Table of contents

Upstream regulators of JNK: MAPKK's immediately upstream

Although in vitro evidence suggests two c-Jun N-terminal kinase (JNK) kinases, MKK4 and MKK7, activate JNK, in vivo confirmation is incomplete. In fact, JNK deficiency may differ from the composite deficiency of MKK4 and MKK7 in Drosophila and mice. Recently, the C. elegans homolog of human JNK, jnk-1, and two MKK-7s, mek-1 and jkk-1, were cloned. jnk-1, which encodes two isoforms JNK-1alpha and JNK-1ß, bas been characterized. A null allele, jnk-1(gk7), yields worms with defective body movement coordination and modest mechanosensory deficits. Similarly to jkk-1 mutants, elimination of GABAergic signals suppresses the jnk-1(gk7) locomotion defect. Like mek-1 nulls, jnk-1(gk7) shows copper and cadmium hypersensitivity. Conditional expression of JNK-1 isoforms rescues these defects, suggesting that they are not due to developmental errors. While jkk-1 or mek-1 inactivation mimic jnk-1(gk7) locomotion and heavy metal stress defects, respectively, mkk-4 inactivation does not, but rather yields defective egg laying. These results delineate at least two different JNK pathways through jkk-1 and mek-1 in C. elegans, and define interaction between MKK7, but not MKK4, and JNK (Villanueva, 2001).

Stress-activated protein kinase (SAPK)/c-Jun N-terminal kinase (JNK), a member of the MAP kinase (MAPK) superfamily, is thought to play a key role in a variety of cellular responses. To date, SEK1/MKK4, one of the MAP kinase kinase (MAPKK) family of molecules, is the only SAPK/JNK kinase that has been cloned. A novel member of the mammalian MAPKKs, designated MKK7, has now been cloned, identified and characterized. MKK7 is most similar to the Drosophila mediator of morphogenesis, hemipterous (hep). Immunochemical studies have identified MKK7 as one of the major SAPK/JNK-activating kinases in osmotically shocked cells. While SEK1/MKK4 can activate both the SAPK/JNK and p38 subgroups of the MAPK superfamily, MKK7 is specific for the SAPK/JNK subgroup. MKK7 is activated strongly by tumour necrosis factor alpha (TNFalpha) as well as by environmental stresses, whereas SEK1/MKK4 is not activated by TNFalpha. Column fractionation studies have shown that MKK7 is a major activator for SAPK/JNK in the TNFalpha-stimulated pathway. It is found that overexpression of MKK7 enhances transcription from an AP-1-dependent reporter construct. Thus, MKK7 is an evolutionarily conserved MAPKK isoform, specific for SAPK/JNK, involved in AP-1-dependent transcription and may be a crucial mediator of TNFalpha signaling (Moriguchi, 1997).

Mitogen-activated protein kinases (MAPKs) mediate many of the cellular effects of growth factors, cytokines and stress stimuli. Their activation requires the phosphorylation of a threonine and a tyrosine residue located in a Thr-X-Tyr motif (where X is any amino acid). This phosphorylation is catalysed by MAPK kinases (MKKs), which are all thought to be 'dual specificity' enzymes that phosphorylate both the threonine and the tyrosine residue of the Thr-X-Tyr motif. The MAPK family member known as stress-activated protein kinase-1c (SAPK1c, also known as JNK1) is activated synergistically in vitro by MKK4 (also called SKK1 and JNKK1) and MKK7 (also called SKK4 and JNKK2). MKK4 has a preference for the tyrosine residue, and MKK7 for the threonine residue, within the Thr-X-Tyr motif. These observations suggest that the full activation of SAPK1c in vivo may sometimes require phosphorylation by two different MKKs, providing the potential for integrating the effects of different extracellular signals. They also raise the possibility that other MAPK family members may be activated by two or more MKKs and that some MKKs may have gone undetected because they phosphorylate the tyrosine residue only, and therefore do not induce any activation unless the threonine has first been phosphorylated by another MKK (Lawler, 1998).

The c-Jun NH2-terminal protein kinase (JNK) is a member of the mitogen-activated protein kinase (MAPK) group and is an essential component of a signaling cascade that is activated by exposure of cells to environmental stress. JNK activation is regulated by phosphorylation on both Thr and Tyr residues by a dual-specificity MAPK kinase (MAPKK). Two MAPKKs, MKK4 and MKK7, have been identified as JNK activators. Genetic studies demonstrate that MKK4 and MKK7 serve nonredundant functions as activators of JNK in vivo. The molecular cloning of the gene that encodes MKK7 is reported and it is demonstrated that six isoforms are created by alternative splicing to generate a group of protein kinases with three different NH2 termini (alpha, beta, and gamma isoforms) and two different COOH termini (1 and 2 isoforms). The MKK7alpha isoforms lack an NH2-terminal extension that is present in the other MKK7 isoforms. This NH2-terminal extension binds directly to the MKK7 substrate JNK. Comparison of the activities of the MKK7 isoforms demonstrates that the MKK7alpha isoforms exhibit lower activity, but a higher level of inducible fold activation, than the corresponding MKK7beta and MKK7gamma isoforms. Immunofluorescence analysis demonstrates that these MKK7 isoforms are detected in both cytoplasmic and nuclear compartments of cultured cells. The presence of MKK7 in the nucleus is not, however, required for JNK activation in vivo. These data establish that the MKK4 and MKK7 genes encode a group of protein kinases with different biochemical properties that mediate activation of JNK in response to extracellular stimuli (Tournier, 1999).

