Recruitment of JNK to JIP1 and JNK-dependent JIP1 phosphorylation regulates JNK module dynamics and activation

JIP1 is a scaffold protein that assembles and facilitates the activation of the mixed lineage kinase-dependent JNK module. Results of earlier work led us to propose a model for JIP1-JNK complex regulation that predicts that under basal conditions, JIP1 maintains DLK in a monomeric, unphosphorylated, and catalytically inactive state. Upon appropriate module stimulation, JNK-JIP1 binding affinity increases and DLK-JIP1 affinity decreases. Dissociation of DLK from JIP1 results in subsequent DLK oligomerization, autophosphorylation, and ultimately module activation. Our previous published results suggested the hypothesis that recruitment of JNK to JIP1 and phosphorylation of JIP1 by JNK is prerequisite for activation of the JNK module (Nihalani, D., Meyer, D., Pajni, S., and Holzman, L. B. (2001) EMBO J. 20, 3447-3458). The present study corroborated this hypothesis by demonstrating that JNK binding to JIP1 is necessary for stimulus-induced dissociation of DLK from JIP1, for DLK oligomerization, and for JNK activation. After mapping JNK-dependent JIP1 phosphorylation sites and testing their functional significance, it was observed that phosphorylation by JNK of JIP1 on Thr-103 and not other phosphorylated JIP1 residues is necessary for the regulation of DLK association with JIP1, DLK activation, and subsequent module activation. A refined model of JIP1-JNK module regulation is presented in which JNK phosphorylation of JIP1 is necessary prior to module activation.     

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.     

Disassembly of the JIP1/JNK molecular scaffold by caspase-3-mediated cleavage of JIP1 during apoptosis

We report here the cleavage of the c-Jun N-terminal Kinase (JNK) pathway scaffold protein, JNK Interacting Protein-1 (JIP1), by caspases during both Tumour Necrosis Factor-Related Apoptosis-Inducing Ligand (TRAIL) and staurosporine-induced apoptosis in HeLa cells. During the initiation of apoptosis, maximal JNK activation is observed when JIP1 is intact, whereas cleavage of JIP1 correlates with JNK inactivation and progression of apoptosis. JIP1 is cleaved by caspase-3 at two sites, leading to disassembly of the JIP1/JNK complex. Inhibition of JIP1 cleavage by the caspase-3 inhibitor DEVD.fmk inhibits this disassembly, and is accompanied by sustained JNK activation. These data suggest that TRAIL and staurosporine induce JNK activation in a caspase-3-independent manner and that caspase-3-mediated JIP1 cleavage plays a role in JNK inactivation via scaffold disassembly during the execution phase of apoptosis. Caspase-mediated cleavage of JIP scaffold proteins may therefore represent an important mechanism for modulation of JNK signalling during apoptotic cell death.     

Siah1 interacts with the scaffold protein POSH to promote JNK activation and apoptosis

Siah proteins are ubiquitin-protein isopeptide ligases (E3) that have been implicated in a variety of cellular actions, including promotion of apoptotic death. Here, we show that Siah1 is a binding partner for POSH (plenty of SH3s), a scaffold component of the apoptotic JNK pathway, and that Siah contributes to death of neurons and other cell types by activating the JNK pathway. Such proapoptotic activity requires the E3 ligase activity of Siah1. Moreover, apoptotic stimuli markedly elevate cellular Siah1 levels by a mechanism reliant on Siah1 protein stabilization. This stabilization requires JNK pathway activation and interaction with POSH and is enhanced by phosphorylation of SIAH1 at tyrosines 100 and 126. Depletion of intracellular Siah proteins via small interference RNA partially protects cells from death evoked by apoptotic stimuli such as trophic factor deprivation and DNA damage. These findings thus reveal a "loop" mechanism in which the JNK pathway promotes SIAH1 stabilization and in which SIAH1 in turn activates the JNK pathway and, ultimately, contributes to cell death.     

