Jun Phosphorylation Research Articles

R-Ras3/(M-Ras) is involved in thermal adaptation in the critical period of thermal control establishment

Neuroanatomically, the body temperature is balanced by the preoptic anterior hypothalamus (PO/AH) and controlled by thermosensitive neurons. Hot or cold exposure during the critical period of temperature control development causes a plastic change in the ratio between hot- and cold-sensitive cells and can modulate temperature tolerance. In this project, mRNA fingerprinting was used to identify the proteins involved in thermal adaptation in 3-day-old chicks. Fifteen genes were induced, among which were NADH dehydrogenase, protocadherin, anolase alpha, 14-3-3epsilon, and R-Ras3. The role of each of these genes is potentially interesting and requires detailed evaluation, but since the present working hypothesis assumed neuronal remodeling, we concentrated on the role of R-Ras3/(M-Ras), which is uniquely expressed in the brain and whose physiological role has not been described. In the present study, R-Ras3 expression during thermal conditioning was investigated by several molecular techniques and its mRNA was found to be induced in the PO/AH with a tenfold peak after 12 h of heat conditioning and a fourfold increase after 6 h of cold conditioning. To improve our understanding of thermal adaptation-related signal transduction, we screened for changes in the expression of transcription factors that were implicated with the Ras gene family, and found that both jun mRNA expression and Jun phosphorylation were induced after 30 min of temperature conditioning. Taken together, the present findings correlate the R-Ras3-jun pathway with thermal-control establishment.

Regulatory Paradigm Revealed

A well-established mechanism for the controlled degradation of proteins involves phosphorylation of the target protein, which marks the protein as a target for ubiquitin E3 ligases. Ubiquitination then leads to proteasomal degradation of the protein. Gao et al. show that controlled degradation of the transcription factor c-Jun is regulated not by Jun phosphorylation per se, but by the phosphorylation-dependent activation of the ubiquitin E3 ligase Itch. The c-Jun N-terminal kinase (JNK), which phosphorylates and activates Jun, also phosphorylates and activates Itch, which then mediates enhanced degradation of Jun. This feedback mechanism appears to attenuate the signaling pathway that leads to the production of T helper 2-type cytokines and may help to control T cell activation. M. Gao, T. Labuda, Y. Xia, E. Gallagher, D. Fang, Y.-C. Liu, M. Karin, Jun turnover is controlled through JNK-dependent phosphorylation of the E3 ligase Itch. Science 306 , 271-275 (2004). [Abstract] [Full Text]

An effector peptide from glutathione-S-transferase-pi strongly and selectively blocks mitotic signaling by oncogenic ras-p21.

Oncogenic ras-p21 directly activates jun-N-terminal kinase (JNK) and its substrate, jun as a unique step on its mitogenic signal transduction pathway. This activation is blocked by the specific JNK-jun inhibitor, glutathione-S-transferase-pi (GST-pi). Four domains of GST-pi have been implicated in this regulatory function: 34-50, 99-121, 165-182, and 194-201. The 34-50 domain is unique in that it does not affect GST-pi binding to JNK-jun but blocks jun phosphorylation by JNK. We now find that it completely blocks oncogenic (Val 12-) ras-p21-induced oocyte maturation but has no effect on insulin-induced oocyte maturation. Because the latter process requires activation of wild-type ras-p21, this peptide appears to be specific for inhibiting only the oncogenic form of ras-p21, suggesting its use in treating ras-induced tumors.

Relevance of mitogen activated protein kinase (MAPK) and phosphotidylinositol-3-kinase/protein kinase B (PI3K/PKB) pathways to induction of apoptosis by curcumin in breast cells

Following observations that curcumin inhibited proliferation ( ic 50=1 –5 μM), invasiveness and progression through S/G2/M phases of the cell cycle in the non-tumourigenic HBL100 and tumourigenic MDA-MB-468 human breast cell lines, it was noted that apoptosis was much more pronounced in the tumour line. Therefore, the ability of curcumin to modulate signalling pathways which might contribute to cell survival was investigated. After pre-treatment of cells for 20 min, curcumin (40 μM) inhibited EGF-stimulated phosphorylation of the EGFR in MDA-MB-468 cells and phosphorylation of extracellular signal regulated kinases (ERKs) 1 and 2, as well as ERK activity and levels of nuclear c- fos in both cell lines. At a lower dose (10 μM), it also inhibited the ability of anisomycin to activate JNK, resulting in decreased c- jun phosphorylation, although it did not inhibit JNK activity directly. In contrast, the activation of p38 mitogen activated protein kinase (MAPK) by anisomycin was not inhibited. Curcumin inhibited basal phosphorylation of Akt/protein kinase B (PKB) in both cell lines, but more consistently and to a greater extent in the MDA-MB-468 cells. The MAPK kinase (MKK) inhibitor U0126 (10 μM), while preventing ERK phosphorylation in MDA-MB-468 cells, did not induce apoptosis. The PI3K inhibitor LY294002 (50 μM) inhibited PKB phosphorylation in both cells lines, but only induced apoptosis in the MDA-MB-468 line. These results suggest that while curcumin has several different molecular targets within the MAPK and PI3K/PKB signalling pathways that could contribute to inhibition of proliferation and induction of apoptosis, inhibition of basal activity of Akt/PKB, but not ERK, may facilitate apoptosis in the tumour cell line.

Structurally distinct modes of recognition of the KIX domain of CBP by Jun and CREB.

