Differential phosphorylation of c-Jun and JunD in response to the epidermal growth factor is determined by the structure of MAPK targeting sequences

MAPK phosphorylation of various substrates is mediated by the presence of docking sites, including the D domain and the DEF motif. Depending on the number and sequences of these domains, substrates are phosphorylated by specific subsets of MAPKs. For example, a D domain targets JNK to c-Jun, whereas a DEF motif is required for ERK phosphorylation of c-Fos. JunD, in contrast, contains both D and DEF domains. Here we show that these motifs mediate JunD phosphorylation in response to either ERK or JNK activation. An intact D domain is required for phosphorylation and activation of JunD by both subtypes of MAPK. The DEF motif acts together with the D domain to elicit efficient phosphorylation of JunD in response to the epidermal growth factor (EGF) but has no function on JunD phosphorylation and activation by JNK signaling. Furthermore, we show that conversion of a c-Jun sequence to a canonical DEF domain, as it is present in JunD, elicits c-Jun activation in response to EGF. Our results suggest that evolution of a particular modular system of MAPK targeting sequences has determined a differential response of JunD and c-Jun to ERK activation.     

Identification of a specific domain responsible for JNK2alpha2 autophosphorylation

c-Jun N-terminal kinases (JNKs) are a group of mitogen-activated protein kinase family members that are important in regulating cell growth, proliferation, and apoptosis. Activation of the JNK pathway has been implicated in the formation of several human tumors. We have previously demonstrated that a 55-kDa JNK isoform is constitutively activated in 86% of human brain tumors and more recently demonstrated that this isoform is either JNK2alpha2 or JNK2beta2. Importantly, we have also found that among the 10 known JNK isoforms, the JNK2 isoforms are unique in their ability to autophosphorylate in vitro and in vivo. This does not require the participation of any upstream kinases and also leads to substrate kinase activity in vitro and in vivo. To clarify the mechanism of JNK2alpha2 autoactivation, we have generated a series of chimeric cDNAs joining portions of JNK1alpha2, which does not have detectable autophosphorylation activity, with portions of JNK2alpha2, which has the strongest autophosphorylation activity. Through in vivo and in vitro kinase assays, we were able to define a domain ranging from amino acids 218 to 226 within JNK2alpha2 that is required for its autophosphorylation. Mutation of JNK2alpha2 to its counterpart of JNK1alpha2 in this region abrogated the autophosphorylation activity and c-Jun substrate kinase activity in vivo and in vitro. Notably, switching of JNK1alpha2 to JNK2alpha2 at this 9-amino acid site enabled JNK1alpha2 to gain the autophosphorylation activity in vivo and in vitro. We also found two other functional sites that participate in JNK2alpha2 activity. One site ranging from amino acids 363 to 382 of JNK2alpha2 is required for efficient c-Jun binding in vitro, and a site ranging from amino acids 383 to 424 enhances autophosphorylation intensity, although it is not required for triggering the autophosphorylation in vitro. These findings have uncovered the regions required for JNK2alpha2 autophosphorylation, and this information could be used as potential targets to block JNK2alpha2 activation.     

JNK3 contributes to c-Jun activation and apoptosis but not oxidative stress in nerve growth factor-deprived sympathetic neurons

The stress activated protein kinase pathway culminates in c-Jun phosphorylation mediated by the Jun Kinases (JNKs). The role of the JNK pathway in sympathetic neuronal death is unclear in that apoptosis is not inhibited by a dominant negative protein of one JNK kinase, SEK1, but is inhibited by CEP-1347, a compound known to inhibit this overall pathway but not JNKs per se. To evaluate directly the apoptotic role of the JNK isoform that is selectively expressed in neurons, JNK3, we isolated sympathetic neurons from JNK3-deficient mice and quantified nerve growth factor (NGF) deprivation-induced neuronal death, oxidative stress, c-Jun phosphorylation, and c-jun induction. Here, we report that oxidative stress in neurons from JNK3-deficient mice is normal after NGF deprivation. In contrast, NGF-deprivation-induced increases in the levels of phosphorylated c-Jun, c-jun, and apoptosis are each inhibited in JNK3-deficient mice. Overall, these results indicate that JNK3 plays a critical role in activation of c-Jun and apoptosis in a classic model of cell-autonomous programmed neuron death.     