Interaction of a mitogen-activated protein kinase signaling module with the neuronal protein JIP3

The c-Jun NH(2)-terminal kinase (JNK) group of mitogen-activated protein kinases (MAPKs) is activated in response to the treatment of cells with inflammatory cytokines and by exposure to environmental stress. JNK activation is mediated by a protein kinase cascade composed of a MAPK kinase and a MAPK kinase kinase. Here we describe the molecular cloning of a putative molecular scaffold protein, JIP3, that binds the protein kinase components of a JNK signaling module and facilitates JNK activation in cultured cells. JIP3 is expressed in the brain and at lower levels in the heart and other tissues. Immunofluorescence analysis demonstrated that JIP3 was present in the cytoplasm and accumulated in the growth cones of developing neurites. JIP3 is a member of a novel class of putative MAPK scaffold proteins that may regulate signal transduction by the JNK pathway.     

TGF-beta-induced apoptosis is mediated by the adapter protein Daxx that facilitates JNK activation

Transforming growth factor-beta (TGF-beta) is a multifunctional growth factor that has a principal role in growth control through both its cytostatic effect on many different epithelial cell types and its ability to induce programmed cell death in a variety of other cell types. Here we have used a screen for proteins that interact physically with the cytoplasmic domain of the type II TGF-beta receptor to isolate the gene encoding Daxx - a protein associated with the Fas receptor that mediates activation of Jun amino-terminal kinase (JNK) and programmed cell death induced by Fas. The carboxy-terminal portion of Daxx functions as a dominant-negative inhibitor of TGF-beta-induced apoptosis in B-cell lymphomas, and antisense oligonucleotides to Daxx inhibit TGF-beta-induced apoptosis in mouse hepatocytes. Furthermore, Daxx is involved in mediating JNK activation by TGF-beta. Our findings associate Daxx directly with the TGF-beta apoptotic-signalling pathway, and make a biochemical connection between the receptors for TGF-beta and the apoptotic machinery.     

A beta-arrestin-dependent scaffold is associated with prolonged MAPK activation in pseudopodia during protease-activated receptor-2-induced chemotaxis

Cell motility during wound healing and inflammation is often dependent on the ability of the cell to sense a gradient of agonist. The first step in this process is the extension of a pseudopod in the direction of the agonist, and a diverse set of signals mediate pseudopod extension by different receptors. We have reported previously that protease-activated receptor-2 (PAR-2), a proinflammatory receptor that is highly expressed in motile cells such as neutrophils, macrophages, and tumor cells, is one of a growing family of receptors that utilizes a beta-arrestin-dependent mechanism for activation of the 42-44-kDa members of the MAPK family (extracellular signal-regulated kinases 1 and 2; ERK1/2). beta-Arrestin-bound PAR-2 serves as a scaffold to sequester a pool of activated ERK1/2 in the cytosol; however, a specific role for the sequestered kinase activity has not been established. We now show that PAR-2 activation promotes ERK1/2- and beta-arrestin-dependent reorganization of the actin cytoskeleton, polarized pseudopodia extension, and chemotaxis. Using subcellular fractionation, confocal microscopy, and physical isolation of pseudopodial proteins, we demonstrate that the previously identified PAR-2/beta-arrestin/ERK1/2 scaffolding complex is enriched in the pseudopodia, where it appears to prolong ERK1/2 activation. These studies suggest that the formation of a beta-arrestin/ERK1/2 signaling complex at the leading edge may be involved in localized actin assembly and chemotaxis and provide the first example of a distinct cellular consequence of beta-arrestin-sequestered ERK1/2 activity.     

