Kit Contents:

Component	Specification	Concentration (total volume)
AP1 Reporter	A mixture of inducible AP1-responsive firefly luciferase construct and constitutively expressing Renilla luciferase construct (40:1).	(100 ng/µl; 500 µl)*
Negative control	A mixture of non-inducible firefly luciferase construct and constitutively expressing Renilla luciferase construct (40:1).	(100 ng/µl; 500 µl)
Positive control	A mixture of constitutively expressing GFP, constitutively expressing firefly luciferase, and constitutively expressing Renilla luciferase constructs (40:1:1).	(100 ng/µl; 250 µl)

* Supplied material provides sufficient reporter for 500 assays, using recommended 96-well plate transfection protocol. The number of assays per kit is a function of the assay plate format used (refer to Cignal Reporter Assay User Manual).

Storage Conditions: The Cignal reporter assay constructs are shipped ambient. Store all tubes at -20  ºC.

1. Dilute transfection-ready reporter, negative control, and positive control construct formulations.
2. Dilute relevant test nucleic acids (siRNA, shRNA, miRNA, expression vector).
3. Prepare appropriate combinations of reporter constructs, controls, and test nucleic acids.
4. Transfect plasmid mixtures separately into replicate wells of your cell line of interest using an optimized transfection procedure for the cell line under study.
5. If applicable, 16 to 24 hours post-transfection, treat the transfected cells with test proteins, peptides, or compounds of interest.
6. Two (2) to three (3) days post-transfection, assay the activities of the signaling pathways under study, utilizing the dual luciferase assay.

The Cignal Reporter Assays include pre-formulated, transfection-ready reporter, negative control, and positive control. The transcription factor reporter and negative control are transfected and subjected to experimental treatments, in parallel. Dual-luciferase results are calculated for each transfectant. The impact of the experimental treatments is determined by comparing the normalized luciferase activities of the reporter to the identically treated negative control, across the complete treatment regimen. The positive control serves as a control for transfection efficiency, by monitoring GFP expression, as well as a positive control for both the firefly and Renilla luciferase assays.

General performance

Average maximum response rate = 42.6
Average Z' factor at maximum response rate = 0.73
Average coefficient of variation (CV%) = 8.7%

Excellent signal to noise ratio

Cignal AP-1 reporter assay showed activation of MAPK signaling: 293-H cells were transfected with AP-1 reporter, negative control and positive control (for transfection protocol refer our user manual). After 24 hours of transfection, medium was changed to assay medium (Opti-MEM + 0.5% FBS + 0.1mM NEAA + 1mM Sodium pyruvate + 100 U/ml penicillin + 100 µg/ml streptomycin) and cells were treated with 10 ng/ml of PMA or an equal volume of DMSO for 18 hours. Dual Luciferase assay was performed 42 hours after transfection, and promoter activity values are expressed as arbitrary units using a Renilla reporter for internal normalization. Experiments were done in triplicates, and the standard deviation is indicated. Cignal AP-1 reporter assay measured 43 fold increase in the AP-1 transcription activity and, in turn, in corresponding MAPK signaling by 10 ng.ml PMA.

