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Scientific Reports Volume 6, Article Number: 21025 (2016) Cite this article

The tri-kinase mitogen-activated protein kinase (MAPK) signal cascade exists in almost all eukaryotic cells. The MAPK cascade is organized by scaffold proteins, which assemble homologous kinases into productive signaling complexes. Arrestin-3 promotes the activation of JNK in cells, and a short 25-residue arrestin-3 peptide has been identified as a key JNK3 binding element. Here we demonstrate that the peptide also binds to MKK4, MKK7 and ASK1, which are upstream JNK3 activated kinases. This peptide is sufficient to enhance the activity of JNK3 in cells. Homologous inhibitory protein 2 peptides that differ only in the four positions bind to MKK4, but do not bind to MKK7 or JNK3, and are not effective in enhancing the activation of JNK3 in the cell. The arrestin-3 peptide is the smallest known MAPK scaffold. This peptide or its mimetics can regulate MAPK and affect the life and death decisions of cells.

The spatial and temporal organization of proteins in cells is essential for coordinating basic activities. Appropriate cellular responses to external or internal stimuli usually require precise coordination of the scaffold protein, which determines the specificity of the signal and the precise time course. In particular, the specificity of signal transduction via the mitogen-activated protein kinase (MAPK) cascade is highly dependent on the scaffold protein 2, 3, and 4. MAPK signaling is involved in the regulation of key cell behaviors, from proliferation to differentiation and apoptotic death4. The overall structure of the tri-kinase MAPK cascade is conserved from yeast to mammals5. Most cells have multiple MAPK, MAPK kinase (MAPKK) and MAPKK kinase (MAPKKK), so the signal result is usually determined by the scaffold of the tissue-specific MAPKKK-MAPKK-MAPK complex2,3,4,6.

c-Jun NH2 terminal protein kinase (JNK) belongs to the MAPK family. JNK regulates the normal physiological processes of cell proliferation, apoptosis, differentiation and migration. JNK is also involved in many diseases, from cancer to neurological and immune diseases8,9,10. The complete activation of all JNK requires two upstream kinases, MKK4 (tyrosine) and MKK7 (threonine) 11 to double phosphorylate the TXY motif in the activation loop. Similar to other MAPKs, JNK activation depends on scaffold proteins, such as JIPs12. Arrestins specifically bind to active phosphorylated G protein-coupled receptors (GPCRs), and were first discovered to be negative regulators of GPCR signaling through G protein13,14. Among the four inhibitory protein subtypes expressed in vertebrates, only inhibitory protein-3 promotes the activation of JNK316, and JNK1/217, which is ubiquitous in cells, serves as the aggregation of MAPKKK ASK116,18, MAPKKs MKK416,18,19 and MKK717 The bracket 20 together, and several isoforms of JNK1/2/316, 17, 18, 21, 22. Recently we have determined that the first 25 residues of arrestin-3 are the key JNK3 binding sites23. Here, we demonstrated that this short inhibin 3-derived peptide also binds ASK1 and MKK4/7 and promotes JNK3 activation in intact cells. This is the smallest JNK cascade stent found so far. Its size paves the way for the design of small molecule mimics, which can be used as tools for targeted manipulation of anti-proliferative and usually pro-apoptotic JNK signals in cells.

We recently discovered that although all three elements in the two inhibitory protein 3 domains are involved in JNK3 binding, the peptide representing the first 25 residues of the inhibitory protein 3 (T1A) is the key interaction site. This opens up three possibilities. First, if T1A only binds to JNK3 and not other kinases in the cascade, it can recruit JNK3 from the functional scaffold, thereby inhibiting JNK3 activation. Second, if T1A binds to several kinases in the JNK3 activation module, but does not promote JNK3 phosphorylation, it may serve as a dominant-negative silencing scaffold, similar to the inhibitory protein-3-KNC mutant we recently described24. Finally, if T1A binds to the same kinase as inhibitory protein 3 and promotes signaling in the JNK3 cascade, it will become the smallest active MAPK scaffold known, which opens up new ways to manipulate MAPK signaling in cells for research and therapeutic purposes way.

