as a potential component functioning upstream of MLK-1 MAPKKK in the KGB-1 pathway. MAX-2 physically associates with and phosphorylates MLK-1 at a Ser residue in the activation loop located between kinase subdomains VII and VIII of MLK-1, resulting in its activation. Additionally, we found that MIG-2, a member of the Rac family of small GTPases, functions as an upstream regulator of MAX-2. Our results thus identify the in vivo machinery regulating the JNK-mediated stress response pathway via a Ste20-related kinase and Rac-type GTPase.     

Regulation of c-Jun N-terminal kinase by MEKK-2 and mitogen-activated protein kinase kinase kinases in rheumatoid arthritis

The mitogen-activated protein kinase (MAPK) c-Jun N-terminal kinase (JNK) is a critical regulator of collagenase-1 production in rheumatoid arthritis (RA). The MAPKs are regulated by upstream kinases, including MAPK kinases (MAPKKs) and MAPK kinase kinases (MAP3Ks). The present study was designed to evaluate the expression and regulation of the JNK pathway by MAP3K in arthritis. RT-PCR studies of MAP3K gene expression in RA and osteoarthritis synovial tissue demonstrated mitogen-activated protein kinase/ERK kinase kinase (MEKK) 1, MEKK2, apoptosis-signal regulating kinase-1, TGF-beta activated kinase 1 (TAK1) gene expression while only trace amounts of MEKK3, MEKK4, and MLK3 mRNA were detected. Western blot analysis demonstrated immunoreactive MEKK2, TAK1, and trace amounts of MEKK3 but not MEKK1 or apoptosis-signal regulating kinase-1. Analysis of MAP3K mRNA in cultured fibroblast-like synoviocytes (FLS) showed that all of the MAP3Ks examined were expressed. Western blot analysis of FLS demonstrated that MEKK1, MEKK2, and TAK1 were readily detectable and were subsequently the focus of functional studies. In vitro kinase assays using MEKK2 immunoprecipitates demonstrated that IL-1 increased MEKK2-mediated phosphorylation of the key MAPKKs that activate JNK (MAPK kinase (MKK)4 and MKK7). Furthermore, MEKK2 immunoprecipitates activated c-Jun in an IL-1 dependent manner and this activity was inhibited by the selective JNK inhibitor SP600125. Of interest, MEKK1 immunoprecipitates from IL-1-stimulated FLS appeared to activate c-Jun through the JNK pathway and TAK1 activation of c-Jun was dependent on JNK, ERK, and p38. These data indicate that MEKK2 is a potent activator of the JNK pathway in FLS and that signal complexes including MEKK2, MKK4, MKK7, and/or JNK are potential therapeutic targets in RA.     

Activation of MTK1/MEKK4 by GADD45 through induced N-C dissociation and dimerization-mediated trans autophosphorylation of the MTK1 kinase domain

The mitogen-activated protein kinase (MAPK) module, composed of a MAPK, a MAPK kinase (MAPKK), and a MAPKK kinase (MAPKKK), is a cellular signaling device that is conserved throughout the eukaryotic world. In mammalian cells, various extracellular stresses activate two major subfamilies of MAPKs, namely, the Jun N-terminal kinases and the p38/stress-activated MAPK (SAPK). MTK1 (also called MEKK4) is a stress-responsive MAPKKK that is bound to and activated by the stress-inducible GADD45 family of proteins (GADD45alpha/beta/gamma). Here, we dissected the molecular mechanism of MTK1 activation by GADD45 proteins. The MTK1 N terminus bound to its C-terminal segment, thereby inhibiting the C-terminal kinase domain. This N-C interaction was disrupted by the binding of GADD45 to the MTK1 N-terminal GADD45-binding site. GADD45 binding also induced MTK1 dimerization via a dimerization domain containing a coiled-coil motif, which is essential for the trans autophosphorylation of MTK1 at Thr-1493 in the kinase activation loop. An MTK1 alanine substitution mutant at Thr-1493 has a severely reduced activity. Thus, we conclude that GADD45 binding induces MTK1 N-C dissociation, dimerization, and autophosphorylation at Thr-1493, leading to the activation of the kinase catalytic domain. Constitutively active MTK1 mutants induced the same events, but in the absence of GADD45.     