A pleiotropic cytokine, tumor necrosis factor-alpha (TNF alpha), regulates the expression of multiple macrophage gene products and thus contributes a key role in host defense. The specificity and mechanism of activation of members of the c-Jun-NH2-terminal kinase/stress-activated protein kinase (JNK/SAPK) subfamily of mitogen-activated protein kinases (MAPKs) in mouse macrophages has been investigated in response to stimulation with TNF alpha. Exposure of macrophages to TNF alpha stimulates a preferential increase in catalytic activity of the p46 JNK/SAPK isoform, as compared with the p54 JNK/SAPK isoform. To investigate the level of regulation of p46 JNK/SAPK activation, an examination was made of the ability of MKK4/SEK1/JNKK (an upstream regulator of JNK/SAPKs) to phosphorylate recombinant kinase-inactive p46 and p54 JNK/SAPKs. Endogenous MKK4 is able to transphosphorylate both isoforms. Both the p46 and p54 JNK/SAPK isoforms are phosphorylated on their TPY motif in response to TNF alpha stimulation. Collectively, these results suggest that the level of control of p46 JNK/SAPK activation is distal not only to MKK4 but also to the p54 JNK/SAPK. Preferential isoform activation within the JNK/SAPK subfamily of MAPKs may be an important mechanism through which TNF alpha regulates macrophage phenotypic heterogeneity and differentiation (Chan, 1997).

Genetic evidence suggests that pheromone-induced cascades of mitogen-activated protein kinase (MAPK) in two strains of evolutionarily distant yeasts (S. cerevisiae and S. pombe) involve the function of the p21cdc42/racl-activated protein kinases (PAKs) Ste20 and Shk1, respectively. Purified Ste20 and Shk1 are each capable of inducing p42MAPK activation in cell-free extracts of Xenopus oocytes, while a mammalian Ste20/Shk1-related protein kinase, p65pak (Pak1), does not induce activation of p42MAPK. In contrast to p42MAPK, activation of JNK/SAPK (homolog of Drosophila basket.JNK) is induced in Xenopus oocyte extracts by both the yeast Ste20 and Shk1 kinases, as well as by mammalian Pak1. These results demonstrate that MAPK cascades that are responsive to PAKs are conserved in higher eukaryotes and suggest that distinct PAKs may regulate distinct MAPK modules (Polverino, 1995).

The mammalian Rac and Cdc42 proteins control formation of lamellipodia and filopodia, respectively. These proteins also activate MAP kinase (MAPK) cascades that regulate gene expression. Constitutively activated forms of Rac and Cdc42Hs are efficient activators of a cascade leading to JNK and p38/Mpk2 activation. RhoA does not exhibit this activity, and none of the proteins activate the extracellular signal-regulated kinase (ERK) subgroup of MAPKs. JNK (but not ERK) activation is also observed in response to Dbl, an oncoprotein that acts as a nucleotide exchange factor for Cdc42Hs. Results with dominant interfering alleles place Rac1 in an intermediate position, between Ha-Ras (See Drosophila Ras) and MEKK in the signaling cascade leading from growth factor receptors and v-Src to JNK activation. JNK and p38 activation are likely to contribute to the biological effects of Rac, Cdc42Hs, and Dbl on cell growth and proliferation (Minden, 1995).

MEK kinases (MEKKs) 1, 2, 3 and 4 are members of sequential kinase pathways that regulate MAP kinases including c-Jun NH2-terminal kinases (JNKs) and extracellular regulated kinases (ERKs). Confocal immunofluorescence microscopy of COS cells demonstrates differential MEKK subcellular localization: MEKK1 is nuclear and found in post-Golgi vesicular-like structures; MEKK2 and 4 are localized to distinct Golgi-associated vesicles that are dispersed by brefeldin A. MEKK1 and 2 are activated by EGF; kinase-inactive mutants of each MEKK partially inhibit EGF-stimulated JNK activity. Kinase-inactive MEKK1, but not MEKK2, 3 or 4, strongly inhibits EGF-stimulated ERK activity. In contrast to MEKK2 and 3, MEKK1 and 4 specifically associates with Rac and Cdc42; kinase-inactive mutants block Rac/Cdc42 stimulation of JNK activity. Inhibitory mutants of MEKK1-4 do not affect p21-activated kinase (PAK) activation of JNK, indicating that the PAK-regulated JNK pathway is independent of MEKKs. Thus, in different cellular locations, specific MEKKs are required for the regulation of MAPK family members, and MEKK1 and 4 are involved in the regulation of JNK activation by Rac/Cdc42, independent of PAK. Differential MEKK subcellular distribution and interaction with small GTP-binding proteins provides a mechanism to regulate MAP kinase responses in localized regions of the cell and to different upstream stimuli (Fanger, 1997).

JNKK, a dual-specificity kinase that activates JNK functions between MEKK and JNK. JNKK activates the JNKs but does not activate the ERKs, and is unresponsive to Raf-1 in transfected HeLa cells. JNKK also activates another MAPK, p38 (Mpk2; the mammalian homolog of HOG1 in yeast), whose activity is regulated similarly to that of the JNKs (Lin, 1995).