GST pi modulates JNK activity through a direct interaction with JNK substrate, ATF2

Human GSTpi, an important detoxification enzyme, has been shown to modulate the activity of JNKs by inhibiting apoptosis and by causing cell proliferation and tumor growth. In this work, we describe a detailed analysis of the interaction in vitro between GSTpi and JNK isoforms (both in their inactive and active, phosphorylated forms). The ability of active JNK1 or JNK2 to phosphorylate their substrate, ATF2, is inhibited by two naturally occurring GSTpi haplotypes (Ile105/Ala114, WT or haplotype A, and Val105/Val114, haplotype C). Haplotype C of GSTpi is a more potent inhibitor of JNK activity than haplotype A, yielding 75-80% and 25-45% inhibition, respectively. We show that GSTpi is not a substrate of JNK, as was earlier suggested by others. Through binding studies, we demonstrate that the interaction between GSTpi and phosphorylated, active JNKs is isoform specific, with JNK1 being the preferred isoform. In contrast, GSTpi does not interact with unphosphorylated, inactive JNKs unless a JNK substrate, ATF2, is present. We also demonstrate, for the first time, a direct interaction: between GSTpi and ATF2. GSTpi binds with similar affinity to active JNK + ATF2 and to ATF2 alone. Direct binding experiments between ATF2 and GSTpi, either alone or in the presence of glutathione analogs or phosphorylated ATF2, indicate that the xenobiotic portion of the GSTpi active site and the JNK binding domain of ATF2 are involved in this interaction. Competition between GSTpi and active JNK for the substrate ATF2 may be responsible for the inhibition of JNK catalysis by GSTpi.     

Adrenomedullin promotes cell cycle transit and up-regulates cyclin D1 protein level in human glioblastoma cells through the activation of c-Jun/JNK/AP-1 signal transduction pathway

Adrenomedullin is a secreted peptide hormone with multiple functions. Although a number of reports have indicated that adrenomedullin may be involved in tumor progression, its mechanism of action remains obscure. In this study, we have analysed the signal transduction pathway activated by adrenomedullin in human glioma cells. Our results revealed that adrenomedullin induced the phosphorylation of both c-Jun and JNK in glioblastoma cells. Silencing JNK expression with siRNA reversed the phosphorylation of c-Jun induced by adrenomedullin, indicating that JNK is responsible of c-Jun activation. In addition, electrophoretic mobility-shift assays showed that the increase in phosphorylation of c-Jun was associated with increased AP-1 DNA binding activity. Supershift assays and co-immunoprecipitation demonstrated that c-Jun and JunD are part of the AP-1 complex, indicating that activated c-Jun is dimerized with JunD in response to adrenomedullin. Furthermore, adrenomedullin was shown to promote cell transit beyond cell cycle phases with a concomittant increase in cyclin D1 protein level, suggesting that adrenomedullin effect cell proliferation through up-regulation of cyclin D1. The inhibition of JNK activation or the suppression of c-Jun or JunD expression with siRNA impaired the effects of adrenomedullin on cell proliferation and on cyclin D1. Taken together, these data demonstrate that activation of cJun/JNK pathway is involved in the growth regulatory activity of adrenomedullin in glioblastoma cells.     

Concurrent protective and destructive signaling of JNK2 in neuroblastoma cells

Investigation of the c-Jun N-terminal kinases (JNKs) has mainly focused on their response to stress and their pro-apoptotic effects. In this regard, JNKs are crucial mediators of chemotherapy-induced killing of tumor cells. Importantly, however, JNKs also have physiological functions in cancer involving cell cycle regulation or oncogenesis. Hypothetically, the composition of JNK signalosomes determines the signaling outcome which, in turn, implies a multitude of different, sometimes opposing and interfering functions. In the present study, the well-characterized human neuroblastoma cell line SH-SY5Y served as a model system to separate physiological and pro-apoptotic JNK actions in the response to the cytoskeleton-interfering substances colchicine, cytochalasin D and taxol. Basically, JNKs mediated both cell death and proliferation. Using the chemical JNK inhibitor SP600125 as well as compartment-specific JNK-inhibiting constructs and dominant-negative isoform mutants, we show that the nuclear subgroup of JNK2 is the dominant effector in colchicine- and taxol-induced apoptosis, while cell cycle promotion is mediated by both cytoplasmic and nuclear JNK2. In contrast, cytochalasin D-triggered apoptosis is independent of JNK signaling. Interestingly, the data of the present study demonstrate for the first time that both cell protective (cell cycle progression) and destructive mechanisms (apoptosis) are simultaneously controlled by a single JNK isoform in the same cell system even under the influence of one stimulus. This has implications for the therapeutic application of JNK inhibitors and cytoskeleton-interfering substances in oncologic disorders.     

