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.     

A scaffold protein in the c-Jun NH2-terminal kinase signaling pathways suppresses the extracellular signal-regulated kinase signaling pathways

We previously reported that c-Jun NH(2)-terminal kinase (JNK)/stress-activated protein kinase-associated protein 1 (JSAP1) functions as a putative scaffold factor in the JNK mitogen-activated protein kinase (MAPK) cascades. In that study we also found MEK1 and Raf-1, which are involved in the extracellular signal-regulated kinase (ERK) MAPK cascades, bind to JSAP1. Here we have defined the regions of JSAP1 responsible for the interactions with MEK1 and Raf-1. Both of the binding regions were mapped to the COOH-terminal region (residues 1054-1305) of JSAP1. We next examined the effect of overexpressing JSAP1 on the activation of ERK by phorbol 12-myristate 13-acetate in transfected COS-7 cells and found that JSAP1 inhibits ERK's activation and that the COOH-terminal region of JSAP1 was required for the inhibition. Finally, we investigated the molecular mechanism of JSAP1's inhibitory function and showed that JSAP1 prevents MEK1 phosphorylation and activation by Raf-1, resulting in the suppression of the activation of ERK. Taken together, these results suggest that JSAP1 is involved both in the JNK cascades, as a scaffolding factor, and the ERK cascades, as a suppressor.     

JSAP1/JIP3 cooperates with focal adhesion kinase to regulate c-Jun N-terminal kinase and cell migration

c-Jun N-terminal kinase (JNK)/stress-activated protein kinase-associated protein 1 (JSAP1) (also termed JNK-interacting protein 3; JIP3) is a member of a family of scaffold factors for the mitogen-activated protein kinase (MAPK) cascades, and it also forms a complex with focal adhesion kinase (FAK). Here we demonstrate that JSAP1 serves as a cooperative scaffold for activation of JNK and regulation of cell migration in response to fibronectin (FN) stimulation. JSAP1 mediated an association between FAK and JNK, which was induced by either co-expression of Src or attachment of cells to FN. Complex formation of FAK with JSAP1 and p130 Crk-associated substrate (p130(Cas)) resulted in augmentation of FAK activity and phosphorylation of both JSAP1 and p130(Cas), which required p130(Cas) hyperphosphorylation and was abolished by inhibition of Src. JNK activation by FN was enhanced by JSAP1, which was suppressed by disrupting the FAK/p130(Cas) pathway by expression of a dominant-negative form of p130(Cas) or by inhibiting Src. We also documented the co-localization of JSAP1 with JNK and phosphorylated FAK at the leading edge and stimulation of cell migration by JSAP1 expression, which depended on its JNK binding domain and was suppressed by inhibition of JNK. The level of JSAP1 mRNA correlated with advanced malignancy in brain tumors, unlike other JIPs. We propose that the JSAP1.FAK complex functions cooperatively as a scaffold for the JNK signaling pathway and regulator of cell migration on FN, and we suggest that JSAP1 is also associated with malignancy in brain tumors.     

Phosphorylation-dependent scaffolding role of JSAP1/JIP3 in the ASK1-JNK signaling pathway. A new mode of regulation of the MAP kinase cascade

JSAP1 (also termed JIP3) is a scaffold protein that interacts with specific components of the JNK signaling pathway. Apoptosis signal-regulating kinase (ASK) 1 is a MAP kinase kinase kinase that activates the JNK and p38 mitogen-activated protein (MAP) kinase cascades in response to environmental stresses such as reactive oxygen species. Here we show that JSAP1 bound ASK1 and enhanced ASK1- and H(2)O(2)-induced JNK activity. ASK1 phosphorylated JSAP1 in vitro and in vivo, and the phosphorylation facilitated interactions of JSAP1 with SEK1/MKK4, MKK7 and JNK3. Furthermore, ASK1-dependent phosphorylation was required for JSAP1 to recruit and thereby activate JNK in response to H(2)O(2). We thus conclude that JSAP1 functions not only as a simple scaffold, but it dynamically participates in signal transduction by forming a phosphorylation-dependent signaling complex in the ASK1-JNK signaling module.     