The mammalian p38 MAP kinase signal transduction pathway is activated by proinflammatory cytokines and environmental stress. The detection of p38 MAP kinase in the nucleus of activated cells suggests that p38 MAP kinase can mediate signaling to the nucleus. To test this hypothesis, expression vectors were constructed for activated MKK3 and MKK6, two MAP kinase kinases that phosphorylate and activate p38 MAP kinase. Expression of activated MKK3 and MKK6 in cultured cells cause a selective increase in p38 MAP kinase activity. Cotransfection experiments demonstrate that p38 MAP kinase activation causes increased reporter gene expression mediated by the transcription factors ATF2 and Elk-1. These data demonstrate that the nucleus is one target of the p38 MAP kinase signal transduction pathway (Raingeaud, 1995).

The identities of upstream activators of the MAP kinase homologs [stress-activated-protein (SAP) kinase-1 (also known as JNK or SAPK), and SAP kinase-2 (also known as p38, RK and CSBP)] have been investigated in rat PC12 cells and human KB cells after exposure to cellular stresses and cytokines. In PC12 cells, the same two upstream activators [SAP kinase kinase-1 (SAPKK-1) and SAPKK-2] are activated after exposure to osmotic shock, ultraviolet irradiation or the protein synthesis inhibitor, anisomycin, and more weakly in response to sodium arsenite. SAPKK-1 is capable of activating both SAP kinase-1 and SAP kinase-2 and is similar, if not identical, to the MAP kinase kinase homolog MKK4, as judged by immunological criteria and by its ability to be activated by MEK kinase in vitro. In contrast, SAPKK-2 activatesSAP kinase-2, but not SAP kinase-1 in vitro. In KB cells, five distinct upstream activators of SAP kinase-1 and SAP kinase-2 were induced, namely SAPKK-1, SAPKK-2, SAPKK-3, SAPKK-4 and SAPKK-5; their appearance depends on the nature of the stimulus. These results demonstrate unexpected complexity in the upstream regulation of stress and cytokine-stimulated kinase cascades and indicate that the selection of the appropriate SAPKK varies with both the stimulus and the cell type (Meier, 1996).

The activity of c-Jun, the major component of the transcription factor AP-1, is potentiated by amino-terminal phosphorylation on serines 63 and 73 (Ser-63/73). This phosphorylation is mediated by the Jun amino-terminal kinase (JNK) and required to recruit the transcriptional coactivator CREB-binding protein (CBP). AP-1 function is antagonized by activated members of the steroid/thyroid hormone receptor superfamily. Recently, a competition for CBP has been proposed as a mechanism for this antagonism. Hormone-activated nuclear receptors prevent c-Jun phosphorylation on Ser-63/73, and consequently, AP-1 activation as well, by blocking the induction of the JNK signaling cascade. Consistently, nuclear receptors also antagonize other JNK-activated transcription factors such as Elk-1 and ATF-2. It is shown here that dexamethasone, a glucocorticoid receptor agonist, and two other nuclear hormone receptors, the retinoic acid receptor and the thyroid hormone receptor, also block c-Jun activation by a mechanism that is (1) independent of the c-Jun DNA binding domain and is one which (2) relies specifically on the c-Jun amino-terminal phosphorylation step. Interference with the JNK signaling pathway represents a novel mechanism by which nuclear hormone receptors antagonize AP-1. This mechanism is based on the blockade of the AP-1 activation step, which is a requisite for interaction with CBP. In addition to acting directly on gene transcription, regulation of the JNK cascade activity constitutes an alternative mode whereby steroids and retinoids may control cell fate and conduct their pharmacological actions as immunosupressive, anti-inflammatory, and antineoplastic agents. Nuclear receptor interference would rely on the inhibition of MEKK activity or a downstream step in the pathway. Dexamethasone can also inhibit a constitutively active MAPK pathway (Caelles, 1997).

The c-Jun NH2-terminal kinase (JNK) group of mitogen-activated protein (MAP) kinases is activated by the exposure of cells to multiple forms of stress. A putative scaffold protein has been identified that interacts with multiple components of the JNK signaling pathway, including the mixed-lineage group of MAP kinase kinase kinases (MLK), the MAP kinase kinase MKK7, and the MAP kinase JNK. This scaffold protein selectively enhanced JNK activation by the MLK signaling pathway. These data establish that a mammalian scaffold protein can mediate activation of a MAP kinase signaling pathway (Whitmarsh, 1998).

Regulation of JNK upstream of MAPKK: The cytoskeleton connection

The PAK family of protein kinases has been suggested as a potential target of the Cdc42 and Rac GTPases, based on studies in vitro. PAK-3 is activated by Cdc42 in vivo. Both the activated (GTPase-defective) Cdc42 and a constitutively active PAK-3 mutant stimulate the activity of Jun kinase 1 (JNK1) in transfected cells. Activated Cdc42 also stimulates the activity of the related p38 mitogen-activated protein kinase but was a less effective activator of ERK2. The effect of Cdc42 on JNK activity is similar to that of the potent inflammatory cytokine interleukin-1 (IL-1). The observation that a dominant-negative Cdc42 mutant inhibits IL-1 activation of JNK1 indicates a role for Cdc42 in IL-1 signaling. These results suggest that Cdc42 and PAK may mediate the effects of cytokines on transcriptional regulation (Bagrodia, 1995).