Exploring the function of the JNK (c-Jun N-terminal kinase) signalling pathway in physiological and pathological processes to design novel therapeutic strategies

JNK (c-Jun N-terminal kinase) is a member of the MAPK (mitogen-activated protein kinase) family that regulates a range of biological processes implicated in tumorigenesis and neurodegenerative disorders. For example, genetic studies have demonstrated that the removal of specific Jnk genes can reduce neuronal death associated with cerebral ischaemia. As such, targeting JNK signalling constitutes an obvious opportunity for therapeutic intervention. However, MAPK inhibitors can display toxic effects. Consequently, dual-specificity MKKs (MAPK kinases) may represent more attractive targets. In particular, evidence that blocking JNK activation by removing MKK4 offers an effective therapy to treat pathological conditions has started to emerge. MKK4 was the first JNK activator identified. The remaining level of JNK activity in cells lacking MKK4 expression led to the discovery of a second activator of JNK, named MKK7. Distinct phenotypic abnormalities associated with the targeted deletion of Mkk4 and Mkk7 in mice have revealed that MKK4 and MKK7 have non-redundant function in vivo. Further insights into the specific functions of the JNK activators in cancer cells and in neurons will be of critical importance to validate MKK4 and MKK7 as promising drug targets.     

The p85alpha regulatory subunit of phosphoinositide 3-kinase potentiates c-Jun N-terminal kinase-mediated insulin resistance

Insulin resistance is a defining feature of type 2 diabetes and the metabolic syndrome. While the molecular mechanisms of insulin resistance are multiple, recent evidence suggests that attenuation of insulin signaling by c-Jun N-terminal kinase (JNK) may be a central part of the pathobiology of insulin resistance. Here we demonstrate that the p85alpha regulatory subunit of phosphoinositide 3-kinase (PI3K), a key mediator of insulin's metabolic actions, is also required for the activation of JNK in states of insulin resistance, including high-fat diet-induced obesity and JNK1 overexpression. The requirement of the p85alpha regulatory subunit for JNK occurs independently of its role as a component of the PI3K heterodimer and occurs only in response to specific stimuli, namely, insulin and tunicamycin, a chemical that induces endoplasmic reticulum stress. We further show that insulin and p85 activate JNK by via cdc42 and MKK4. The activation of this cdc42/JNK pathway requires both an intact N terminus and functional SH2 domains within the C terminus of the p85alpha regulatory subunit. Thus, p85alpha plays a dual role in regulating insulin sensitivity and may mediate cross talk between the PI3K and stress kinase pathways.     

Role of the Caenorhabditis elegans Shc adaptor protein in the c-Jun N-terminal kinase signaling pathway

Mitogen-activated protein kinases (MAPKs) are integral to the mechanisms by which cells respond to physiological stimuli and a wide variety of environmental stresses. In Caenorhabditis elegans, the stress response is controlled by a c-Jun N-terminal kinase (JNK)-like mitogen-activated protein kinase (MAPK) signaling pathway, which is regulated by MLK-1 MAPK kinase kinase (MAPKKK), MEK-1 MAPK kinase (MAPKK), and KGB-1 JNK-like MAPK. In this study, we identify the shc-1 gene, which encodes a C. elegans homolog of Shc, as a factor that specifically interacts with MEK-1. The shc-1 loss-of-function mutation is defective in activation of KGB-1, resulting in hypersensitivity to heavy metals. A specific tyrosine residue in the NPXY motif of MLK-1 creates a docking site for SHC-1 with the phosphotyrosine binding (PTB) domain. Introduction of a mutation that perturbs binding to the PTB domain or the NPXY motif abolishes the function of SHC-1 or MLK-1, respectively, thereby abolishing the resistance to heavy metal stress. These results suggest that SHC-1 acts as a scaffold to link MAPKKK to MAPKK activation in the KGB-1 MAPK signal transduction pathway.     

Metabolic stress signaling mediated by mixed-lineage kinases

Saturated free fatty acid (FFA) is a major source of metabolic stress that activates the c-Jun NH(2)-terminal kinase (JNK). This FFA-stimulated JNK pathway is relevant to hallmarks of metabolic syndrome, including insulin resistance. Here we used gene ablation studies in mice to demonstrate a central role for mixed-lineage protein kinases (MLK) in this signaling pathway. Saturated FFA causes protein kinase C (PKC)-dependent activation of MLK3 that subsequently causes increased JNK activity by a mechanism that requires the MAP kinase kinases MKK4 and MKK7. Loss of PKC, MLK3, MKK4, or MKK7 expression prevents FFA-stimulated JNK activation. Together, these data establish a signaling pathway that mediates effects of metabolic stress on insulin resistance.