To determine the functional capacity of the T1A peptide, we took advantage of the availability of purified MKK4 and MKK7, both of which activate JNK311 and show binding to full-length inhibitor protein 320. We expressed T1A as an MBP fusion in E. coli and purified it on an amylose column. The ability of purified GST-MKK4 or GST-MKK7 (Figure 1A) to bind to MBP-T1A immobilized on an amylose column was tested in an in vitro pull-down assay, with MBP and MBP-arrestin-3 as negative and positive Control separately (Figure 1B). MBP-T1A, instead of the control MBP, effectively retained both kinases (Figure 1B). Interestingly, similar to full-length inhibitory protein 3, the interaction of T1A peptide with MKK4 is stronger than with MKK7 (Figure 1B, bottom panel). Therefore, in addition to JNK323, the T1A peptide also binds to two MKKs that are known to be phosphorylated. Since the pull-down is performed with purified protein, the data proves that the interaction of T1A with MKK4 and MKK7 is direct and does not involve any intermediates or assistants. Next, we tested whether T1A binds to the top kinase ASK1 in the cascade. Since ASK1 cannot be obtained in purified form, we co-expressed HA-labeled ASK1 and YFP-labeled T1A in COS7 cells (using YFP as a control), lysed the cells, and immunoprecipitated the YFP construct with anti-GFP antibody (Figure 1C)). We found that HA-ASK1 and YFP-T1A co-immunoprecipated effectively, but not with the control YFP (Figure 1C). Therefore, in addition to JNK323, the T1A peptide also binds to all the upstream kinases of its activation cascade, ASK1, MKK4, and MKK7 (Figure 1).

(A) Purified GST (control), GST-MKK4 and GST-MKK7 (Coomassie staining) (B) Top: MBP (control), MBP-T1A and MBP-arrestin- eluted from amylose beads 3 Coomassie staining of bait. Lower imprinting: GST-MKK4 and GST-MKK7 retained by MBP-T1A and MBP-arrestin-3. Pull down as described in the method. (C) HA-ASK1 was co-immunoprecipitated with YFP-T1A, but not the YFP control, from cells co-expressing these proteins (top blot: lysate; bottom blot: protein immunoprecipitated with anti-GFP antibody). The complete blot is shown in Supplementary Figure S1.

Next, we tested whether T1A acts as a scaffold for signal transduction. For this, we used experiments with purified proteins because they provide the clearest data supporting direct interactions. Since purified ASK1 is not available, we reconstructed the MKK4-JNK3 and MKK7-JNK3 modules in the absence and presence of different concentrations of synthetic purified T1A peptides, and measured JNK3 phosphorylation (Figure 2A). In both cases, we obtained a bell curve reflecting the phosphorylation level of JNK3 as a function of T1A concentration (Figure 2B). This dependence, that the signal increases at lower scaffold concentrations and decreases at higher concentrations, has also been found to be used for scaffolds of full-length inhibitor protein 320 to these modules. It is considered to be a feature of simple scaffolds. It works by combining enzymes and substrates, because a lower scaffold concentration may form a complete scaffold-enzyme-substrate ternary complex, while a higher scaffold The concentration increases the possibility of forming incomplete enzyme-scaffold and substrate-scaffold binary complexes, inhibiting signal transduction25,26. The optimal concentration of scaffold protein for signal transduction depends on its affinity for the scaffold protein20,25,26.

(A) Representative autoradiograph showing that JNK3α2 is phosphorylated by purified MKK4 (upper image) and MKK7 (lower image) at the specified concentration of synthetic purified T1A peptide (10 seconds incubation). (B) Quantification of JNK3α2 phosphorylation by MKK4 and MKK7. (C) The phosphorylation of JNK3α2 by MKK4 and MKK7 in COS7 cells co-expressing JNK3α2 with MKK4 or MKK7 (control) and YFP, YFP-T1A or Inhibitor protein 3. The complete blot is shown in Supplementary Figure S2.

Our previous work showed that in a complex containing MKK4 and inhibin-3, the efficiency of phosphorylation of JNK3 is higher than the efficiency of phosphorylation of JNK3 by MKK4 in the absence of inhibin-320. Since these experiments were performed with pure ingredients, the data showed that T1A (Figure 1A) and its substrate JNK323, which directly bind to MKK4/7 at the same time, placed MKK in a favorable position to phosphorylate JNK3 (Figure 2A, B). Previously, we found that the optimal concentration of arrestin-3 for scaffolds MKK4-JNK3 and MKK7-JNK3 modules is ~0.6 μM and ~6 μM20, which reflects that the affinity of arrestin-3 for MKK7 is lower than that for MKK420. Interestingly, the optimal concentration of T1A to promote the phosphorylation of JNK3 by MKK4 is lower than that of the MKK7-JNK3 module (Figure 2B), which is consistent with the stronger binding of this peptide to MKK4 (Figure 1B). Interestingly, in both cases, the optimal T1A concentration is about 10 times lower than the concentration of full-length inhibitory protein 3. These results indicate that T1A has a higher affinity for MKK and/or JNK3, which is consistent with its ability to retain more MKK than Arrestin 3 (Figure 1B). This phenomenon may be due to the fact that the T1A peptide is more accessible when it is free, rather than in the case of full-length inhibitory protein 3, where it is partially shielded by other elements of the N domain27.