Contractile activity plays a critical role in the regulation of gene transcription in skeletal muscle; in turn, this determines muscle functional capabilities. However, little is known about the molecular signaling mechanisms that convert contractile activity into gene regulatory responses in skeletal muscle. In the current study, the effects of contractile activity in vivo were determined on the c-Jun NH2-terminal kinase (JNK) pathway, a signaling cascade that has been implicated in the regulation of transcription. Electrical stimulation of the sciatic nerve to produce contractions in anaesthetized rats increases JNK activity by up to 7-fold above basal. Maximal enzyme activity occurs at 15 min of contraction and remains elevated at 60 min of contraction. The upstream activators of JNK, the mitogen-activated protein kinase kinase 4 and the mitogen-activated protein kinase kinase kinase 1 follow a similar time course of activation in response to contractile activity. In contrast, contraction induces a rapid and transient activation of the extracellular-regulated kinase pathway, indicating that the regulation of JNK signaling is distinct from that of extracellular-regulated kinase. The activation of the JNK signaling cascade is temporally associated with an increased expression of c-jun mRNA. These results demonstrate that contractile activity regulates JNK activity in skeletal muscle and suggest that activation of JNK may regulate contraction-induced gene expression in skeletal muscle (Aronson, 1997).

In COS-7 cells, activated Ras effectively stimulates MAPK but poorly induces JNK activity. In contrast, mutationally activated Rac1 and Cdc42 GTPases potently activate JNK without affecting MAPK; oncogenic guanine nucleotide exchange factors for these Rho-like proteins selectively stimulate JNK activity. Furthermore, expression of inhibitory molecules for Rho-related GTPases and dominant negative mutants of Rac1 and Cdc42 block JNK activation by oncogenic exchange factors or after induction by inflammatory cytokines and growth factors. Taken together, these findings strongly support a critical role for Rac1 and Cdc42 in controlling the JNK signaling pathway (Coso, 1995).

c-Jun NH2-terminal protein kinase (JNK), a distant member of the mitogen-activated protein (MAP) kinase family, regulates gene expression in response to various extracellular stimuli. JNK is activated by JNK-activating kinase 1 (JNKK1), a dual specificity protein kinase that phosphorylates JNK on threonine 183 and tyrosine 185 residues. JNKK2, a novel member of the MAP kinase kinase family, is phosphorylated and activated by MEKK1, a MAP kinase kinase kinase in the JNK signaling cascade. JNKK2 activity is also stimulated by constitutively active forms of Rac and Cdc42Hs, members of the Rho small GTP-binding protein family. Unlike JNKK1, which activates both JNK and p38 MAP kinases, JNKK2 stimulates only JNK. Transient transfection assays demonstrate that JNKK2 potentiates the stimulation of c-Jun transcriptional activity by MEKK1. The existence of multiple JNK-activating kinases may contribute to the specificity of the JNK signaling cascade (Lu, 1997).

Several members of the Rho family of small guanosine triphosphatases (GTPases) regulate the organization of the actin cytoskeleton: Rho controls the assembly of actin stress fibers and focal adhesion complexes; Rac regulates actin filament accumulation at the plasma membrane to produce lamellipodia and membrane ruffles, and Cdc42 stimulates the formation of filopodia. When microinjected into quiescent fibroblasts, Rho, Rac, and Cdc42 stimulate cell cycle progression through G1 and subsequent DNA synthesis. Furthermore, microinjection of the dominant negative forms of Rac and Cdc42 or of the Rho inhibitor C3 transferase blocks serum-induced DNA synthesis. Unlike Ras, none of the Rho GTPases activate the MAPK cascade that contains the protein kinases c-Raf1, MEK (MAPK or ERK kinase), and ERK. Instead, Rac and Cdc42, but not Rho, stimulate a distinct MAP kinase, the c-Jun kinase JNK/SAPK (Jun NH2-terminal kinase or stress-activated protein kinase). In this manner, Rho, Rac, and Cdc42 control signal transduction pathways essential for cell growth (Olson, 1995).

The DH domain protein mNET1, a Rho-family guanine nucleotide exchange factor (GEF) has been characterized. N-terminal truncation of mNET1 generates an activated transforming form of the protein, mNET1DeltaN, which acts as a GEF for RhoA but not Cdc42 or Rac1. In NIH 3T3 cells, activated mNET1 induces formation of actin stress fibers and potentiates activity of the transcription factor serum response factor. Inhibitor studies show that these processes are dependent on RhoA and independent of Cdc42 or Rac1. However, in contrast to the GTPase-deficient RhoA.V14 mutant, expression of activated mNET1 also activates the SAPK/JNK pathway. This requires mNET1 GEF activity, since activation is blocked by point mutations in mNET1's DH domain and its C-terminal pleckstrin homology (PH) domain, and by the dominant-interfering RhoA mutant RhoA.N19. Although mNET1DeltaN-induced SAPK/JNK activation requires a C3 transferase-sensitive GTPase, activation occurs independent of the generation of titratable GTP-bound RhoA. Thus, mNET1 can activate signaling pathways in addition to those directly controlled by activated RhoA (Alberts, 1998b).