In vitro development of mouse embryonic stem cells lacking JNK/stress-activated protein kinase-associated protein 1 (JSAP1) scaffold protein revealed its requirement during early embryonic neurogenesis

The Jsap1 gene encodes a scaffold protein for c-Jun N-terminal kinase cascades. We established c-Jun N-terminal kinase (JNK)/stress-activated protein kinase-associated protein 1 (JSAP1)-null mouse embryonic stem cell lines by homologous recombination. The JSAP1-null embryonic stem cells were viable, however, exhibited hyperplasia of the ectoderm during embryoid body formation, and spontaneously differentiated into neurons more efficiently than did wild type. The expression of components of c-Jun N-terminal kinase cascades and a subset of marker mRNAs during early embryogenesis was altered in the JSAP1-null mutants. Retinoic acid dramatically increased the expression of JSAP1 and JNK3, which were co-precipitated with anti-JNK3 in the neuroectoderm of wild type but not JSAP1-null embryoid bodies. In the neurons differentiated from the wild type embryoid bodies, JSAP1 was localized in the soma, neurites, and growth cone-like structure of the neurites, and neurite outgrowth from the JSAP1-null embryoid bodies was apparently less efficient than from wild type. JSAP1 and c-Jun N-terminal kinase 3 were coexpressed in the embryonic ectoderm of E7.5 mouse embryo, whereas Wnt1 and Pax2 were coexpressed with JSAP1 at the midbrain-hindbrain junction in E12.5 mouse embryo, thus suggesting that JSAP1 is required for early embryonic neurogenesis.     

Protein kinase D specifically mediates apoptosis signal-regulating kinase 1-JNK signaling induced by H2O2 but not tumor necrosis factor

Although both tumor necrosis factor (TNF) and H2O2 induce activation of c-Jun N-terminal kinase (JNK) kinase cascades, it is not known whether they utilize distinct intracellular signaling pathways. In this study, we first examined a variety of pharmacological inhibitors on TNF and H2O2-induced JNK activation. Go6983 or staurosporine, which inhibits protein kinase C isoforms had no effects on TNF or H2O2-induced JNK activation. However, Go6976 and calphostin, which can inhibit protein kinase C as well as protein kinase D (PKD), blocked H2O2- but not TNF-induced JNK activation, suggesting that PKD may be specifically involved in H2O2-induced JNK activation. Consistently, H2O2, but not TNF, induced phosphorylation of PKD and translocation of PKD from endothelial cell membrane to cytoplasm where it associates with the JNK upstream activator, apoptosis signal-regulating kinase 1 (ASK1). The association is mediated through the pleckstrin homology domain of PKD and the C-terminal domain of ASK1. Inhibition of PKD by Go6976 or by small interfering RNA of PKD blocked H2O2-induced ASK1-JNK activation and endothelial cell apoptosis. Interestingly, H2O2 induced 14-3-3 binding to PKD via the phospho-Ser-205/208 and phospho-Ser-219/223 and H2O2-induced 14-3-3 binding of PKD was specifically blocked by Go6976 but not by Go6983. More significantly, the 14-3-3-binding defective forms of PKD failed to associate with ASK1 and to activate JNK signaling, highlighting the importance of 14-3-3 binding of PKD in H2O2-induced activation of ASK1-JNK cascade. Thus, our data have identified PKD as a critical mediator in H2O2- but not TNF-induced ASK1-JNK signaling.     

Expression and distribution of JNK/SAPK-associated scaffold protein JSAP1 in developing and adult mouse brain

The c-Jun N-terminal kinase (JNK) is one of the three major mitogen-activated protein kinases (MAPKs) playing key roles in various cellular processes in response to both extracellular and intracellular stimuli. JNK/SAPK-associated protein 1 (JSAP1 also referred to as JIP3) is a JNK-associated scaffold that controls the specificity and efficiency of JNK signaling cascades. Here we studied its expression in mouse brains. JSAP1 mRNA was expressed in developing and adult brains, showing spatial patterns similar to JNK1-3 mRNAs. In embryos, JSAP1 immunolabeling was intense for progenitor cells in the ventricular zone throughout the brain and in the external granular layer of the cerebellum, and for neurons and glial cells differentiating in the mantle zone. In adults, JSAP1 was distributed in various neurons and Bergmann glia, with higher levels in striatal cholinergic interneurons, telencephalic parvalbumin-positive interneurons and cerebellar Purkinje cells. In these neurons, JSAP1 was observed as tiny particulate staining in spines, dendrites, perikarya and axons, where it was often associated with the smooth endoplasmic reticulum (sER) and cell membrane. Immunoblots revealed enriched distribution in the microsomal fraction and cytosolic fraction. Therefore, the characteristic cellular expression and subcellular distribution of JSAP1 might be beneficial for cells to efficiently link external stimuli to the JNK MAPK pathway and other intracellular machineries.     