Gene expression is coordinated in part by interactions between transcriptional activators and other transcription factors such as coactivators. The KIX domain of the coactivator and histone acetyltransferase CREB binding protein (CBP) binds numerous mammalian and viral transcriptional activators such as BRCA1, CREB, c-Jun, c-Myb, p53, papillomavirus E2, and HTLV-1 Tax. Formation of the CREB-CBP complex depends on phosphorylation of the KID region of CREB and involves induced folding of KID upon binding a hydrophobic groove of the KIX domain of CBP. Here we investigate the formation of the complex formed by human KIX and the N-terminal activation domain of human c-Jun. The c-Jun activation domain and KID do not share significant sequence similarity. Circular dichroism spectroscopy shows that the Jun N-terminal activation domain is intrinsically disordered in isolation and that KIX binding is independent of Jun phosphorylation. In contrast to the mode of binding exhibited by CREB, NMR chemical shift mapping indicates that the c-Jun activation domain binds to a distinctly different surface of KIX than used by CREB. Moreover, NMR and sedimentation equilibrium studies show that the activation domains of c-Jun and CREB can simultaneously bind the KIX domain of CBP. The results illustrate a new mode of binding and combinatorial recruitment via the KIX domain of CBP by multiple transcriptional activators.

Phosphorylation of Drosophila Jun by the MAP kinase rolled regulates photoreceptor differentiation.

Drosophila Jun (D-Jun) is a nuclear component of the receptor tyrosine kinase/Ras signal transduction pathway which triggers photoreceptor differentiation during eye development. Here we show that D-Jun is a substrate for the ERK-related Drosophila MAP kinase Rolled, which has previously been shown to be a part of this pathway. A D-Jun mutant that carries alanines in place of the Rolled phosphorylation sites acts as a dominant suppressor of photoreceptor cell fate if expressed in the eye imaginal disc. In contrast, a mutant in which the phosphorylation sites are replaced by phosphate-mimetic Asp residues, as well as a VP16-D-Jun fusion protein, can promote photoreceptor differentiation. These data implicate Jun phosphorylation in the choice between neuronal and non-neuronal fate during Drosophila eye development.

The Transformation of C-Jun-Overexpressing Cells Is Correlated with IGFS-Induced C-Jun Phosphorylation

We have analyzed, in parental and c-jun-transfected rat liver epithelial (REL) cells, the capacity of the insulin-like growth factors (IGF1 and 2) to stimulate growth under anchorage dependent and independent conditions and the role of the c-jun protein in the response to these factors. Although the increase of the proliferation of cells in monolayer was similar for the two lines, only the c-jun-transfected cells formed colonies in soft agar after IGFs exposure. We showed that IGF1 and 2 did not modify the expression of c-fos or c-jun proteins during short-time exposures but both factors enhanced c jun phosphorylation which suggests that this phenomenon could be involved in IGfs-regulated gene expression. Moreover, we showed that both the overexpression and the increase of phosphorylation status of c-jun protein were necessary to transform REL cells.

UV Irradiation and Heat Shock Mediate JNK Activation via Alternate Pathways

To elucidate cellular pathways involved in Jun-NH2-terminal kinase (JNK) activation by different forms of stress, we have compared the effects of UV irradiation, heat shock, and H2O2. Using mouse fibroblast cells (3T3-4A) we show that while H2O2 is ineffective, UV and heat shock (HS) are potent inducers of JNK. The cellular pathways that mediate JNK activation after HS or UV exposure are distinctly different as can be concluded from the following observations: (i) H2O2 is a potent inhibitor of HS-induced but not of UV-induced JNK activation; (ii) Triton X-100-treated cells abolish the ability of UV, but not HS, to activate JNK; (iii) the free radical scavenger N-acetylcysteine inhibits UV- but not HS-mediated JNK activation; (iv) N-acetylcysteine inhibition is blocked by H2O2 in a dose-dependent manner; (v) a Cockayne syndrome-derived cell line exhibits JNK activation upon UV exposure, but not upon HS treatment. The significance of Jun phosphorylation by JNK after treatment with UV, HS, or H2O2 was evaluated by measuring Jun phosphorylation in vivo and also its binding activity in gel shifts. HS and UV, which are potent inducers of JNK, increased the level of c-Jun phosphorylation when this was measured by [32P]orthophosphate labeling of 3T3-4A cultures. H2O2 had no such effect. Although H2O2 failed to activate JNK in vitro and to phosphorylate c-Jun in vivo, all three forms of stress were found to be potent inducers of binding to the AP1 target sequence. Overall, our data indicate that both membrane-associated components and oxidative damage are involved in JNK activation by UV irradiation, whereas HS-mediated JNK activation, which appears to be mitochondrial-related, utilizes cellular sensors.

JNK is involved in signal integration during costimulation of T lymphocytes

T lymphocyte activation and interleukin-2 (IL-2) production require at least two signals, generated by phorbol ester (TPA) and Ca 2+ ionophore or costimulation of the T cell receptor (TCR) and the CD28 auxiliary receptor. We investigated how these stimuli affect mitogen activated protein (MAP) kinases. Full activation of the MAP kinases that phosphorylate the Jun activation domain, JNK1 and JNK2, required costimulation of T cells with either TPA and Ca 2+ ionophore or antibodies to TCR and CD28. Alone, each stimulus resulted in little or no activation. Similar to its effect on IL-2 induction, cyclosporin A (CsA) inhibited the synergistic activation of JNK, and a competitive inhibitor of Jun phosphorylation by JNK inhibited IL-2 promoter activation. By contrast, the MAP kinases ERK1 and ERK2 were fully activated by TPA or TCR stimulation and were not affected by Ca 2+, CD28, or CsA. Hence, integration of signals that lead to T cell activation occurs at the level of JNK activation.