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The mechanisms by which cells rapidly polarize in the direction of external signals are not yet well understood. Helper T cells, when contacted by an antigen-presenting cell, polarize their cytoskeletons toward the antigen-presenting cell within minutes. In T cells, the mammalian Ras-related GTPase CDC42 (the homologue of yeast CDC42, a protein involved in budding polarity) can regulate the polarization of both actin and microtubules toward antigen-presenting cells but is not involved in other T-cell signaling processes such as those which culminate in interleukin 2 production. Although T-cell polarization appears dispensable for signaling leading to interleukin 2 production, polarization may direct lymphokine secretion towards the correct antigen-presenting cell in a crowded cellular environment. Inhibitor experiments suggest that phosphatidylinositol 3-kinase is required for cytoskeletal polarization but that calcineurin activity, known to be important for other aspects of signaling, is not. Apparent conservation of CDC42 function between yeast and T cells suggests that this GTPase is a general regulator of cytoskeletal polarity in many cell types (Stowers, 1995).
T lymphocyte activation and interleukin-2 (IL-2) production require at least two signals, generated either by phorbol ester (TPA) and Ca2+ ionophore, or costimulation of the T cell receptor (TCR) and the CD28 auxiliary receptor. Full activation of the MAP kinases that phosphorylate the Jun activation domain, JNK1 and JNK2, requires costimulation of T cells with either TPA and Ca2+ ionophore or antibodies to TCR and CD28. Alone, each stimulus results in little or no activation. Similar to its effect on IL-2 induction, cyclosporin A (CsA) inhibits the synergistic activation of JNK, and a competitive inhibitor of Jun phosphorylation by JNK inhibits IL-2 promoter activation. In contrast, the MAP kinases ERK1 and ERK2 are fully activated by TPA or TCR stimulation and are not affected by Ca2+, CD28, or CsA. Hence, integration of signals that lead to T cell activation occurs at the level of JNK activation (Su, 1994).
The Jun N-terminal kinase (JNK) signaling pathway has been implicated in cell proliferation and apoptosis, but its function seems to depend on the cell type and inducing signal. In T cells, JNK has been implicated in both antigen-induced activation and apoptosis. Initially, mice lacking the JNK2 isozymes were generated. The mutant mice were healthy and fertile but defective in peripheral T-cell activation induced by antibody to the CD3 component of the T-cell receptor (TCR) complex: proliferation and production of interleukin-2 (IL-2), IL-4 and interferon-gamma (IFN-gamma) are all reduced. The proliferation defect is restored by exogenous IL-2. B-cell activation is normal in the absence of JNK2. Activation-induced peripheral T-cell apoptosis is comparable between mutant and wild-type mice, but immature [CD4(+) CD8(+)] thymocytes lacking JNK2 are resistant to apoptosis induced by administration of anti-CD3 antibody in vivo. The lack of JNK2 also results in partial resistance of thymocytes to anti-CD3 antibody in vitro, but has little or no effect on apoptosis induced by anti-Fas antibody, dexamethasone or ultraviolet-C (UVC) radiation. It is concluded that JNK2 is essential for efficient activation of peripheral T cells but not B cells. Peripheral T-cell activation is probably required indirectly for induction of thymocyte apoptosis resulting from administration of anti-CD3 antibody in vivo. JNK2 functions in a cell-type-specific and stimulus-dependent manner, being required for apoptosis of immature thymocytes induced by anti-CD3 antibody but not for apoptosis induced by anti-Fas antibody, UVC or dexamethasone. JNK2 is not required for activation-induced cell death of mature T cells (Sabapathy, 1999a).