Interaction of c-Jun amino-terminal kinase interacting protein-1 with p190 rhoGEF and its localization in differentiated neurons

c-Jun amino-terminal kinase (JNK) interacting protein-1 (JIP-1) was originally identified as a cytoplasmic inhibitor of JNK. More recently, JIP-1 was proposed to function as a scaffold protein by complexing specific components of the JNK signaling pathway, namely JNK, mitogen-activated protein kinase kinase 7, and mixed lineage kinase 3. We have identified the human homologue of JIP-1 that contains a phosphotyrosine binding (PTB) domain in addition to a JNK binding domain and an Src homology 3 domain. To identify binding targets for the hJIP-1 PTB domain, a mouse embryo cDNA library was screened using the yeast two-hybrid system. One clone encoded a 191-amino acid region of the neuronal protein rhoGEF, an exchange factor for rhoA. Overexpression of rhoGEF promotes cytoskeletal rearrangement and cell rounding in NIE-115 neuronal cells. The interaction of JIP-1 with rhoGEF was confirmed by coimmunoprecipitation of these proteins from lysates of transiently transfected HEK 293 cells. Using glutathione S-transferase rhoGEF fusion proteins containing deletion or point mutations, we identified a putative PTB binding site within rhoGEF. This binding site does not contain tyrosine, indicating that the JIP PTB domain, like that of Xll alpha and Numb, binds independently of phosphotyrosine. Several forms of endogenous JIP-1 protein can be detected in neuronal cell lines. Indirect immunofluorescence analysis localized endogenous JIP-1 to the tip of the neurites in differentiated NIE-115 and PC12 cells. The interaction of JIP-1 with rhoGEF and its subcellular localization suggests that JIP-1 may function to specifically localize a signaling complex in neuronal cells.     

Study of calmodulin binding to the alternatively spliced C-terminal domain of the plasma membrane Ca2+ pump.

The C-terminal regions of the four human plasma membrane Ca2+ pump isoforms 1a-d generated from alternatively spliced RNA have been expressed in Escherichia coli, and the recombinant proteins have been purified to a very high degree. The C-termini of isoforms 1a, 1c, and 1d contain an insert encoded by an alternatively spliced exon which is homologous to the calmodulin binding domain of isoform 1b. In isoforms 1c and 1d (29 and 38 amino acid insertions, respectively), subdomain A of the original calmodulin binding site of isoform 1b is followed by the spliced-in domain, which is then followed by subdomain B of the original calmodulin binding site. The positive charges of histidine residues at positions 27, 28, and 38 of the alternatively spliced sequence are likely to be responsible for the observed pH-dependent calmodulin binding to the novel "duplicated" binding site. The affinity of calmodulin for the C-terminal domains of isoforms 1a, 1c, and 1d, which contain the histidine-rich inserts, is much higher at pH 5.9 than at pH 7.2. A synthetic peptide (I31) containing 31 amino acids of the alternatively spliced sequence (from residue 9 to 40) also binds calmodulin with strong pH dependency. Alternative splicing in the C-terminal domain is proposed to confer pH dependence to the regulation of the activity of Ca2+ pump isoforms.     

The axon guidance defect of the telencephalic commissures of the JSAP1-deficient brain was partially rescued by the transgenic expression of JIP1

The JNK interacting protein, JSAP1, has been identified as a scaffold protein for mitogen-activated protein kinase (MAPK) signaling pathways and as a linker protein for the cargo transport along the axons. To investigate the physiological function of JSAP1 in vivo, we generated mice lacking JSAP1. The JSAP1 null mutation produced various developmental deficits in the brain, including an axon guidance defect of the corpus callosum, in which phospho-FAK and phospho-JNK were distributed at reduced levels. The axon guidance defect of the corpus callosum in the jsap1-/- brain was correlated with the misplacement of glial sling cells, which reverted to their normal position after the transgenic expression of JNK interacting protein 1(JIP1). The transgenic JIP1 partially rescued the axon guidance defect of the corpus callosum and the anterior commissure of the jsap1-/- brain. The JSAP1 null mutation impaired the normal distribution of the Ca+2 regulating protein, calretinin, but not the synaptic vesicle marker, SNAP-25, along the axons of the thalamocortical tract. These results suggest that JSAP1 is required for the axon guidance of the telencephalic commissures and the distribution of cellular protein(s) along axons in vivo, and that the signaling network organized commonly by JIP1 and JSAP1 regulates the axon guidance in the developing brain.     

Regulation of N-cadherin-based cell-cell interaction by JSAP1 scaffold in PC12h cells

We previously reported that the level of c-Jun NH2-terminal kinase (JNK)/stress-activated protein kinase-associated protein 1 (JSAP1), a scaffold protein for JNK signaling, increases dramatically during nerve growth factor (NGF)-induced differentiation of PC12h cells. In the present study, we investigated the function of JSAP1 during PC12h cell differentiation by knocking down the level of JSAP1. The depletion of JSAP1 caused NGF-treated PC12h cells to form aggregates and impaired their differentiation. The aggregation was not observed in JSAP1-depleted cells that were untreated or treated with epidermal growth factor. Immunocytochemical studies indicated that N-cadherin, but not E-cadherin, was localized to sites of cell-cell contact in the aggregated cells. Furthermore, an inhibitory anti-N-cadherin antibody completely blocked the aggregation. Taken together, these results suggest that JSAP1 regulates cell-cell interactions in PC12h cells specifically in the NGF-induced signaling pathway, and does so by modulating N-cadherin.     