Identification of the critical features of a small peptide inhibitor of JNK activity.

The c-Jun N-terminal kinases (JNKs) are a subfamily of the mitogen-activated protein kinases (MAPKs). Although progress in evaluating the functions of other MAPKs has been facilitated by the characterization of specific inhibitors, no JNK-directed inhibitor is commercially available. We have identified a 21-amino acid peptide inhibitor of activated JNKs, based on amino acids 143-163 of the JNK-binding domain (JBD) of the JNK scaffolding protein, JNK-interacting protein-1 (JIP-1). This peptide, I-JIP (Inhibitor of JNK-based on JIP-1), inhibited JNK activity in vitro toward recombinant c-Jun, Elk, and ATF2 up to 90%. A truncated I-JIP (TI-JIP), the C-terminal 11 amino acids of I-JIP, directly interacted with recombinant JNKs but not its substrates as shown by surface plasmon resonance analysis. Scanning alanine replacement within truncated I-JIP identified 4 residues (Arg-156, Pro-157, Leu-160, or Leu-162) as independently critical for inhibition. JBD peptide sequences from JIP-2 and JIP-3 shared these critical residues and accordingly were effective JNK inhibitors. In contrast, peptides based on the JBDs of ATF2 and c-Jun inhibited JNK activity by <40%, which agreed with their lack of homology to the critical Arg-156 and Pro-157. These studies thus define a small peptide inhibitor sequence of JNKs based on the JIP proteins.     

Activation by phosphorylation and purification of human c-Jun N-terminal kinase (JNK) isoforms in milligram amounts

c-Jun N-terminal kinases (JNKs) are part of the mitogen-activated protein kinase (MAPK) signaling cascade. They are activated through dual phosphorylation of two residues in the activation loop, a threonine and a tyrosine, by MAP2 kinases (MKK4 and 7) in response to various extracellular stresses such as UV or osmotic shock, as well as by cytokines and growth factors. Only small amounts of phosphorylated, active JNKs have previously been produced because of difficulties in expressing these phosphorylated kinases in Escherichia coli, which lack the appropriate upstream kinases. We have now established a novel activation and purification method that allows for reproducible production of milligram amounts of active, phosphorylated JNKs suitable for a variety of enzymatic, biophysical and structural characterizations. We utilize N-terminally His-tagged MKK4 that is coexpressed in E. coli with a constitutively active form of MEKK1. This phosphorylated, active His-MKK4 is purified by Ni–NTA chromatography and used to phosphorylate milligram amounts of three different isoforms of human JNKs (JNK1α1, JNK1α2 and JNK2α2) that had separately been expressed and purified from E. coli in their inactive forms. These in vitro activated JNKs are phosphorylated on both residues (T183, Y185) in their activation loops and are active towards their substrate, ATF2.     

Enzyme-linked immunosorbent assay for the measurement of JNK activity in cell extracts.

A colorimetric enzyme-linked immunosorbent assay (ELISA) for the measurement of kinase activity of c-Jun N-terminal kinases (JNKs) in cell extracts is described. The assay involves passive immobilisation of the substrate GST-cJun on the surface of a microtiter plate, selection of JNK protein kinases directly in substrate-coated wells, kinase reaction, and detection of substrate phosphorylation by a phosphoepitope-specific antibody. The ability of this assay to selectively measure JNK activity relies on the high-affinity interaction between JNKs and c-Jun. Accordingly, we found that JNKs could be captured on the microtiter plate surface through binding to the immobilised GST-cJun. Moreover, JNKs retained the specificity of their interaction with and phosphorylation of c-Jun with respect to the dependence on both intact docking domain and the dimerisation state of c-Jun. This novel procedure represents a marked improvement on conventional radioactive assays in terms of sensitivity, accuracy of evaluation, low time consumption, high throughput and amenability to automation. It is expected to be useful forthe acceleration and facilitation of JNK activity measurement in cell extracts, in particular for large-scale screening of clinical samples.     

The tumor suppressor p16(INK4a) prevents cell transformation through inhibition of c-Jun phosphorylation and AP-1 activity

Inactivation of the p16(INK4a) tumor suppressor protein is critical for the development of human cancers, including human melanoma. However, the molecular basis of the protein's inhibitory effect on cancer development is not clear. Here we investigated a possible mechanism for p16(INK4a) inhibition of neoplastic transformation and UV-induced skin cancer. We show that p16(INK4a) suppresses the activity of c-Jun N-terminal kinases (JNKs) and that it binds to the glycine-rich loop of the N-terminal domain of JNK3. Although p16(INK4a) does not affect the phosphorylation of JNKs, its interaction with JNK inhibits c-Jun phosphorylation induced by UV exposure. This, in turn, interferes with cell transformation promoted by the H-Ras-JNK-c-Jun-AP-1 signaling axis.     