CD40 is a 45- to 50-kDa transmembrane glycoprotein that plays an important role in B cell proliferation, survival, memory, and Ig isotype switching. How does CD40 engagement couple to these distal events in B cells? In comparison to signaling via the B cell Ag receptor (BCR), CD40 cross-linking is less effective at activating protein tyrosine kinases. CD40 engagement results in the phosphorylation of both extracellular signal-regulated protein kinase (ERK) and the Ras guanine nucleotide exchange factor, Son of sevenless. In addition to this noteworthy observation, both ERK and c-Jun NH2-terminal kinase activities are increased after both CD40 and BCR ligation. Overnight treatment of cells with phorbol ester as well as pharmacologic inhibitors of protein kinase C abrogates these signaling events after BCR treatment; however, no effect is seen on CD40-mediated activation of ERK or c-Jun NH2-terminal kinase, suggesting that the BCR and CD40 differentially utilize protein kinase C to couple with these signaling pathways (Li, 1996a).
In the absence of accessory cell-derived costimulatory signals, T cells activated by antigen receptor stimulation lose the capacity to synthesize the growth factor interleukin-2 (IL-2), and enter a state termed clonal anergy. An analysis of CD3- and CD28-induced signal transduction reveals reduced ERK and JNK enzyme activities in murine anergic T cells. The amounts of ERK and JNK proteins remain unchanged, and the kinases could be fully activated in the presence of phorbol ester. Dephosphorylation of the calcineurin substrate NFATp (preexisting nuclear factor of activated T cells) also remains inducible. These results suggest that a specific block in the activation of ERK and JNK contributes to defective IL-2 production in clonal anergy (Li, 1996b).
The c-Jun NH2-terminal kinase (JNK) signaling pathway has been implicated in the immune response that is mediated by the activation and differentiation of CD4 helper T (TH) cells into TH1 and TH2 effector cells. JNK activity observed in wild-type activated TH cells is severely reduced in TH cells from Jnk1-/- mice. The Jnk1-/- T cells hyperproliferate, exhibit decreased activation-induced cell death, and preferentially differentiate to TH2 cells. The enhanced production of TH2 cytokines by Jnk1-/- cells is associated with increased nuclear accumulation of the transcription factor NFATc. Thus, the JNK1 signaling pathway plays a key role in T cell receptor-initiated TH cell proliferation, apoptosis, and differentiation (Dong, 1998).
Mitogen-activated protein (MAP) kinases can be grouped into three structural families, ERK, JNK, and p38, each of which are thought to carry out unique functions within cells. ERK, JNK, and p38 are activated by distinct combinations of stimuli in T cells that simulate full or partial activation through the T cell receptor. These kinases are regulated by reversible phosphorylation on Tyr and Thr, and the dual specific phosphatases PAC1 and MKP-1 previously have been implicated in the in vivo inactivation of ERK, or of ERK and JNK, respectively. A new MAP kinase phosphatase, MKP-2, is induced in human peripheral blood T cells with phorbol 12-myristate 13-acetate and is expressed in a variety of nonhematopoietic tissues as well. PAC1, MKP-2, and MKP-1 recognize ERK and p38, ERK and JNK, and ERK, p38, and JNK, respectively. Thus, individual MAP kinase phosphatases can differentially regulate the potential for cross-talk between the various MAP kinase pathways. A hyperactive allele of ERK2, analogous to the Drosophila sevenmaker gain-of-function mutation of rolled, has significantly reduced sensitivity to all three MAP kinase phosphatases in vivo (Chu, 1996).
A novel member of the tumor necrosis factor (TNF) cytokine family, designated TRANCE, was cloned during a search for apoptosis-regulatory genes using a somatic cell genetic approach in T cell hybridomas. The TRANCE gene encodes a type II membrane protein of 316 amino acids with a predicted molecular mass of 35 kDa. Its extracellular domain is most closely related to TRAIL, FasL, and TNF. TRANCE is an immediate early gene, up-regulated by TCR stimulation, and controlled by calcineurin-regulated transcription factors. TRANCE is most highly expressed in thymus and lymph nodes but not in nonlymphoid tissues and is abundantly expressed in T cells but not in B cells. Cross-hybridization of the mouse cDNA to a human thymus library yielded the human homolog, which encodes a protein 83% identical to the mouse ectodomain. Human TRANCE maps to chromosome 13q14, while mouse TRANCE is located on the portion of mouse chromosome 14 syntenic with human chromosome 13q14. A recombinant soluble form of TRANCE composed of the entire ectodomain induces c-Jun N-terminal kinase (JNK) activation in T cells but not in splenic B cells or in bone marrow-derived dendritic cells. These results suggest a role for this TNF-related ligand in the regulation of the T cell-dependent immune response (Wong, 1997).
