Jnk activation assay

The activity of JNK in the immunoprecipitates was assessed by an immunokinase reaction using c-Jun as a substrate. Protein extracts were obtained at the indicated time points following UV treatment.

JNK activity was assessed by an immunokinase reaction using c-Jun as a substrate. The contribution of PKC to the overall level of JNK activity is expected to affect the biological activities regulated by JNK, including its regulation of programmed cell death. The mechanisms underlying the regulation of JNK activity are of great importance given the key role these kinases play in the regulation of cell survival or death in response to diverse extra-cellular stimuli.

Along these lines, finding the contribution of MKK-independent signaling pathways to JNK activation and function allows better understanding of the network of signaling cascades engaged in the activation of these important protein kinases. This background JNK2 activity was recently described by Pimienta et al. Pimienta et al. Thus, conditions that cause the activation of PKC may be part of the environment cell type, growth conditions rather than the actual response to stimuli, and as such, may result in basal phosphorylation of JNK on S This two tier control mechanism is expected to be important in regard to the degree of JNK activity, and consequently, the degree of the biological output following its activation.

Our finding also implies that physiological conditions under which PKC is constitutively active would be more prone to result in greater activation of JNK in response to stress, but also, under conditions where JNK kinases are constitutively upregulated.

The particular contribution of different PKC isoforms to JNK activation can not be established from our in vitro experiments since we used a complex mixture of different PKCs. Further experiments are needed to clarify the nature of these differences. Recently, Ventura et al. These findings may indicate that by affecting JNK activity, PKC phosphorylation of JNK could contribute to both the anti-apoptotic early phase and the pro-apoptotic late phase response.

In all, the present study provides important insight into the mechanism underlying PKC phosphorylation of JNK as well as the implication of such phosphorylation for JNK activities. Purification of the proteins used for the kinase reactions. We thank I. Weinstein for PKC constructs, R. Davis and M. We thank members of the Ronai lab for active discussions. Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript.

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National Center for Biotechnology Information , U. Int J Biochem Cell Biol. Author manuscript; available in PMC Apr Pablo Lopez-Bergami a, b and Ze'ev Ronai a.

Find articles by Pablo Lopez-Bergami. Find articles by Ze'ev Ronai. Author information Copyright and License information Disclaimer. Mediation of cell death by poly ADP-ribose polymerase Pharmacol Res ; 52 : 5— Mediation of poly ADP-ribose polymerasedependent cell death by apoptosis-inducing factor. Johnson GL, Lapadat R. The Bax subfamily of Bcl2-related proteins is essential for apoptotic signal transduction by c-Jun NH 2 -terminal kinase.

Mol Cell Biol ; 22 : — Novel role for JNK as a stress-activated Bcl2 kinase. J Biol Chem ; : — Essential roles of receptor-interacting protein and TRAF2 in oxidative stress-induced cell death. Mol Cell Biol ; 24 : — Exp Hematol ; 31 : — Overexpression of human poly ADP-ribose polymerase in transfected hamster cells leads to increased poly ADP-ribosyl ation and cellular sensitization to gamma irradiation.

Eur J Biochem ; : 15— Cell ; : 61— Embo J ; 14 : — Trends Biochem Sci ; 20 : — Leppa S, Bohmann D. Diverse functions of JNK signaling and c-Jun in stress response and apoptosis. Oncogene ; 18 : — Cell Death Differ ; 13 : — EMBO Rep ; 2 : — Cell ; : — Tumor necrosis factor-induced nonapoptotic cell death requires receptor-interacting protein-mediated cellular reactive oxygen species accumulation.

Genes Dev ; 19 : — Chiarugi A. Poly ADP-ribose polymerase: killer or conspirator? Trends Pharmacol Sci ; 23 : — Zahradka P, Ebisuzaki K.

A shuttle mechanism for DNA-protein interactions. The regulation of poly ADP-ribose polymerase. Eur J Biochem ; : — Biochem Biophys Res Commun ; : — Poly ADP-ribose synthetase is phosphorylated by protein kinase C in vitro. Poly ADP-ribose polymerase-1 regulates activation of activator protein-1 in murine fibroblasts.

J Immunol ; : — Genes Dev ; 9 : — The end of the cell line: methods for the study of apoptosis in vitro. Methods Cell Biol ; 46 : — Download references. We thank Prof. One prime example is in HeLa cells. In this study, we present several lines of evidence that Daxx and FADD bind to Fas independently and activate distinct cell death signals. Although both Daxx and FADD cannot bind to the lpr cg point mutant of Fas, NMR structural analysis of the Fas death domain suggest that this mutant may be unfolded and is thus uninformative for mapping the exact site of binding Huang et al.