Constitutive activity of JNK2 alpha2 is dependent on a unique mechanism of MAPK activation

c-Jun N-terminal kinases (JNKs) are part of the mitogen-activated protein kinase (MAPK) family and are important regulators of cell growth, proliferation, and apoptosis. Typically, a sequential series of events are necessary for MAPK activation: phosphorylation, dimerization, and then subsequent translocation to the nucleus. Interestingly, a constitutively active JNK isoform, JNK2alpha2, possesses the ability to autophosphorylate and has been implicated in several human tumors, including glioblastoma multiforme. Because overexpression of JNK2alpha2 enhances several tumorigenic phenotypes, including cell growth and tumor formation in mice, we studied the mechanisms of JNK2alpha2 autophosphorylation and autoactivation. We find that JNK2alpha2 dimerization in vitro and in vivo occurs independently of its autophosphorylation but is dependent on nine amino acids, known as the alpha-region. Alanine scanning mutagenesis of the alpha-region reveals that five specific mutants (L218A, K220A, G221A, I224A, and F225A) prevent JNK2alpha2 dimerization rendering JNK2alpha2 inactive and incapable of stimulating tumor formation. Previous studies coupled with additional mutagenesis of neighboring isoleucines and leucines (I208A, I214A, I231A, and I238A) suggest that a leucine zipper may play an important role in JNK2alpha2 homodimerization. We also show that a kinase-inactive JNK2alpha2 mutant can interact with and inhibit wild type JNK2alpha2 autophosphorylation, suggesting that JNK2alpha2 undergoes trans-autophosphorylation. Together, our results demonstrate that JNK2alpha2 differs from other MAPK proteins in two major ways; its autoactivation/autophosphorylation is dependent on dimerization, and dimerization most likely precedes autophosphorylation. In addition, we show that dimerization is essential for JNK2alpha2 activity and that prevention of dimerization may decrease JNK2alpha2 induced tumorigenic phenotypes.     

Differential requirement of MKK4 and MKK7 in JNK activation by distinct scaffold proteins

Different scaffold proteins play distinct roles in various signaling pathways by recruiting different downstream molecules. Here, using MKK4(-/-) and MKK4(-/-)/7(-/-) murine embryonic fibroblast cells, we examined differential employment of MKK4 and MKK7 by scaffold proteins Axin, Dvl, and Epstein-Barr virus latent membrane protein-1 (LMP-1) in mediating JNK activation. We present evidence that Axin depends mainly on MKK7 for activation of JNK, while Dvl depends almost equally on MKK4 and MKK7 for JNK activation, In contrast, LMP-1-induced JNK activation is primarily dependent on MKK4. Our results demonstrate that Axin, Dvl, and LMP-1 differentially utilize MKK4 and MKK7 for JNK activation.     

JNK-interacting protein 3 associates with Toll-like receptor 4 and is involved in LPS-mediated JNK activation

Lipopolysaccharide (LPS) is recognized by Toll-like receptor (TLR) 4 and activates NF-kappaB and a set of MAP kinases. Here we have investigated proteins associated with the cytoplasmic domain of mouse TLR4 by yeast two-hybrid screening and identified JNK-interacting protein 3 (JIP3), a scaffold protein for JNK, as a TLR4-associated protein. In mammalian cells, JIP3, through its N-terminal region, constitutively associates with TLR4. The association is specific to JIP3, as the two other JIPs, JIP1 and JIP2, failed to bind TLR4. In HEK 293 cells exogenously expressing TLR4, MD2 and CD14, co-expression of JIP3 significantly increased the complex formation of TLR4-JNK and LPS-mediated JNK activation. In contrast, expression of C-terminally truncated forms of JIP3 impaired LPS-induced JNK activation in a mouse macrophage cell line, RAW264.7. Moreover, RNA interference of JIP3 inhibited LPS-mediated JNK activation. In RAW264.7 cells, JIP3 associates MEKK-1, but not with TAK-1. Finally, JIP3 also associates with TLR2 and TLR9, but not with TLR1 or TLR6. Altogether, our data indicate the involvement of JIP3 in JNK activation in downstream signals of some TLRs.     