MAPKs (Mitogen-Activated Protein Kinases) are Serine-threonine protein Kinases that are activated in response to a variety of extracellular stimuli and mediate signal transduction from the cell surface to the nucleus. MAPKs are expressed in multiple cell types including Cardiomyocytes, Vascular Endothelial cells, and Vascular Smooth Muscle Cells. Three major MAPKs include ERKs (Extracellular signal-Regulated Kinases), JNKs (c-Jun NH(2)-terminal protein Kinases), and p38 Kinases. Members of the JNK/SAPK (Stress-Activated Protein Kinase) family of MAPKs are strongly stimulated by numerous Environmental Stresses, but also more modestly stimulated by Mitogens, Inflammatory Cytokines, Oncogenes, and inducers of Cell differentiation and morphogenesis. Ten mammalian JNK isoforms have been identified and are encoded by three distinct genes, JNK1, JNK2, and JNK3, the transcripts of which are alternatively spliced to yield four JNK1 isoforms, four JNK2 isoforms, and two JNK3 isoforms. JNK1 and JNK2 are the products of alternative splicing of a single gene and are expressed in many tissues, but JNK3 is specifically expressed in brain. Members of the JNK family play crucial roles in regulating responses to various Stresses, and in Neural Development, Inflammation, and Apoptosis. JNK activation is much more complex than that of ERK1/ERK2 owing to inputs by a greater number of MAPKKKs (Mitogen-Activated Protein Kinase Kinase Kinases) (at least 13, including MEKK1 (MAP/ERK Kinase-Kinase-1)-MEKK4 (MAP/ERK Kinase-Kinase-4), ASK (Apoptosis Signal-regulating Kinase) and MLKs (Mixed-Lineage Kinases), which are activated by upstream Rho-family GTPases). These activate JNK MAPKKs MEK4 (MAPK/ERK Kinase-4) and MEK7 (MAPK/ERK Kinase-7), which further activate JNKs. The JNK MAPK modules are regulated by a number of different scaffold proteins, including JIP1 (JNK Interacting Protein-1), JIP2 (JNK Interacting Protein-2), JIP3 (JNK Interacting Protein-3), JIP4 (JNK Interacting Protein-4), Beta-Arrestin-2, Filamin and CrkII. The scaffold proteins presumably target the MAPK modules to different sites in the cell and play roles in kinase activation and/or substrate selection (Ref.1 & 2).

Stress or Genotoxic agents are the most powerful inducers of JNK. Different forms of stress have been shown to mediate JNK activation via various cellular pathways. JNK activation in response to UV irradiation is mediated by upstream signaling components, including Rac (Ras-Related C3 Botulinum Toxin Substrate), CDC42 (Cell Division Cycle-42), PAK (p21/CDC42/Rac1-Activated Kinase), ASK1 (Apoptosis Signal-regulating Kinase-1), MLK, MEKK1, SEK1 (SAPK/ERK Kinase-1)/MKK4, MKK7 and p21Ras, in concert with nuclear DNA lesions. Besides Stress, JNKs can also be activated via GPCRs (G-Protein Coupled Receptors), RTKs (Receptor Tyrosine Kinases) and Cytokine Receptors. How GPCRs activate the JNKs is still an unanswered question. Free Beta-Gamma dimers and GN-Alpha12 and GN-Alpha13 proteins are able to activate JNK in a Rac1-CDC42 or p115RhoGEF and RhoA-dependent manner. However, the nature of the GEFs (Guanine nucleotide Exchange Factors) that connect Beta-Gamma and GN-Alpha12/ GN-Alpha13 to Rac1 and CDC2 is still unclear. Interestingly, GN-Alpha12 can also activate JNK by activating the MEKK (MEK kinase). The activation of JNK by Cytokine receptors appears to be mediated by the TRAF (TNF Receptor-Associated Factor) group of Adaptor proteins. Activation of the TNF receptor leads to recruitment of TRAF2 (TNF Receptor-Associated Factor-2), which is required for JNK activation. These adaptor proteins (TRADD (Tumor Necrosis Factor Receptor-1-Associated Death Domain Protein), RIP (Receptor-Interacting Protein) and Daxx) have been reported to bind MEKK1 and ASK1. TRAF2 activates MAPK4Ks like GCK (Germinal Center Kinase), GCKR (GCK-Related Kinase), GLK (GCK-Like Kinase) and HGK (HPK/GCK-like Kinase), which further activates JNKs via MEKK1 and MKK4/7 respectively. ASK1 also interacts with TRAF2 and activates JNK via MKK4/7 (Ref. 3, 4 & 5).