The cytoskeletal reorganizing functions of Rac and Cdc-42 can be shown to be separate from their abilities to activate downstream targets. Besides activating JNK and SAPK MAP kinase cascades, Rac and Cdc-42 regulate formation of lamellipodia and filopodia, both of which involve formation of polymerized actin structures and the assembly of associated integrin complexes. The modification of cytoskeleton mediated by Rac and Cdc-42 is activated by extracellular factors such as lysophosphatidic acid, bombesin, PDGF, EGF (Drosophila homolog: Spitz) and insulin. Rac and Cdc-42 control MAP kinase pathways and actin cytoskeleton organization independently through distinct downstream targets. Rac and Cdc42 contain a Serine 40C effector site. Mutations of this site result in proteins that can no longer interact with the Serine/Threonine kinase p65PAK; these are proteins that are unable to activate the JNK MAP kinase pathway. However, these specific mutants of Rac and Cdc42 can still induce cytoskeletal changes and G1 cell cycle progression. Rac containing an F37A effector site substitution, on the other hand, no longer interacts with the Ser/Thr kinase p160ROCK and is unable to induce lamellipodia or G1 progression. Thus Rac and Cdc42 induce actin polymerization and G1 cell cycle progression independently of p65PAK and the JNK/SAPK MAP kinase cascade (Lamarche, 1996).

The MLK (mixed lineage) ser/thr kinases are most closely related to the MAP kinase kinase kinase family. In addition to a kinase domain, MLK1, MLK2 and MLK3 each contain an SH3 domain, a leucine zipper domain and a potential Rac/Cdc42 GTPase-binding (CRIB) motif. The C-terminal regions of the proteins are essentially unrelated. Using yeast two-hybrid analysis and in vitro dot-blots, it has been shown that MLK2 and MLK3 interact with the activated (GTP-bound) forms of Rac and Cdc42, with a slight preference for Rac. Transfection of MLK2 into COS cells leads to strong and constitutive activation of the JNK (c-Jun N-terminal kinase) MAP kinase cascade, but also to activation of ERK (extracellular signal-regulated kinase) and p38. When expressed in fibroblasts, MLK2 co-localizes with active, dually phosphorylated JNK1/2 to punctate structures along microtubules. In an attempt to identify proteins that affect the activity and localization of MLK2, a yeast two-hybrid cDNA library has been screened. MLK2 and MLK3 interact with members of the KIF3 family of kinesin superfamily motor proteins and with KAP3A, the putative targeting component of KIF3 motor complexes, suggesting a potential link between stress activation and motor protein function (Nagata, 1998).

Most transformed cells have lost anchorage and serum dependence for growth and survival. When serum is absent, fibronectin survival signals transduced by focal adhesion kinase (FAK), suppress p53-regulated apoptosis in primary fibroblasts and endothelial cells. This study sought to identify survival sequences in FAK and signaling molecules downstream of FAK required for anchorage-dependent survival of primary fibroblasts. Binding of the SH3 domain of p130Cas (see CAS/CSE1 segregation protein) to proline-rich region 1 of FAK is required to support survival of fibroblasts on fibronectin when serum is withdrawn. The FAK-p130Cas complex activates c-Jun NH2-terminal kinase (JNK) via a Ras/Rac1/Pak1/MAPK kinase 4 (MKK4) pathway. Activated (phospho-) JNK colocalizes with FAK in focal adhesions of fibroblasts cultured on fibronectin, which supports their survival, but JNK does not colocalize with FAK in fibroblasts cultured on collagen. which does not support survival. Cells often survive in the absence of extracellular matrix if serum factors are provided. In that case, survival signals are transduced by FAK, phosphatidylinositol 3'-kinase (PI3-kinase), and Akt/protein kinase B (PKB). However, when serum is absent, PI3-kinase and Akt/PKB are not involved in the fibronectin-FAK-JNK survival pathway documented here. Thus, survival signals from extracellular matrix and serum are transduced by FAK via two distinct pathways (Almeida, 2000).

G-protein coupled receptors regulate JNK

ß-arrestins, originally discovered in the context of heterotrimeric guanine nucleotide binding protein-coupled receptor (GPCR) desensitization, also function in internalization and signaling of these receptors. c-Jun amino-terminal kinase 3 (JNK3) has been identified as a binding partner of ß-arrestin 2 using a yeast two-hybrid screen and by coimmunoprecipitation from mouse brain extracts or cotransfected COS-7 cells. The upstream JNK activators apoptosis signal-regulating kinase 1 (ASK1) and mitogen-activated protein kinase (MAPK) kinase 4 were also found in complex with ß-arrestin 2. Cellular transfection of ß-arrestin 2 causes cytosolic retention of JNK3 and enhanced JNK3 phosphorylation stimulated by ASK1. Moreover, stimulation of the angiotensin II type 1A receptor activates JNK3 and triggers the colocalization of ß-arrestin 2 and active JNK3 to intracellular vesicles. Thus, ß-arrestin 2 acts as a scaffold protein, which brings the spatial distribution and activity of this MAPK module under the control of a GPCR (McDonald, 2000).

In budding yeast, Ste5 is a scaffold protein that forms a multicomponent complex with the Fus3 (Kss1) MAPK, Ste7 MAPKK, and Ste11 MAPKKK to facilitate the specific and efficient activation of the mating pheromone pathway. Also in yeast, Pbs2, which itself is a MAPKK, has been proposed as a possible scaffold protein in the HOG (high-osmolarity glycerol response) signal transduction pathway. An intriguing similarity between Ste5 and ß-arrestin 2 is that both are recruited to the plasma membrane as a consequence of agonist stimulation of a GPCR. In mammalian cells, a group of JNK-interacting proteins (JIP1, -2 and -3) have been identified as scaffolding proteins for specific JNK signaling modules (McDonald, 2000 and references therein).