Structural basis for the selective inhibition of JNK1 by the scaffolding protein JIP1 and SP600125

The c-jun N-terminal kinase (JNK) signaling pathway is regulated by JNK-interacting protein-1 (JIP1), which is a scaffolding protein assembling the components of the JNK cascade. Overexpression of JIP1 deactivates the JNK pathway selectively by cytoplasmic retention of JNK and thereby inhibits gene expression mediated by JNK, which occurs in the nucleus. Here, we report the crystal structure of human JNK1 complexed with pepJIP1, the peptide fragment of JIP1, revealing its selectivity for JNK1 over other MAPKs and the allosteric inhibition mechanism. The van der Waals contacts by the three residues (Pro157, Leu160, and Leu162) of pepJIP1 and the hydrogen bonding between Glu329 of JNK1 and Arg156 of pepJIP1 are critical for the selective binding. Binding of the peptide also induces a hinge motion between the N- and C-terminal domains of JNK1 and distorts the ATP-binding cleft, reducing the affinity of the kinase for ATP. In addition, we also determined the ternary complex structure of pepJIP1-bound JNK1 complexed with SP600125, an ATP-competitive inhibitor of JNK, providing the basis for the JNK specificity of the compound.     

The cellular response to neuregulins is governed by complex interactions of the erbB receptor family.

Deregulated signaling by the four members of the epidermal growth factor receptor tyrosine kinase family (erbB family) is implicated in the genesis or progression of human cancers. However, efforts to analyze signaling by these receptors have been hampered by the diversity of ligands and extensive interreceptor cross talk. We have expressed the four human erbB family receptors, singly and in pairwise combinations, in a pro-B-lymphocyte cell line (Ba/F3) and investigated the range of interactions activated by the epidermal growth factor homology domain of the agonist neuregulin beta. The results provide the first comprehensive analysis of the response of this receptor family to a single peptide agonist. This peptide induced complex patterns of receptor tyrosine phosphorylation and regulation of Ba/F3 cell survival and proliferation. These data demonstrate the existence of several previously undocumented receptor interactions driven by neuregulin.     

Marrow-derived stem cell motility in 3D synthetic scaffold is governed by geometry along with adhesivity and stiffness

Design of 3D scaffolds that can facilitate proper survival, proliferation, and differentiation of progenitor cells is a challenge for clinical applications involving large connective tissue defects. Cell migration within such scaffolds is a critical process governing tissue integration. Here, we examine effects of scaffold pore diameter, in concert with matrix stiffness and adhesivity, as independently tunable parameters that govern marrow‐derived stem cell motility. We adopted an “inverse opal” processing technique to create synthetic scaffolds by crosslinking poly(ethylene glycol) at different densities (controlling matrix elastic moduli or stiffness) and small doses of a heterobifunctional monomer (controlling matrix adhesivity) around templating beads of different radii. As pore diameter was varied from 7 to 17 µm (i.e., from significantly smaller than the spherical cell diameter to approximately cell diameter), it displayed a profound effect on migration of these stem cells—including the degree to which motility was sensitive to changes in matrix stiffness and adhesivity. Surprisingly, the highest probability for substantive cell movement through pores was observed for an intermediate pore diameter, rather than the largest pore diameter, which exceeded cell diameter. The relationships between migration speed, displacement, and total path length were found to depend strongly on pore diameter. We attribute this dependence to convolution of pore diameter and void chamber diameter, yielding different geometric environments experienced by the cells within. Bioeng. 2011; 108:1181–1193. © 2010 Wiley Periodicals, Inc.     

The MAPK scaffold kinase suppressor of Ras is involved in ERK activation by stress and proinflammatory cytokines and induction of arthritis

The MAPK ERK is required for LPS-induced TNF production by macrophages. Although the scaffold kinase suppressor of Ras (KSR)1 is required for efficient Erk activation by mitogenic stimuli, the role of KSR1 in ERK activation by inflammatory and stress stimuli is unknown. In this study, we examined the effects of KSR deficiency on ERK activation by stress stimuli and show that ERK activation by TNF, IL-1, and sorbitol is attenuated in the absence of KSR1. To determine the significance of this defect in vivo, we tested KSR-deficient mice using a passive transfer model of arthritis. We found that the induction of arthritis is impaired in the absence of KSR. Thus, KSR plays a role in ERK activation during inflammatory and stress responses both in vitro and in vivo.