Growth Factors also activate JNKs. Although the Signaling cascade from Growth Factor Receptors to ERKs is relatively well understood, the pathway leading to JNK activation is more obscure. Activation of JNK by EGF (Epidermal Growth Factor) or NGF (Nerve Growth Factor) is dependent on H-Ras activation. Growth Factors and Growth Factor Receptors stimulate Ras by recruiting SOS (Son of Sevenless), GRB2 (Growth Factor Receptor-Bound Protein-2) and SHC to the membrane. PI3K (Phosphatidylinositde-3-Kinase) also activate Ras. Ras activates two protein kinases, Raf1 and MEKK (MEK (MAPK, or ERK, kinase) Kinase). Raf1 contributes directly to ERK activation but not to JNK activation, whereas MEKK participated in JNK activation but caused ERK activation only after overexpression. Recently, Raf1 is found to interact with the proapoptotic, stress-activated protein kinase ASK1 in vitro and in vivo. This interaction allows Raf1 to act independently of the MEK–ERK pathway to activate JNK pathway (Ref.6 & 7). The Rho family GTPases, CDC42 (Cell Division Cycle-42) and Rac also initiate a cascade leading to JNK/SAPK, presumably by binding and activating the protein kinase PAK (p21-Activated Kinases), a kinase that phosphorylates and promotes activation of MEKK1. Rac/CDC42 are also involved in JNK activation via a pathway consisting of a sequential cascade MLKs and MKK4/7 (MAP Kinase Kinase-4/7. MLK2 (Mixed-Lineage Kinase-2) and MLK3 (Mixed-Lineage Kinase-3) interact with the activated (GTP-bound) forms of Rac and CDC42, with a slight preference for Rac. Besides MLKs, MEKK1/4 and Posh (Plenty of SH3) are also activated by Rac/CDC42 to activate MKK4/7 and thus JNKs. Adaptor proteins such as Crk (v-Crk Avian Sarcoma Virus Ct10 Oncogene Homolog) and CrkL (v-Crk Avian Sarcoma Virus Ct10 Oncogene Homolog-Like) also leads to activation of JNKs in response to RTK. HPK1 (Hematopoietic Progenitor Kinase-1) associates with Crk and CrkL through binding to the SH3 (Src-Homology Domain-3) of these proteins. Furthermore, association of HPK1 with these proteins increases HPK1's kinase activity. HPK1 then act as upstream of MEKK1 and TAK1 (Transforming Growth Factor-Beta-activated Kinase-1) in the JNK kinase cascade. JNKs are negatively regulated by MKP (MAP Kinase Phosphatase) (Ref.2, 8 & 9).

The activated JNK/SAPKs translocate to the nucleus where they phosphorylate transcription factors such as c-Jun, Elk1, DPC4 (Deleted In Pancreatic Carcinoma 4)/ SMAD4 (Sma and MAD (Mothers Against Decapentaplegic) Related Protein-4), p53, ATF2 (Activating Transcription Factor-2), NFAT4 (Nuclear Factor of Activated T-Cell-4) and NFAT1 (Nuclear Factor of Activated T-Cell-1). JNK1 directly phosphorylates Bcl2 (B-Cell CLL/Lymphoma-2) in vitro, co-localizes and collaborates with Bcl2 to mediate prolonged cell survival. JNK cascade also activates TCF (Ternary Complex Factor) protein. JNK also phosphorylate HSF1 (Heat Shock Factor-1) and JNK-mediated phosphorylation of HSF1 selectively stabilize the HSF1 protein and confers protection to cells under conditions of severe stress. DCX is also a substrate of JNK and interacts with both JNK and JIP. MAPs (Microtubule-Associated Proteins), both MAP1B and MAP2B are also found to be the substrates of JNK. Ser-727 phosphorylation of STAT3 (Signal Transducer and Activator of Transcription-3) can also be induced by JNK. JNK also regulates Insulin signaling by negatively regulating IRS1 (Insulin Receptor Substrate-1). JNK is generally thought to be involved in inflammation, proliferation and Apoptosis. Accordingly, its substrates are transcription factors and Anti-apoptotic proteins. However, JNK also phosphorylates Serine 178 on Paxillin and regulate cell migration. Despite extensive progress in the understanding of the JNK MAP kinase pathway, the mechanisms by which the pathway contributes to the many cellular programs where JNKs are activated are poorly defined. The JIP1 proteins have been proposed to act as molecular scaffolds that organize the JNK signal transduction pathway in response to specific stimuli. The JNK stress pathway is thought to be important in many pathological conditions including the progression of some neurodegenerative diseases such as Huntington’s and also in cancer. This pathway therefore offers potential targets for therapeutic intervention. The identification of critical components of this signaling pathway, such as JIP1, offers new routes to understand how this pathway is regulated and potential ways of manipulating it to combat disease (Ref.10, 11 & 12).