To test whether T1A can promote JNK3 phosphorylation through MKK in cells, we co-expressed MKK4 or MKK7 with YFP-T1A in COS7 cells, using YFP and full-length inhibitory protein 3 as negative and positive controls, respectively (Figure 2C). We found that YFP-T1A, but not YFP, increased the phosphorylation of JNK3 in both MKKs, similar to inhibin 3. Therefore, in vitro and in intact cells, T1A is necessary and sufficient to promote signal transduction in the MKK4/7-JNK3 module.

T1A constitutes a small part of arrestin-3 N-domain27. All inhibitory proteins are composed of two independently folded domains 27, 28, 29, 30, which can be expressed separately and retain certain functions 18, 31, 32. Therefore, we tested whether the isolating domain of arrestin-3, as well as other ubiquitously expressed non-visual subtypes of arrestin-2, which also binds to kinase 18 of the JNK3 activation cascade, can promote JNK3 phosphorylation in cells. To this end, we expressed inhibin and its single domain with the same HA tag (to compare their expression on the same blot) as well as ASK1 and JNK3 in COS7 cells (Figure 3). We confirmed that the expression of arrestin-3 significantly increased the phosphorylation of JNK3 in cells, while arrestin-2 did not 16, 18, 33, and did not find any effect of the isolated domain of the inhibitory protein (Figure 3).

Western blots of COS7 cell lysates that co-express HA-ASK1 and HA-JNK3α2 (control) or HA-labeled full-length inhibitor protein 3 (Arr3), inhibitor protein 2 (Arr2), or isolated N- and C- A domain of non-visual inhibitory proteins (Arr3N, Arr2N, Arr3C, Arr2C). Phospho-JNK blots showed that in these constructs, only full-length inhibitor protein 3 promoted the phosphorylation of JNK3α2 in cells. The complete blot is shown in Supplementary Figure S3.

T1A effectively enhances JNK3 phosphorylation through MKK4 and MKK7 (Figure 1, 2), while full-length inhibitory protein 3 promotes JNK3 phosphorylation when co-expressed with ASK116,18,21,33, which phosphorylates and activates MKK. If arrestin-3 N-domain cannot promote the activation of JNK3, because it can only support MKK4/7-JNK3 module, then T1A peptide only forms part of arrestin-3 N-domain, and it would not be expected to have ASK1 in the cell Under circumstances promote JNK3 phosphorylation. To test this, we co-expressed a YFP fusion of T1A and two other JNK3 binding peptides T3 and T6 (Figure 4A)23, with JNK3 and ASK1 in COS7 cells and monitored JNK3 phosphorylation, using YFP and inhibin- 3 As negative and positive controls respectively (Figure 4B). As expected, arrestin-3 increased the level of JNK3 phosphorylation (Figure 4B). T1A, but not other peptides, is also active, and the effect of T1A is greater than that of full-length inhibitory protein 3 (Figure 4B). To test whether T1A still functions as a simple scaffold in intact cells when ASK1 is overexpressed, as in the case of MKK4/7-JNK3 signaling module (Figure 2), we combined ASK1 and JNK3 with YFP (control) and Different amounts of YFP-T1A (Figure 4C). The dose-response curve of T1A in these experiments is also biphasic, with a clear optimal value, indicating that T1A is a simple scaffold of the tri-kinase cascade, similar to full-length inhibitory protein 319.

(A) The structure of arrestin-3 (PDB: 3P2D;27), in which the three peptides involved in JNK3 binding 23 are shown in red (T1A), blue (T3) and green (T6). (B) COS7 cells co-express HA-ASK1 and HA-JNK3α2, without (control) or full-length inhibitory protein 3 (Arr3), YFP or designated YFP-labeled JNK3 binding peptide. The top three blots show the expression level of the specified protein; the bottom blot shows that T1A promotes JNK3α2 phosphorylation more effectively than full-length inhibitory protein 3. (C) COS7 cells co-express HA-ASK1 and HA-JNK3α2 with YFP (control) or YFP-T1A at different concentrations. The upper two blots show the expression level of the specified protein; the lower blot shows the biphasic dependence of JNK3α2 phosphorylation on T1A levels. The complete blot is shown in Supplementary Figures S4b (Panel B) and S4c (Panel C).

In order to test the specificity of T1A, we compared its ability to bind purified MKK4, MKK7 and JNK3 with the ability of B1A. B1A is a homologous N-terminal peptide derived from the closely related inhibitory protein 2. This position is different from T1A (Figure 2). 5). We chose B1A as the “natural” negative control because, despite its 78% sequence identity with Inhibitor 334, Inhibitor 2 does not promote JNK3 activation in cells16, 18, 21. We found that although the two peptides have the same binding ability to MKK4, the interaction of B1A with JNK3 and its other upstream activator MKK7 is weak (Figure 5). To test whether this difference in binding translates to differential activity in cells, we used YFP-arrestin-3 and YFP as positive and negative controls, and compared YFP-T1A and YFP-B1A in promoting JNK3 activation in cells overexpressing ASK1 Abilities, respectively. We found that compared with T1A, arrestin-2 derived B1A does not act as a scaffold for the ASK1-MKK4/7-JNK3 cascade in cells (Figure 5e), indicating that this specific sequence is not a simple accessibility inhibition of this part Protein determines its function.