However, Daxx has no evident death domain and interacts with Fas death domain via a novel domain contained in the amino acids of DaxxC, while FADD interacts with Fas via homotypic death domain—death domain interactions. Moreover, dominant-negative forms of Daxx and FADD each consisting of the minimal Fas-binding domains did not inhibit the heterologous protein's biologic function even at saturating levels Figure 7.

Daxx and FADD not only appear to bind differently to Fas but transduce physiologically distinct death signals. However, this pathway cannot activate JNK. Although either the Daxx- or the FADD-induced pathway alone is sufficient to activate cell death, these two pathways probably work together in vivo. Why expand the number of potentially dangerous cell suicide signals if one signal is sufficient?

As in the case of growth regulation, the collaboration of multiple signaling pathways allows fine-tuned regulation and creates multiple checkpoints for control. In this instance, incorporation of the Daxx pathway may allow the Fas death signal to be regulated by Bcl Anti-murine Fas Jo2 antibody was the generous gift of S.

Nagata Ogasawara et al. Resistant cells were selected in media containing 2. DNA fragments for most plasmid constructs were obtained by PCR amplification using Pfu polymerase Stratagene and primers incorporated with appropriate restriction sites and epitope tags as needed. Each construct was confirmed by partial DNA sequence and by immunoblot analysis.

Lutz, Apoptosis Inc. The two-hybrid screen was performed essentially as described Gyuris et al. The cDNA inserts of the library plasmids within those colonies were isolated and grouped. Representative plasmids from each group were retransformed back into yeast to test their interaction with different baits.

Filter lift assay for colony color and quantitative liquid assay were done as described Yang et al. Choi, Rockefeller Univ. Sequences of the longest cDNA clone 2. For Northern analysis, the C-terminal 0. GST fusions were purified as described Smith and Johnson, A fraction of the reaction mixture was analyzed by Coomassie staining to visualize GST fusion proteins.

Thirty-six hours after cotransfection, cells were solubilized in E1A buffer, precipitated with glutathione beads Molecular Probes , washed three times, and immunoblotted for HA using ECL Amersham.

The wells were precoated with 0. X-Gal staining was done for 4 hr to overnight. The percentage of apoptotic cells was determined by the number of blue cells with apoptotic morphology divided by the total number of blue cells.

Specific apoptosis was calculated as the percentage of blue cells with apoptotic morphology in each experimental condition minus the percentage of blue cells with apoptotic morphology in pEBB vector—transfected cells.

At least cells from four random fields were counted in each experiment, and the data shown are the average and SD of at least three independent experiments. Magnetic beads 1. HeLa and cells were transfected in 60 mm dishes with Flag-JNK plus the indicated expression plasmids by the calcium phosphate method. Approximately 24 hr after transfection, cells were serum starved for 14—16 hours. Cells transfected with Fas were treated with 0.

To test for the effect of protease inhibitors, cells were treated with 0. After an incubation of 24 hr, cells were switched to 0. We are indebted to Dr. Genhong Cheng for his help in the initial phase of this project. We thank Drs. Brent, Y. Choi, C. Der, D. Goeddel, A. Hoffmann, M. Karin, R. Kolesnick, P. Krammer, R. Lutz, S. Nagata, R. Treisman, D. Wallach, and J.

Yuan their generous gifts of reagents, and S. Fesik for examining the Daxx sequence. We thank P. Svec and Y. Li for excellent technical assistance, and Drs. Koleske, B. Chen, Z. Songyang, Y. Yamanashi, I. Stancovsky and other members of Baltimore lab for valuable advice. It functions in the control of a number of cellular processes, including proliferation, embryonic development and apoptosis. The JNK pathway is activated by a bewildering number of mechanisms.

Landmark discoveries in the area of JNK research are summarized in Figure 1. The JNK module plays an important role in apoptosis, inflammation, cytokine production, and metabolism. Those receptors or receptor-independent stress-induced membranal changes further transmit the signals to adaptor proteins that can by themselves activate kinases in the MAP4K, and sometime, MAP3K tiers of the JNK cascade.

MKK4 is involved in a variety of physiological and pathophysiological processes. The loss of mkk7 leads to decreased proliferation. The jnk1 and jnk2 are widely expressed, whereas expression of jnk3 is mainly confined to brain, heart and testis. It control the cell response to the harmful extracellular stimulus such as inflammatory cytokines, UV-irradiation gamma-irradiation etc..

Cells under these harmful stimulus may have DNA mutation or damage.