The Epstein-Barr virus latent membrane protein-1 (LMP-1) is an integral membrane protein that transforms fibroblasts and is essential for EBV-mediated B-cell immortalization. LMP-1 has been shown to trigger cellular NF-kappaB activity; however, this cannot fully explain the oncogenic potential of LMP-1. LMP-1 induces the activity of the AP-1 transcription factor, a dimer of Jun/Jun or Jun/Fos proteins. LMP-1 effects on AP-1 are mediated through activation of the c-Jun N-terminal kinase (JNK) cascade, but not the extracellular signal-regulated kinase (Erk) pathway. Consequently, LMP-1 triggers the activity of the c-Jun N-terminal transactivation domain, which is known to be activated upon JNK-mediated phosphorylation. Deletion analysis indicates that the 55 C-terminal amino acids of the LMP-1 molecule, but not its TRAF interaction domain, are essential for AP-1 activation. JNK-mediated transcriptional activation of AP-1 is the direct output of LMP-1-triggered signaling, as shown by an inducible LMP-1 mutant. Using a tetracycline-regulated LMP-1 allele, it has been demonstrated that JNK is also an effector of non-cytotoxic LMP-1 signaling in B cells, the physiological target cells of EBV. In summary, these data reveal a novel effector of LMP-1, the SEK/JNK/c-Jun/AP-1 pathway, which contributes to an understanding of the immortalizing and transforming potential of LMP-1 (Kieser, 1997).
The c-Jun NH(2)-terminal kinase (JNK) has been implicated in regulating cell survival, apoptosis, and transformation. However, the distinct role of JNK isoforms in regulating tumor development is not yet clear. It has been found that skin tumor formation induced by the tumor promoter, 12-O-tetradecanoylphorbol-13-acetate (TPA), is suppressed in JNK2-deficient (Jnk2-/-) mice. This study shows that JNK1-deficient (Jnk1-/-) mice are more susceptible to TPA-induced skin tumor development than wild-type mice. The rate of tumor development in Jnk1-/- mice is significantly more rapid than that observed in wild-type mice. At the end of 33 weeks of TPA promotion, the number of skin tumors and tumors >1.5 mm in diameter per mouse in Jnk1-/- mice was significantly increased by 71% and 82%, respectively, relative to the wild-type mice. Furthermore, the carcinoma incidence and the number of carcinomas per mouse are also higher in Jnk1(-/-) mice. Strikingly, Jnk1-/- mouse skin is more sensitive to TPA-induced AP-1 DNA binding activity and phosphorylation of extracellular signal-regulated kinases and Akt, which are two important survival signaling components. These results suggest that JNK1 is a crucial suppressor of skin tumor development (She, 2002).
NF-kappaB inhibition promotes epidermal tumorigenesis; however, whether this reflects an underlying role in homeostasis or a special case confined to neoplasia is unknown. Embryonic lethality of mice lacking NF-kappaB RelA has hindered efforts to address this. Therefore developmentally mature RelA-/- skin was generated. RelA-/- epidermis displays hyperplasia without abnormal differentiation, inflammation, or apoptosis. Hyperproliferation is TNFR1-dependent because Tnfr1 deletion normalizes cell division. TNFR1-dependent JNK activation occurs in RelA-/- epidermis, and JNK inhibition abolishes hyperproliferation due to RelA deficiency. Thus, RelA antagonizes TNFR1-JNK proliferative signals in epidermis and plays a nonredundant role in restraining epidermal growth (Zhang, 2004).