Characterization of the interaction of phorbol esters with the C1 domain of MRCK (myotonic dystrophy kinase-related Cdc42 binding kinase) alpha/beta

C1 domains mediate the recognition and subsequent signaling response to diacylglycerol and phorbol esters by protein kinase C (PKC) and by several other families of signal-transducing proteins such as the chimerins or RasGRP. MRCK (myotonic dystrophy kinase-related Cdc42 binding kinase), a member of the dystrophia myotonica protein kinase family that functions downstream of Cdc42, contains a C1 domain with substantial homology to that of the diacylglycerol/phorbol ester-responsive C1 domains and has been reported to bind phorbol ester. We have characterized here the interaction of the C1 domains of the two MRCK isoforms alpha and beta with phorbol ester. The MRCK C1 domains bind [20-(3)H]phorbol 12,13-dibutyrate with K(d) values of 10 and 17 nm, respectively, reflecting 60-90-fold weaker affinity compared with the protein kinase C delta C1b domain. In contrast to binding by the C1b domain of PKCdelta, the binding by the C1 domains of MRCK alpha and beta was fully dependent on the presence of phosphatidylserine. Comparison of ligand binding selectivity showed resemblance to that by the C1b domain of PKCalpha and marked contrast to that of the C1b domain of PKCdelta. In intact cells, as in the binding assays, the MRCK C1 domains required 50-100-fold higher concentrations of phorbol ester for induction of membrane translocation. We conclude that additional structural elements within the MRCK structure are necessary if the C1 domains of MRCK are to respond to phorbol ester at concentrations comparable with those that modulate PKC.     

A novel Jun N-terminal kinase (JNK)-binding protein that enhances the activation of JNK by MEK kinase 1 and TGF-beta-activated kinase 1.

We have identified a novel Jun N-terminal kinase (JNK)-binding protein, termed JNKBP1, and examined its binding affinity for JNK1, JNK2, JNK3, and extracellular signal-regulated kinase 2 (ERK2) in COS-7 cells. JNKBP1 preferentially interacted with the JNKs, but not with ERK2. Furthermore, we investigated the effect of overexpressing JNKBP1 on the JNK and ERK signaling pathways in COS-7 cells. JNKBP1 alone had only a marginal effect on JNK activity. However, the activation of JNK by MEK kinase 1 and TGF-beta-activated kinase 1 was significantly enhanced in the presence of JNKBP1. In contrast, JNKBP1 had no or very little effect on the ERK signaling pathway. These results suggest that JNKBP1 functions to facilitate the specific and efficient activation of the JNK signaling pathways.     

The MKK7 Gene Encodes a Group of c-Jun NH2-Terminal Kinase Kinases

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. We report here the molecular cloning of the gene that encodes MKK7 and demonstrate that six isoforms are created by alternative splicing to generate a group of protein kinases with three different NH₂ termini (α, β, and γ isoforms) and two different COOH termini (1 and 2 isoforms). The MKK7α 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 MKK7α isoforms exhibit lower activity, but a higher level of inducible fold activation, than the corresponding MKK7β and MKK7γ 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 was 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.     

Rb protein down-regulates the stress-activated signals through inhibiting c-Jun N-terminal kinase/stress-activated protein kinase

The Rb protein is the product of the retinoblastoma susceptibility gene and loss of Rb function is detected in many types of human cancers. Rb plays important roles in the regulation of cell proliferation, differentiation, senescence, and apoptotic cell death. Here we show that Rb can physically interact with c-Jun NH(2)-terminal kinase/stress-activated protein kinase (JNK/SAPK), thereby inhibiting intracellular signals mediated by JNK/SAPK. Both in vitro binding and in vitro kinase studies suggest that a carboxyl-terminal domain of Rb containing amino acids 768-928 might be crucial for inhibiting JNK/SAPK. In comparison, Rb did not affect enzymatic activity of either extracellular signal-regulated kinase 1 or p38. Ectopically expressed Rb also abrogated the apoptotic cell death induced by ultraviolet radiation or the activation of MEKK1, an upstream kinase that can stimulate the JNK/SAPK cascade. JNK/SAPK inhibition highlights a novel function of Rb, which may provide a new mechanism by which Rb regulates cell death. JNK/SAPK is a major protein kinase that can be stimulated in response to a variety of cellular stresses. Our results, therefore, suggest that Rb, by inhibiting JNK/SAPK, may act as a negative regulator in stress-activated intracellular signaling cascades.