(a) Sequence comparison of T1A and B1A derived from arrestin-3, derived from arestin-2, does not promote JNK3 activation. The residues that differ between T1A and B1A are shown in magenta. (b–d) MBP (negative control), MBP-arrestin-3 (positive control), MBP-T1A and MBP-B1A pull down purified JNK3 (b), MKK4 (c) and MKK7 (d), as in the method The proceeding. Bottom, Coomassie gel loaded with MBP fusion; middle, reserved Western blots of JNK3 (b), MKK4 (c) and MKK7 (d); top, Western blots from 3-4 independent experiments Quantify. Please note that B1A binds to MKK4 like T1A, but has no obvious interaction with JNK3 or MKK7. (E) COS7 cells co-express HA-ASK1 and HA-JNK3α2 with YFP (negative control), YFP-arrestin-3 (Arr3, positive control), YFP-T1A or YFP-B1A. The lower two blots show the expression level of the specified protein; the upper blot and bar graph (quantification of JNK3α2 phosphorylation in four independent experiments) indicate that T1A promotes JNK3α2 phosphorylation, while B1A does not. * p <0.05; ns, meaningless. The complete blot and gel are shown in Supplementary Figure S5.

Therefore, the first 25 residues contained in the T1A peptide are mainly responsible for inhibiting the ability of the protein-3 scaffold ASK1-MKK4/7-JNK3 signal module. The lack of activity in the Arrestin 3 N domain (Figure 3) containing the T1A peptide, and the promotion of JNK3 phosphorylation in exactly the same T1A experimental paradigm (Figure 4), clearly shows that this peptide is better than arrestin- 3 or its N domain is easier to obtain in the context of. It is easy to speculate that the receptor binding of arrestin-3 stimulates its ability to promote JNK3 activation by increasing the accessibility of T1A elements to related kinases. The remarkable flexibility of receptor binding inhibitor proteins demonstrated by biophysical methods35,36 supports this idea. Although it is not possible to obtain the crystal structure of Arrestin 3 complexed with any GPCR, the only existing structure of Arrestin receptor complex, namely the structure of visual arrestin 1 bound to rhodopsin, is consistent with this hypothesis. There is also a need to identify Arrestin-3 elements that promote signal transduction and lead to activation of other MAPKs, such as ERK1/238 and p3839. The activation of these two kinases is strictly dependent on the binding of inhibitory proteins to receptors38, 39, which suggests that inhibitory protein elements that change conformation and/or become more exposed during GPCR interaction may be the main suspects.

Our data identified a relatively short inhibin 3-derived peptide as an effective scaffold for the ASK1-MKK4/7-JNK3 signaling cascade, making it the smallest MAPK scaffold ever. However, T1A can be large enough to simultaneously bind all related kinases. Each residue has the structure -NH-CH(R)-CO-, and each bond>1.4A. Therefore, considering the angle, the length of a single dwell is about 3A. Therefore, a 25-residue peptide in a fully extended conformation can be up to ~75A in length, which is similar to the maximum "wingspan" (the distance between the distal tips of two inhibitory protein domains) of all inhibitory proteins 27,28,29 ,30. Nevertheless, the limited size of T1A opens up prospects for the design of peptide and/or non-peptide small molecule mimics, which can be used as tools for manipulating MAPK signals for research and therapy. Many human diseases are caused by excessive cell proliferation (for example, cancer) or death (for example, Alzheimer's disease, Parkinson's disease, and other neurodegenerative diseases). Targeted activation of JNK family kinases usually has an anti-proliferative effect, while activation of ERK1/2 promotes cell survival. Scaffolds that promote signal transduction in these pathways may be easier to administer than direct pharmacological activators, making them safer intervention tools. Determining the shortest active form of the T1A-derived peptide must be the next step in this direction.

All restriction enzymes and DNA modification enzymes (T4 DNA ligase, Vent DNA polymerase, and calf intestinal alkaline phosphatase) are from New England Biolabs (Ipswich, MA). Other chemicals come from recently described sources17,23.

We use the systemic names of inhibitory proteins: arrestin-1 (historical name S-antigen, 48 kDa protein, visual or rod inhibitory protein), arrestin-2 (β-arrestin or β-arrestin1), arrestin-3 (β-arrestin2) Or hTHY-ARRX) and arrestin-4 (cone or X-arrestin; for unknown reasons, its gene is called “arrestin 3” in the HUGO database).