Like members of the JIP family, ß-arrestin 2 associates with all three kinase components of a single MAPK cascade, thus enhancing signaling efficiency. However, unlike JIPs, the ß-arrestin 2-MAPK module is regulated by agonist stimulation of GPCRs. It is likely that individual JNK isoforms exhibit distinct patterns of regulation. For example, as demonstrated in this study, JNK3 activity appears to be specifically enhanced by ß-arrestin 2, whereas JNK1 activity is unaffected. Thus, formation of similar complexes may prevent inappropriate cross talk between the various MAPK pathways (McDonald, 2000).

JNK1 is activated in response to several GPCRs. The association of JNK3 with ß-arrestin 2 provides a mechanism whereby ß-arrestin 2 might localize JNK3 to specific subcellular compartments and/or target JNK3 to specific substrates in response to GPCR agonists. The results reported here add to a growing list of functions subserved by ß-arrestins in regulating signaling through heptahelical receptors. By acting as a scaffold for a specific MAPK pathway, ß-arrestin 2 provides a mechanism for bringing this signaling module under the control of such receptors. Other evidence (suggests that ß-arrestins may also play roles in organizing pathways leading from GPCRs to activation of the ERKs (McDonald, 2000).

Non-canonical WNT pathway results in JNK activation

A pathway regulating convergent extension movements during gastrulation in vertebrate embryos has been shown to be a vertebrate equivalent of the planar cell polarity (PCP) pathway. However, it is not known whether the JNK pathway functions in this non-canonical Wnt pathway to regulate convergent extension movements in vertebrates. In addition, it is not known whether JNK is in fact activated by Wnt stimulation. This study shows that Wnt5a is capable of activating JNK in cultured cells, and evidence is presented that the JNK pathway mediates the action of Wnt5a to regulate convergent extension movements in Xenopus. These results thus demonstrate that the non-canonical Wnt/JNK pathway is conserved in both vertebrate and invertebrate and establish that JNK has an activity that regulates morphogenetic cell movements (Yamanaka, 2002).

These results suggest that the appropriate activation of JNK is necessary for correct convergent extension. It has previously been shown that overexpressed Dishevelled inhibits the convergent extension. Antisense JNK MO cancels significantly the overexpressed Dishevelled-induced inhibition of the convergent extension movements of explants, suggesting that Dishevelled lies upstream of JNK. Since recent reports indicate that in zebrafish and Xenopus embryos, the activity of Wnt11, a member of the Wnt5a class ligands, is required for cells to undergo correct convergent extension movements, the JNK pathway in vivo may lie downstream of Wnt11 rather than Wnt5a, or both Wnt5a and Wnt11 may function redundantly. In any case, it can be concluded that the non-canonical Wnt/JNK pathway is conserved evolutionarily in both vertebrates and invertebrates, since previous studies have demonstrated that a pathway regulating convergent extension in developing vertebrate embryos is equivalent to the PCP pathway (also known as the non-canonical Wnt pathway) in Drosophila, and that the PCP pathway in Drosophila signals via the JNK pathway to control cell polarity. The mechanism by which JNK regulates convergent extension movements in vertebrates remains to be established. Preliminary data suggest that activated JNK affects cell-cell adhesion. This is in good agreement with the previous report indicating that Wnt5a is able to decrease the cell-cell adhesion. Furthermore, a study in Drosophila has suggested a role for the transcription factor c-Jun, one of major targets of JNK, in the PCP pathway. It is thus possible that the JNK/c-Jun-mediated gene expression is also important for regulation of the morphogenetic cell movements. Identifying molecular components upstream and downstream of the MKK7/JNK cascade in the non-canonical Wnt pathway in vertebrates is a priority for future research (Yamanaka, 2002 and references therein).

Involving dynamic and coordinated cell movements that cause drastic changes in embryo shape, gastrulation is one of the most important processes of early development. Gastrulation proceeds by various types of cell movements, including convergence and extension, during which polarized axial mesodermal cells intercalate in radial and mediolateral directions and thus elongate the dorsal marginal zone along the anterior-posterior axis. A noncanonical Wnt signaling pathway, which is known to regulate planar cell polarity (PCP) in Drosophila, participates in the regulation of convergent extension movements in Xenopus as well as in the zebrafish embryo. The Wnt5a/Wnt11 signal is mediated by members of the seven-pass transmembrane receptor Frizzled (Fz) and the signal transducer Dishevelled (Dsh) through the Dsh domains that are required for the PCP signal. It has also been shown that the relocalization of Dsh to the cell membrane is required for convergent extension movements in Xenopus gastrulae. Although it appears that signaling via these components leads to the activation of JNK and rearrangement of microtubules, the precise interplay among these intercellular components is largely unknown. In this study, it is shown that Xenopus prickle (Xpk), a Xenopus homolog of a Drosophila PCP gene, is an essential component for gastrulation cell movement. Xpk encodes an 835-amino acid protein with a single PET domain and three repetitive LIM domains in its N-terminal half. Both gain-of-function and loss-of-function of Xpk severely perturbs gastrulation and causes spina bifida embryos without affecting mesodermal differentiation. XPK binds to Xenopus Dsh as well as to JNK. This suggests that XPK plays a pivotal role in connecting Dsh function to JNK activation (Takeuchi, 2003).