References:

1. Himes SR, Sester DP, Ravasi T, Cronau SL, Sasmono T, Hume DA.
The JNK are important for development and survival of macrophages.
J Immunol. 2006 Feb 15;176(4):2219-28.
PubMed ID: 16455978

2. Moulin N, Widmann C.
Islet-brain (IB)/JNK-interacting proteins (JIPs): future targets for the treatment of neurodegenerative diseases?
Curr Neurovasc Res. 2004 Apr;1(2):111-27.
PubMed ID: 16185188

3. Zhou JY, Liu Y, Wu GS.
The role of mitogen-activated protein kinase phosphatase-1 in oxidative damage-induced cell death.
Cancer Res. 2006 May 1;66(9):4888-94.
PubMed ID: 16651445

4. Yang L, Mao L, Chen H, Catavsan M, Kozinn J, Arora A, Liu X, Wang JQ.
A signaling mechanism from G alpha q-protein-coupled metabotropic glutamate receptors to gene expression: role of the c-Jun N-terminal kinase pathway.
J Neurosci. 2006 Jan 18;26(3):971-80.
PubMed ID: 16421317

5. Yang Q, Kim YS, Lin Y, Lewis J, Neckers L, Liu ZG.
Tumour necrosis factor receptor 1 mediates endoplasmic reticulum stress-induced activation of the MAP kinase JNK.
EMBO Rep. 2006 May 5;
PubMed ID: 16680093

6. Kraus S, Benard O, Naor Z, Seger R.
c-Src is activated by the epidermal growth factor receptor in a pathway that mediates JNK and ERK activation by gonadotropin-releasing hormone in COS7 cells.
J Biol Chem. 2003 Aug 29;278(35):32618-30.
PubMed ID: 12750372

7. Matsukawa J, Matsuzawa A, Takeda K, Ichijo H.
The ASK1-MAP kinase cascades in mammalian stress response.
J Biochem (Tokyo). 2004 Sep;136(3):261-5.
PubMed ID: 15598880

8. Yamauchi J, Miyamoto Y, Kokubu H, Nishii H, Okamoto M, Sugawara Y, Hirasawa A, Tsujimoto G, Itoh H.
Endothelin suppresses cell migration via the JNK signaling pathway in a manner dependent upon Src kinase, Rac1, and Cdc42.
FEBS Lett. 2002 Sep 11;527(1-3):284-8.
PubMed ID: 12220675

9. Zhou JY, Liu Y, Wu GS.
The role of mitogen-activated protein kinase phosphatase-1 in oxidative damage-induced cell death.
Cancer Res. 2006 May 1;66(9):4888-94.
PubMed ID: 16651445

10. Baan B, van Dam H, van der Zon GC, Maassen JA, Ouwens DM.
The role of JNK, p38 and ERK MAP-kinases in insulin-induced Thr69 and Thr71-phosphorylation of transcription factor ATF2.
Mol Endocrinol. 2006 Apr 6;
PubMed ID: 16601071

11. Sprowles A, Robinson D, Wu YM, Kung HJ, Wisdom R.
c-Jun controls the efficiency of MAP kinase signaling by transcriptional repression of MAP kinase phosphatases.
Exp Cell Res. 2005 Aug 15;308(2):459-68.
PubMed ID: 15950217

12. Heasley LE, Han SY.
JNK Regulation of Oncogenesis.
Mol Cells. 2006 Apr 30;21(2):167-73.
PubMed ID: 16682809



Back to Top