In order to prepare the MBP fusion protein containing the inhibitory protein-2/3 element, the cDNA encoding the inhibitory protein fragment was subcloned into pMal-p2T (a generous gift from Dr. Keiji Tanaka, Tokyo Institute of Medical Sciences), located at Eco RI and Xho I Point within the framework of MBP, as described in 23. MBP-Arr3 (full-length inhibitory protein-3) was created by subcloning the corresponding cDNA into pMal-p2T between the Eco RI and Not I sites. All MBP-arrestin-3 fusion proteins contain the same TLVPRGSPGF linker between MBP and arrestin-3 or fragments thereof. The MBP protein used as a negative control was purified using an empty pMal-p2T vector, which contains the same linker with 10 additional residues: PGRLERPHRD. As described in 23, the MBP fusion was purified. Perform MBP pull-down as described earlier. In short, the indicated MBP fusion (10-30 μg in 50 μL 20 mM Tris/150 mM NaCl) was immobilized on amylose resin (25 μL, 50% slurry, New England Biolabs), at 4 Rotate slightly for 1 hour at °C. As described in 40, the purified MKK4/7 (10 μg in 50 μL 20 mM Tris/150 mM NaCl) was added to the immobilized MBP fusion and gently rotated at 4°C for 2 hours. The sample was transferred to a centrifugal filter (Durapore®-PVDF-0.65 μm) and washed 3 times with 50 mM HEPES-Na, pH 7.3, 150 mM NaCl. The protein is eluted in 100 μl of elution buffer (wash buffer containing 50 mM maltose) by gently swirling at 4°C for 5 minutes. The eluate was analyzed by SDS-PAGE and Western blot.

JNK3a220,23 and MKK4 and MKK717,20 were expressed in E. coli and purified as previously described. As mentioned in 19 and 20, the effects of arrestin-3 and MBP-T1A on MKK7 or MKK4 on JNK3α2 phosphorylation were analyzed by in vitro kinase assay. In short, the assay is performed in 10 μL containing the following final concentrations: 50 nM active MKK7 or MKK4, 1 μM JNK3α2, and inhibitory protein 3 or T1A at the indicated concentration. The mixture was incubated individually for 10 seconds at 30°C. The reaction was stopped by adding 15 μL Laemmli SDS sample buffer (Sigma), 2 μl of the total reaction sample was subjected to SDS-PAGE (8%) and transferred to a polyvinylidene fluoride (PVDF) membrane (Millipore, Bedford, MA). Phosphorylation The JNK3α2 was visualized by rabbit anti-phospho-JNK antibody (Cell Signaling), and the level of JNK phosphorylation was quantified.

T1A peptide (MGEKPGTRVFKKSSPNCKLTVYLGK) represents the first 25 residues of inhibin-3. Fluorene was synthesized on NovaSyn® TGR R resin (13.5 μmol, Novabiochem, Darmstadt, Germany) using an automated synthesis robot (SyroI, MultiSyntech, Witten, Germany) Methoxycarbonyl chloride (FMOC)/tert-butyl strategy, as described in 4.1. FMOC-amino acids are from Iris Biotech and Novabiochem (Marktredwitz, Germany). Amino acid side chain protecting groups are used as follows: trityl (Trt) for Asn and Cys; tBu for Glu, Thr, Ser and Tyr; tert-butoxycarbonyl (Boc) for Lys; and pentamethyl-2,3 -Dihydrobenzofuran-5-sulfonyl (Pbf) is used for Arg. Use 40% (v/v) piperidine (Sigma-Aldrich, Taufkirchen, Germany) in N,N-dimethylformamide (DMF; Biosolve, Valkenswaard, Netherlands) for automatic FMOC deprotection for 3 minutes and 20% ( v/v) Piperidine is placed in DMF for 10 minutes. The coupling of amino acids is carried out twice. Pre-incubate FMOC-amino acid with OxymaPure (Iris Biotech) for 2 minutes. After adding N,N'-diisopropylcarbodiimide (DIC; Iris Biotech), the reaction was incubated for 40 minutes. After successful synthesis, the peptide was cleaved from the resin with 90% trifluoroacetic acid (TFA) and 10% 1,2-ethanedithiol/phenylsulfide (v/v 3:7). Reduce methionine with 1,2-ethanedithiol and trimethylsilyl bromide in TFA. Subsequently, the peptide was purified on a reversed-phase C18 column (Phenomenex Jupiter 10u Proteo 90 Å: 250 × 21.2 mm; 7.8 μm; 90 Å) and passed MALDI-TOF mass spectrometry (Ultraflex II, Bruker, Bremen, Germany) and analytical reversed-phase HPLC Column Phenomenex Kinetex 5u XB-C18 100 Å (Phenomenex: 250 × 4.6 mm; 5 μm; 100 Å) and Phenomenex Jupiter 4u Proteo 90 Å (Phenomenex: 250 × 4.6 mm); 904 μm. Eluent A is H2O with 0.1% TFA, and Eluent B is ACN with 0.08% TFA. On two columns, use a gradient from 10% eluent B in A to 60% eluent B in A in 40 minutes. According to this procedure, the purity of the peptide is ≥95% (theoretical Mr = 2766.5 Da, experiment ([MH]) = 2767.6).