The possibility that XPK activates JNK was examined because JNK has been reported to act in the noncanonical Wnt pathway downstream of Dsh. To evaluate JNK activation, the phosphorylation of a target of JNK, c-Jun, was tested in HEK293T cells transfected with Xdsh, Xpk, or both cDNAs. Xpk alone failed to activate JNK, whereas Xdsh activates JNK in a dose-dependent manner. However, cotransfection of Xdsh with Xpk but not with ΔP/L (lacking the PET and LIM domains) cDNAs dramatically increase the level of c-Jun phosphorylation, even at Xdsh levels, which alone cannot activate JNK efficiently. This result suggests that XPK cooperates with Xdsh to activate JNK through its P/L domain. At a high level of Xdsh protein, Xdsh alone can activate JNK to a certain level, and interestingly, wild-type Xpk or ΔP/L but not P/L suppresses the JNK activation by Xdsh, suggesting that the part of XPK excluding the PET/LIM domain may act negatively to Xdsh-mediated JNK activation at high levels of Xdsh. This is consistent with the observation that ΔP/L and Xpk including the LIM domain counteract each other. These observations prompted a test of whether XPK interacts physically with Xdsh. To test this possibility, a GST-pull-down assay of tagged XPK and Xdsh was carried out in HEK293T cells. Xdsh and XPK are each efficiently precipitated with the other's GST fusion protein, indicating that XPK and Xdsh interacted physically with each other. The interaction between XPK and Xdsh is conserved among species; the Drosophila Prickle PET/LIM domain has been reported to bind Dsh. The yeast two-hybrid assay also demonstrates that the PET/LIM domains of XPK are sufficient to bind Xdsh (Takeuchi, 2003).

It is speculated that XPK might act as a scaffold for JNK activation, so whether XPK binds to JNK was tested. Neither the PET/LIM nor the ΔPET/LIM is sufficient to bind JNK, and only wild-type XPK can bind JNK significantly. Nevertheless, this further suggests that XPK forms a ternary complex with Xdsh and JNK. Although this possibility was examined, the formation of the ternary complex could not be demonstrated (Takeuchi, 2003).

It has been proposed that Prickle generates asymmetric Frizzled and Dishevelled localization in the Drosophila wing, through the suppression of Fz and Dsh localization at the proximal cell cortex. In this study, it is shown that XPK is a key component connecting Xdsh to JNK activation during Xenopus gastrulation. It has been predicted from Drosophila genetics that JNK is one of the downstream targets of the PCP pathway. These results reinforce the idea that the noncanonical Wnt (PCP) pathway regulates gastrulation cell movements in vertebrates through JNK activation. To further understand the pathway, attempts are currently being made to identify XPK-interacting components that regulate JNK activation (Takeuchi, 2003).

Protein kinase C (PKC) has been implicated in the Wnt signaling pathway; however, its molecular role is poorly understood. The PKC family is subdivided into three subfamilies: the classical, novel, and atypical PKCs (cPKC, nPKC, and aPKC, respectively). cPKC is activated by Ca2+ and diacylglycerol (DAG), nPKC is activated by DAG but not by Ca2+, and aPKC is not activated by these molecules. Novel genes encoding delta-type PKC have been identified in the Xenopus EST databases. Loss of PKCdelta (a member of the nPKC subfamily) function reveals that it is essential for convergent extension during gastrulation. The relationship between PKCdelta and the Wnt pathway was examined. PKCdelta is translocated to the plasma membrane in response to Frizzled signaling. In addition, loss of PKCdelta function inhibits the translocation of Dishevelled and the activation of c-Jun N-terminal kinase (JNK) by Frizzled. Furthermore, PKCdelta forms a complex with Dishevelled, and the activation of PKCdelta by phorbol ester is sufficient for Dishevelled translocation and JNK activation. Thus, PKCdelta plays an essential role in the Wnt/JNK pathway by regulating the localization and activity of Dishevelled (Kinoshita, 2003).

Xenopus PKCdelta has a highly conserved C1 domain, which binds to DAG and phorbol esters such as PMA, a functional analog of DAG. PKCdelta was translocated to the plasma membrane in animal cap cells in response to both Xfz7 and PMA. These results and other observations suggested that Xfz7 might activate PKCdelta through DAG on the plasma membrane, although there is no direct evidence that activation of the Wnt/Frizzled pathway produces DAG. However, heterotrimeric G proteins have been implicated in the Wnt/Frizzled pathway. It has been shown that certain heterotrimeric G proteins coupled with seven-transmembrane receptors activate phospholipase C-ß, which hydrolyzes phosphatidylinositol phosphate to produce DAG and inositol triphosphate. In addition, Xfz7 function is blocked by pertussis toxin, which inhibits the Gi family. Taken together, these findings suggest that Xfz7 probably activates PKCdelta through a heterotrimeric G protein that produces DAG. It will be important to determine which G protein is involved in this pathway and whether DAG is produced by G protein function (Kinoshita, 2003).