The p53 tumor suppressor protein is a potent transcription factor that is activated in response to various DNA-damaging agents, leading to cell cycle arrest and/or apoptosis. Disruption of this pathway occurs in a wide range of human cancers and is highly correlated with the tumorigenic phenotype. The key to the magnitude and duration of p53 activities lies in its stability. In normally growing cells, p53 half-life is limited to minutes, whereas cellular stress or exposure to DNA-damaging agents prolongs it to hours. Proteins known to alter p53 stability include HPV16-E6, WT-1, E1B/E4orf6, SV40 T-antigen, and Mdm2. Whereas the association of SV40 T antigen, WT1, or E1B/E4orf6 with p53 increases its stability, the binding of E6 or Mdm2 with p53 accelerates its degradation. To date, Mdm2 is the only cellular protein whose direct association with p53 results in its ubiquitination and subsequent degradation. The regulation of p53 stability has been associated with post-translational modifications, including phosphorylation on amino-terminal residues. Jun-N (amino)-terminal kinase (JNK) is known to target the ubiquitination and stability of its associated proteins, c-Jun, JunB, and ATF2. JNK targeting for ubiquitination occurs in a phosphorylation-dependent manner because phosphorylated forms of c-Jun and ATF2 are found to be protected against JNK-targeted ubiquitination. Essential for JNK's ability to target the ubiquitination of ATF2, c-Jun, and JunB is JNK's association with each of these proteins. Recent evidence for the association of JNK with p53 has provided the foundation for the hypothesis that JNK also has a role in the regulation of p53 stability (Fuchs, 1998).
Nonactive JNK plays an active role in regulating p53 stability. The amount of p53-JNK complex is inversely correlated with p53 level. A peptide corresponding to the JNK binding site on p53 efficiently blocks ubiquitination of p53. Similarly, p53 lacking the JNK binding site exhibits a longer half-life than wild-type p53. Outcompeting JNK association with p53 increases the level of p53, whereas overexpression of a phosphorylation mutant form of JNK inhibits p53 accumulation. JNK-p53 and Mdm2-p53 complexes are preferentially found in G0/G1 and S/G2M phases of the cell cycle, respectively. Altogether, these data indicate that JNK is an Mdm2-independent regulator of p53 stability in nonstressed cells (Fuchs, 1998).
In both Mdm2/p53 null cells and BALB/3T3/12.1 cells, JNK appears to be the principal regulator of p53 ubiquitination. These data also suggest that Mdm2 and JNK represent two independent pathways for targeting p53 stability. The fact that Mdm2-p53 complexes are found at S and G2/M phases of the cell cycle, but JNK-p53 complexes are found in G0/G1 phases, suggests that through their targeting of p53 stability in nonstressed cells, Mdm2 and JNK may regulate different cellular functions of p53 during normal cell growth. These data do not preclude the existence of other targeting molecules, as immunodepletion of JNK from Mdm2 null cell lysates does not completely abolish p53 targeting for in vitro ubiquitination. In line with previous studies, stress-mediated JNK activation inversely correlates with targeting of its associated proteins, as shown here for p53. JNK activation via MEKK1 results in p53 phosphorylation, inhibition of Mdm2 association, and p53 ubiquitination as reflected by a prolonged p53 half-life. Yet because JNK from UV-treated cells can still associate with recombinant p53 in vitro, it is possible that in vivo p53 phosphorylation in response to stress requires multiple stress kinases, which mediate sufficient changes to p53 conformation to result in p53 dissociation from its targeting molecules. The emerging model suggests that p53 stability is affected by JNK independently of Mdm2 in a cell cycle-dependent manner. In this model, prolonged half-life, which is characteristic of mutant forms of p53, could be attributed to lack of p53 association with one or both targeting molecules. JNK is likely to be among the growing number of adapter molecules that participate in the formation of the E3 ubiquitin/ligase complex. As a targeting molecule that mediates the stability of the oncogene, c-Jun, and the tumor suppressor p53, JNK emerges as a key regulator of cell growth in normally growing cells (Fuchs, 1998).
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