As described, the HA-labeled inhibitor protein and its isolated domains were constructed. By subcloning the cDNA encoding YFP-arrestin-3 or YFP-labeled arrestin-3 fragment cDNA into pcDNA3.1 between Eco RI and Hind III restriction sites, a full-length arrestin-3 and its carrying N-terminal YFP tag fragment.

The COS7 African green monkey cells were cultured in DMEM supplemented with 10% heat-inactivated FBS (Invitrogen), penicillin and streptomycin at a temperature of 37°C and placed in a humidified incubator with 5% CO2. According to the manufacturer's instructions, the cells were plated at 80-90% confluence and transfected with Lipofectamine 2000 (Invitrogen). The cells were used 48 hours after transfection and serum starved overnight before the experiment.

Incubate COS7 cells with phosphatase inhibitors (50 mM NaF and 10 mM Na3VO4) in serum-free medium at 37°C for 15 minutes; wash with cold PBS; and use 1% SDS, 10 mM Tris (pH 7.4) ), 10 mM NaF, 100 μM Na3VO4, 2 mM EDTA, 2 mM benzamidine and 1 mM PMSF SDS lysis buffer. JNK activity was measured by Western blotting, and phosphorylated (active) JNK17, 20, and 23 were detected using an antibody specific to phosphorylated JNK. The whole cell lysate was boiled for 5 minutes, centrifuged at 10,000 × g for 10 minutes, and the supernatant was used for Western blotting. The protein was measured using the Bio-Rad Coomassie Blue assay. The protein was separated on 8% SDS-PAGE and transferred to a PVDF membrane (Millipore, Bedford, MA). The blot was incubated with the primary antibody (Cell Signaling Technology, Inc) anti-phospho-JNK, anti-JNK, and anti-HA (6E2) (1:1000 to 1:5000), followed by the appropriate HRP-conjugated secondary antibody. The protein bands are detected by enhanced chemiluminescence (ECL, Pierce) and then exposed to X-ray film. To quantify phopho-JNK, we used serial dilutions of HEK-A cell lysates stimulated with anisomycin (1 μg/ml) to ensure that all samples were within the linear range. The values ​​of these proteins are expressed in arbitrary units.

How to cite this article: Zhan, X. etc. Peptide microscaffolds promote JNK3 activation in cells. science. Rep. 6, 21025; doi: 10.1038/srep21025 (2016).

Lim, WA designs customized cell signal circuits. Nat Rev Mol Cell Biol 11, 393–403 (2010).

Burack, WR & Shaw, AS Signal Transduction: Hang on the bracket. Curr Opin Cell Biol 12, 211–216 (2000).

Dhanasekaran, DN, Kashef, K., Lee, CM, Xu, H. & Reddy, EP scaffold protein of MAP-kinase module. Oncogene, 3185-3202 (2007).

Keshet, Y. & Seger, R. MAP kinase signaling cascade: A system composed of hundreds of components that can regulate a variety of physiological functions. Method Mol Biol 661, 3–38 (2010).

Widmann, C., Gibson, S., Jarpe, MB & Johnson, GL Mitogen-activated protein kinase: protection of the tri-kinase module from yeast to humans. Physiology Rev. 79, 143–180 (1999).

Good, MC, Zalatan, JG & Lim, WA Scaffold protein: the hub that controls the flow of cellular information. Science 332, 680–686 (2011).

CAS ADS article Google Scholar 

Davis, RJ MAP kinase signal transduction in the JNK group. Cell 103, 239-252 (2000).

Sabapathy, K. The role of the JNK pathway in human diseases. Prog Mol Biol Transl Sci 106, 145–169 (2012).

Flemming, A. Alzheimer's disease: JNK3 as a new target for AD? Nat Rev Drug Discov 11, 829 (2012).

Yin, SO etc. JNK3 persists the metabolic stress induced by Aβ peptide. Neuron 75, 824–837 (2012).

Lawler, S., Fleming, Y., Goedert, M. & Cohen, P. Cooperative activation of SAPK1/JNK1 by two MAP kinase kinases in vitro. Curr Biol 8, 1387–1390 (1998).