Xdsh and PKCdelta form a complex and the complex formation is not dependent on PKCdelta activity. In addition, the activation of PKCdelta is sufficient and necessary for the membrane localization of Xdsh in response to Xfz7. These findings suggest that Xfz7 may be involved in the translocation of the PKCdelta-Xdsh complex to the plasma membrane through the production of DAG. In other words, PKCdelta recruits Xdsh to the membrane in response to Xfz7 signaling. It will be necessary to determine which domain of Xdsh interacts with PKCdelta and vice versa. Preliminary work shows that a C-terminal fragment including the DEP domain of Xdsh coimmunoprecipitates with PKCdelta as well as the full-length Xdsh protein. This is consistent with the fact that this domain of Dishevelled is sufficient for its membrane translocation and function in the PCP pathway (Kinoshita, 2003 and references therein).

The Dishevelled protein is known to be hyperphosphorylated in response to Wnt and Frizzled. The loss of PKCdelta function blocks this phosphorylation of Xdsh. It has been shown that the phosphorylation and membrane localization of Xdsh are closely related. The simplest model is that DAG activates PKCdelta on the membrane, and PKCdelta phosphorylates Xdsh directly. PKCalpha has been shown to phosphorylate Xdsh in vitro. PKCdelta may have the similar activity. However, Dishevelled is known to interact with other kinases, such as casein kinases 1 and 2, Par-1, and PAK1/MuSK. PKCdelta may regulate such protein kinases and thus indirectly regulate Xdsh phosphorylation. It would be interesting to examine whether PKCdelta phosphorylates Xdsh directly, and to elucidate the role of Xdsh phosphorylation in its localization and in the activation of downstream signaling. Determination of the sites in Xdsh that are phosphorylated by Xfz7 signaling awaits further study (Kinoshita, 2003).

The following three results indicate that PKCdelta mediates the activation of JNK by Xfz7: (1) JNK activation by Xfz7 was inhibited by the loss of PKCdelta function. (2) The activation of PKCdelta by PMA was sufficient for JNK activation. (3) The gastrulation-defective phenotype of PKCdelta MO is rescued by active MKK7, which activates JNK. JNK has been implicated in the noncanonical Wnt pathway, but it is still unknown how Xdsh activates the JNK pathway. The membrane localization and/or phosphorylation of Xdsh may enable other proteins such as Rho to interact with Xdsh to activate the JNK cascade. It will be interesting and important to learn how JNK regulates convergent extension movements during gastrulation (Kinoshita, 2003).

Wnt11 is a secreted protein that signals through the non-canonical planar cell polarity pathway and is a potent modulator of cell behavior and movement. In human, mouse, and chicken, there is a single Wnt11 gene, but in zebrafish and Xenopus, there are two genes related to Wnt11. The originally characterized Xenopus Wnt11 gene is expressed during early embryonic development and has a critical role in regulation of gastrulation movements. A second Xenopus Wnt11-Related gene (Wnt11-R) has been identified that is expressed after gastrulation. Sequence comparison suggests that Xenopus Wnt11-R, not Wnt11, is the ortholog of mammalian and chicken Wnt11. Xenopus Wnt11-R is expressed in neural tissue, dorsal mesenchyme derived from the dermatome region of the somites, the brachial arches, and the muscle layer of the heart, similar to the expression patterns reported for mouse and chicken Wnt11. Xenopus Wnt11-R exhibits biological properties similar to those previously described for Xenopus Wnt11, in particular the ability to activate Jun-N-terminal kinase (JNK) and to induce myocardial marker expression in ventral marginal zone (VMZ) explants. Morpholino inhibition experiments demonstrate, however, that Wnt11-R is not required for cardiac differentiation, but functions in regulation of cardiac morphogenesis. Embryos with reduced Wnt11-R activity exhibit aberrant cell-cell contacts within the myocardial wall and defects in fusion of the nascent heart tube (Garriock, 2005).

In Drosophila, activation of Jun N-terminal Kinase (JNK) mediated by Frizzled and Dishevelled leads to signaling linked to planar cell polarity. A biochemical delineation of WNT-JNK planar cell polarity was sought in mammalian cells, making use of totipotent mouse F9 teratocarcinoma cells that respond to WNT3a via Frizzled-1. The canonical WNT-β-catenin signaling pathway requires both Gαo and Gαq heterotrimeric G-proteins, whereas this study shows that WNT-JNK signaling requires only Gαo protein. Gαo propagates the signal downstream through all three Dishevelled isoforms, as determined by epistasis experiments using the Dishevelled antagonist Dapper1 (DACT1). Suppression of either Dishevelled-1 or Dishevelled-3, but not Dishevelled-2, abolishes WNT3a activation of JNK. Activation of the small GTPases RhoA, Rac1 and Cdc42 operates downstream of Dishevelled, linking to the MEKK 1/MEKK 4-dependent cascade, and on to JNK activation. Chemical inhibitors of JNK (SP600125), but not p38 (SB203580), block WNT3a activation of JNK, whereas both the inhibitors attenuate the WNT3a-β-catenin pathway. These data reveal both common and unique signaling elements in WNT3a-sensitive pathways, highlighting crosstalk from WNT3a-JNK to WNT3a-β-catenin signaling (Bikkavilli, 2008).

Table of contents

basket/JNK: Biological Overview | Regulation | Protein Interactions | Developmental Biology | Effects of Mutation | References

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