Hyperphosphorylation of the retinoid X receptor alpha by activated c-Jun NH2-terminal kinases.

The nuclear receptor mouse retinoid X receptor alpha (mRXRalpha) was shown to be constitutively phosphorylated in its NH2-terminal A/B region, which contains potential phosphorylation sites for proline-directed Ser/Thr kinases. Mutants for each putative site were generated and overexpressed in transfected COS-1 cells. Constitutively phosphorylated residues identified by tryptic phosphopeptide mapping included serine 22 located in the A1 region that is specific to the RXRalpha1 isoform. Overexpression and UV activation of the stress-activated kinases, c-Jun NH2-terminal kinases 1 and 2 (JNK1 and JNK2), hyperphosphorylated RXRalpha, resulting in a marked decrease in its electrophoretic mobility. This inducible hyperphosphorylation involved three residues (serines 61 and 75 and threonine 87) in the B region of RXRalpha and one residue (serine 265) in the ligand binding domain (E region). Binding assays performed in vitro with purified recombinant proteins demonstrated that JNKs did not interact with RXRalpha but bound to its heterodimeric partners, retinoic acid receptors alpha and gamma (RARalpha and RARgamma). Hyperphosphorylation by JNKs did not affect the transactivation properties of either RXRalpha homodimers or RXRalpha/RARalpha heterodimers in transfected cultured cells.     

Distinct roles for JNK1 and JNK2 in regulating JNK activity and c-Jun-dependent cell proliferation

Different c-Jun N-terminal kinases (JNKs) are activated by a plethora of signals and phosphorylate substrates such as c-Jun, which is required for efficient cell cycle progression. Although JNK1 and JNK2 were shown to differentially regulate fibroblast proliferation, the underlying mechanistic basis remains unclear. We found that Jnk2-/- fibroblasts exit G1 and enter S phase earlier than wild-type counterparts, while Jnk1-/- cells show the inverse phenotype. Moreover, Jnk2-/- erythroblasts also exhibit a proliferative advantage. JNK2 deficiency results in elevated c-Jun phosphorylation and stability, whereas the absence of JNK1 reduces c-Jun phosphorylation and stability. Re-expression of JNK2 in Jnk2-/- cells reverses the JNK2 null phenotype, whereas ectopic expression of JNK1 augments it. JNK2 is preferentially bound to c-Jun in unstimulated cells, thereby contributing to c-Jun degradation. In contrast, JNK1 becomes the major c-Jun interacting kinase after cell stimulation. These data provide mechanistic insights into the distinct roles of different JNK isoforms.     

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.     

Designed Ankyrin Repeat Proteins (DARPins) as Novel Isoform-Specific Intracellular Inhibitors of c-Jun N-Terminal Kinases

The c-Jun N-terminal kinases (JNKs) are involved in many biological processes such as proliferation, differentiation, apoptosis, and inflammation and occur in highly similar isoforms in eukaryotic cells. Isoform-specific functions and diseases have been reported for individual JNK isoforms mainly from gene-knockout studies in mice. There is, however, a high demand for intracellular inhibitors with high selectivity to improve the understanding of isoform-specific mechanisms and for use as therapeutic tools. The commonly used JNK inhibitors are based on small molecules or peptides that often target the conserved ATP binding site or docking sites and thus show only moderate selectivity. To target novel binding epitopes, we used designed ankyrin repeat proteins (DARPins) to generate alternative intracellular JNK inhibitors that discriminate two very similar isoforms, JNK1 and JNK2. DARPins are small binding proteins that are well expressed, stable, and cysteine-free, which makes them ideal candidates for applications in the reducing intracellular environment. We performed ribosome display selections against JNK1α1 and JNK2α1 using highly diverse combinatorial libraries of DARPins. The selected binders specifically recognize either JNK1 or JNK2 or both isoforms in vitro and in mammalian cells. All analyzed DARPins show affinities in the low nanomolar range and isoform-specific inhibition of JNK activation in vitro at physiological ATP concentrations. Importantly, DARPins that selectively inhibit JNK activation in human cells were also identified. These results emphasize the great potential of DARPins as a novel class of highly specific intracellular inhibitors of distinct enzyme isoforms for use in biological studies and as possible therapeutic leads.