Yasuda, J., Whitmarsh, AJ, Cavanagh, J., Sharma, M. & Davis, RJ JIP group of mitogen-activated protein kinase scaffold proteins. Mol Cell Biol 19, 7245–7254 (1999).

Gurevich, VV and Gurevich, the structural basis of EV inhibitory protein-mediated G protein-coupled receptor regulation. Pharm Ther 110, 465–502 (2006).

Carman, CV and Benovic, JL G protein-coupled receptors: on and off. Curr Opin Neurobiol 8, 335–344 (1998).

Gurevich, EV & Gurevich, VV Arrestins are ubiquitous cell signaling pathway regulators. Genome Biology 7, 236 (2006).

MacDonald, PH etc. β-arrestin 2: A receptor-regulated MAPK scaffold used to activate JNK3. Science 290, 1574–1577 (2000).

CAS ADS article Google Scholar 

Kook, S. etc. Arrestin-3 binds to JNK1α1 and JNK2α2, and promotes the activation of these ubiquitous JNK isoforms in cells through the scaffold. J Biol Chem 288, 37332–37342 (2013).

Song, X., Coffa, S., Fu, H. & Gurevich, VVarrestin How to assemble MAPK into a signal complex? J Biol Chem 284, 685–695, doi: M806124200 [pii] 10.1074/jbc.M806124200 (2009).

CAS Article PubMed PubMed Central Google Scholar 

Zhan, X., Kaoud, TS, Dalby, KN & Gurevich, VV Non-visual inhibitory protein functions as a simple scaffold for assembling the MKK4-JNK3α2 signaling complex. Biochemistry 50, 10520–10529 (2011).

Zhan, X., Kaoud, TS, Kook, S., Dalby, KN & Gurevich, VV The combination of JNK3 and inhibitor protein 3 has different effects on the recruitment of upstream MAP kinase kinases. J Biol Chem 288, 28535–28547 (2013).

Miller, we wait. The motif responsible for activating the carboxyl terminal of β-arrestin2 of JNK3 was identified. J Biol Chem 276, 27770-27777 (2001).

Song, X., Raman, D., Gurevich, EV, Vishnivetskiy, SA & Gurevich, VV Visual and two non-visual inhibitory proteins bind JNK3 and Mdm2 in their "inactive" conformation and relocate them from the nucleus to Cytoplasm. J Biol Chem 281, 21491-21499 (2006).

Zhan, X., Perez, A., Gimenez, LE, Vishnivetskiy, SA and Gurevich, VV Arrestin-3 binds to the MAP kinase JNK3α2 through multiple sites on two domains. Cell Signal 26, 766–776 (2014).

Breitman, M. et al. Silent scaffolds: Dominant negative inhibitory protein inhibitory protein 3 mutants inhibit the activity of c-Jun N-terminal kinase 3 in cells. J Biol Chem 287, 19653–19664 (2012).

Levchenko, A., Bruck, J. & Sternberg, PW Scaffold protein may biphasically affect the level of mitogen-activated protein kinase signal and reduce its threshold characteristics. Proc Natl Acad Sci USA 97, 5818–5823 (2000).

CAS ADS article Google Scholar 

Levchenko, A., Bruck, J. & Sternberg, PW The regulation module that produces biphasic signal response in biological systems. Systems Biology (Stevenage) 1, 139–148 (2004).

Zhan, X., Gimenez, LE, Gurevich, VV & Spiller, BW The crystal structure of inhibin 3 reveals the basis for the difference in receptor binding between two non-visual inhibins. J Mol Biol 406, 467–478 (2011).

Sutton, RB, etc. The crystal structure of Cone Arrestin is 2.3Å: the evolution of receptor specificity. J Mol Biol 354, 1069–1080 (2005).

Han, M., Gurevich, VV, Vishnivetskiy, SA, Sigler, PB & Schubert, C. 1.9 A β-arrestin crystal structure: possible mechanisms of receptor binding and membrane translocation. Structure 9, 869–880 (2001).

Hirsch, JA, Schubert, C., Gurevich, VV & Sigler, PB 2.8 A The crystal structure of visual inhibitory protein: a model of inhibitory protein regulation. Cell 97, 257–269 (1999).

Ahmed, sir etc. The ubiquitin ligase parkin promotes the Mdm2-inhibitor interaction but inhibits the ubiquitination of the inhibitor. Biochemistry 50, 3749–3763 (2011).

Hansen, SM etc. Arrestin mobilizes the signaling protein to the cytoskeleton and redirects its activity. J Mol Biol 368, 375–387 (2007).

Seo, J., Tsakem, EL, Breitman, M. & Gurevich, VV Identification of inhibitory protein 3 specific residues required for JNK3 activation. J Biol Chem 286, 27894–27901 (2011).

Sterne-Marr, R. etc. Beta-arrestin and polypeptide variants of Arrestin 3. J Biol Chem 268, 15640-15648 (1993).

Kim, M. et al. The receptor binds to the conformation of visual inhibitory protein. Proc Nat Acad Sci USA 109, 18407–18412 (2012).

CAS ADS article Google Scholar 

Zhuang, T. et al. The participation of different inhibitory protein 1 elements in combination with different functional forms of rhodopsin. Proc Nat Acad Sci USA 110, 942–947 (2013).

CAS ADS article Google Scholar 

Kang, Y. et al. Femtosecond X-ray laser measures the crystal structure of rhodopsin bound to inhibitory protein. Nature 523, 561–567 (2015).

CAS ADS article Google Scholar 

Luttrell, LM, etc. The β-arrestin scaffold activates and targets extracellular signal-regulated kinases. Proc Natl Acad Sci USA 98, 2449-2454 (2001).

CAS ADS article Google Scholar 

Bruchas, MR, Macey, TA, Lowe, JD & Chavkin, C. Kappa opioid receptor activation of p38 MAPK is dependent on GRK3 and inhibin in neurons and astrocytes. J Biol Chem 281, 18081-18089 (2006).

Zhan, X. etc. Arrestin-3 dependent activation of c-Jun N-terminal kinase (JNK). Curr Protoc Pharmacol 68, 2.12.11-12.12.26 (2015).

Els, S., Beck-Sickinger, AG & Chollet, C. Ghrelin receptor: high composition activity and methods for developing inverse agonists. Method Enzymol 485, 103–121 (2010).

The author thanks Dr. Keiji Tanaka of the Tokyo Institute of Medical Sciences for providing the pMal-2T plasmid, and Ronny Müller for his assistance in peptide synthesis. Supported in part by NIH grants GM077561, GM081756 and EY011500 (VVG), GM059802, CA167505, Texas Cancer Prevention Institute (CPRIT) (RP140648) and Welch Foundation (F-1390) (KND); GM095633 (TMI); NS065868 (EVG); DFG SFB1052-A3 (ABS), the European Union and the Free State of Saxony ESF100148835 (SHE and ABS). TSK is supported by the CPRIT postdoctoral internship scholarship, HS is supported by the DAAD RISE summer scholarship, QC is supported by the Vanderbilt International Scholars Program, and NAP is supported by NIH T32 GM008320.

Current address: Current address: Department of Chemistry, Tennessee Tech University, PO Box 5055, Cookeville, TN 38505.,

Department of Pharmacology, Vanderbilt University, Nashville, 37232, Tennessee, USA

Xuan Zhi Zhan, Henriette Stoy, Nicole A. Perry, Chen Qiuyan, Alejandro Perez, Jack V. Slagis, Tina M. Iverson, Eugenia V. Gurevich and Vsevolod V. Gurevich

University of Tubingen, Tubingen, 72074, Germany

School of Pharmacy, Minneaminiya University, Egypt

Department of Medicinal Chemistry, University of Texas at Austin, Austin, 78712, Texas, USA

Tamer S. Kaoud & Kevin N. Dalby

Leipzig University, Faculty of Biological Sciences, Pharmacy and Psychology, Institute of Biochemistry, Brüderstrasse 34, Leipzig, 04103, Germany

Sylvia Els-Heindl & Annette G. Beck-Sickinger

Department of Biochemistry, Vanderbilt University, Nashville, 37232, Tennessee, USA

Vanderbilt University Structural Biology Center, Nashville, 37232, Tennessee, USA

Vanderbilt Institute of Chemical Biology, Vanderbilt University, Nashville, 37232, Tennessee, USA

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VVG, EVG, XZ, KND, TMI, QC and AGBS designed the research, analyzed the data, and wrote the manuscript; XZ, HS, EVG, NAP, QC, TSK, AP, SHE and JVS conducted experiments and analyzed the data , And participated in the writing of the manuscript.

Correspondence with Vsevolod V. Gurevich.

The author declares that there are no competing economic interests.

This work has been licensed under the Creative Commons Attribution 4.0 International License Agreement. The images or other third-party materials in this article are included in the Creative Commons license of the article, unless otherwise stated in the credit line; if the material is not included under the Creative Commons license, the user will need permission from the license holder to copy The material. To view a copy of this license, please visit http://creativecommons.org/licenses/by/4.0/

Zhan, X., Stoy, H., Kaoud, T. etc. Peptide microscaffolds promote JNK3 activation in cells. Scientific Report 6, 21025 (2016). https://doi.org/10.1038/srep21025

DOI: https://doi.org/10.1038/srep21025

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