Jesús M. Salvador

Alternative p38 activation pathway mediated by T cell receptor–proximal tyrosine kinases

Signaling-responsive MAP kinases (MAPKs) are key in mediating immune responses and are activated through the phosphorylation of a Thr-X-Tyr motif by upstream MAPK kinases. Here we show that T cells stimulated through the T cell receptor (TCR) used an alternative mechanism in which p38 was phosphorylated on Tyr323 and subsequently autophosphorylated residues Thr180 and Tyr182. This required the TCR-proximal tyrosine kinase Zap70 but not the adaptor protein LAT, which was required for activation of extracellular signal–regulated protein kinase MAPKs. TCR activation of p38 lacking Tyr323 was diminished, and blocking of p38 activity prevented p38 dual phosphorylation in normal T cells but not in B cells. Thus, phosphorylation of Tyr323 dependent on the tyrosine kinase Lck and mediated by Zap70 serves as an important mechanism for TCR activation of p38 in T cells.

Mice Lacking the p53-Effector Gene Gadd45a Develop a Lupus-Like Syndrome

This study addresses the biological function of the p53-effector genes Gadd45a and p21 in the immune system. We find that Gadd45a is a negative regulator of T cell proliferation because, compared to wild-type cells, Gadd45a(-/-) T cells have a lower threshold of activation and proliferate to a greater extent following primary T cell receptor stimulation. Gadd45a(-/-) mice develop an autoimmune disease, similar to human systemic lupus erythematosus (SLE), characterized by high titers of anti-dsDNA, anti-ssDNA, and anti-histone autoantibodies, severe hematological disorders, autoimmune glomerulonephritis, and premature death. Here we show that the lack of both Gadd45a and p21 dramatically accelerates the development of autoimmunity observed in each individual single-gene disruption mutant, demonstrating that these genes play nonredundant roles in the immune response.

Gadd45 in Stress Signaling, Cell Cycle Control, and Apoptosis

The first identified Gadd45 gene, Gadd45a, encodes a ubiquitously expressed protein that is often induced by DNA damage and other stress signals associated with growth arrest and apoptosis. This protein and the other two members of this small gene family, Gadd45b and Gadd45g, have been implicated in a variety of the responses to cell injury including cell cycle checkpoints, apoptosis, and DNA repair. In vivo, many of the prominent roles for the Gadd45 proteins are associated with signaling mediated by p38 mitogen-activated protein kinases (MAPK). Gadd45 proteins can contribute to p38 activation either by activation of upstream kinase(s) or by direct interaction. In vivo, there are important tissue and cell-type-specific differences in the roles for Gadd45 in MAPK signaling. In addition to being p53-regulated, Gadd45a has been found to contribute to p53 activation via p38. Like other stress and signaling proteins, Gadd45 proteins show complex regulation and numerous effectors.

Activating p38 MAPK : New tricks for an old kinase

Mitogen-activated protein kinases (MAPKs) participate in signaling initiated by a wide variety of extracellular stimuli. MAPKs are most commonly activated by a series of phosphorylation events in which one kinase phosphorylates another, the “MAPK cascade”. The cascade concludes with the dual phosphorylation of MAPKs on a conserved Thr-X-Tyr motif. In the case of the p38 MAPK, an exception to this paradigm has been found when signaling via the T cell antigen receptor (TCR). Rather than trigger the MAPK cascade, TCR-mediated stimulation activates proximal tyrosine kinases, which results in the phosphorylation of p38 on a noncanonical activating residue, Tyr-323. This phosphorylation activates p38 to phosphorylate third party substrates as well as its own Thr-X-Tyr motif. Here we discuss the structural and functional implications of this alternative p38 activation pathway, which may provide a new target for tissue-specific pharmacologic inhibition.

The autoimmune suppressor Gadd45α inhibits the T cell alternative p38 activation pathway

The p38 MAP kinase (MAPK) is phosphorylated and activated by upstream MAPK kinases. T cells have an alternative pathway in which T cell receptor–activated tyrosine kinase Zap70 phosphorylates p38 on Tyr323. Mice lacking Gadd45α, a small p38-binding molecule, develop a lupus-like autoimmune disease. Here we show that resting T cells but not B cells from Gadd45a−/− mice had spontaneously increased p38 activity in the absence of upstream MAPK kinase activation. The p38 from resting Gadd45a−/− T cells was spontaneously phosphorylated on Tyr323, and its activity was specifically inhibited by recombinant Gadd45α in vitro. Thus, constitutive activation of T cell p38 through the alternative pathway is prevented by Gadd45α, the absence of which results in p38 activation, T cell hyperproliferation and autoimmunity.

The microRNA miR-148a functions as a critical regulator of B cell tolerance and autoimmunity

Autoreactive B cells have critical roles in a large diversity of autoimmune diseases, but the molecular pathways that control these cells remain poorly understood. We performed an in vivo functional screen of a lymphocyte-expressed microRNA library and identified miR-148a as a potent regulator of B cell tolerance. Elevated miR-148a expression impaired B cell tolerance by promoting the survival of immature B cells after engagement of the B cell antigen receptor by suppressing the expression of the autoimmune suppressor Gadd45α, the tumor suppressor PTEN and the pro-apoptotic protein Bim. Furthermore, increased expression of miR-148a, which occurs frequently in patients with lupus and lupus-prone mice, facilitated the development of lethal autoimmune disease in a mouse model of lupus. Our studies demonstrate a function for miR-148a as a regulator of B cell tolerance and autoimmunity.

Gadd45g Is Essential for Primary Sex Determination, Male Fertility and Testis Development

In humans and most mammals, differentiation of the embryonic gonad into ovaries or testes is controlled by the Y-linked gene SRY. Here we show a role for the Gadd45g protein in this primary sex differentiation. We characterized mice deficient in Gadd45a, Gadd45b and Gadd45g, as well as double-knockout mice for Gadd45ab, Gadd45ag and Gadd45bg, and found a specific role for Gadd45g in male fertility and testis development. Gadd45g-deficient XY mice on a mixed 129/C57BL/6 background showed varying degrees of disorders of sexual development (DSD), ranging from male infertility to an intersex phenotype or complete gonadal dysgenesis (CGD). On a pure C57BL/6 (B6) background, all Gadd45g−/− XY mice were born as completely sex-reversed XY-females, whereas lack of Gadd45a and/or Gadd45b did not affect primary sex determination or testis development. Gadd45g expression was similar in female and male embryonic gonads, and peaked around the time of sex differentiation at 11.5 days post-coitum (dpc). The molecular cause of the sex reversal was the failure of Gadd45g−/− XY gonads to achieve the SRY expression threshold necessary for testes differentiation, resulting in ovary and Müllerian duct development. These results identify Gadd45g as a candidate gene for male infertility and 46,XY sex reversal in humans.

Tyr323‐dependent p38 activation is associated with rheumatoid arthritis and correlates with disease activity

OBJECTIVEnThe p38 MAPK is important in the pathogenic immune response in rheumatoid arthritis (RA). The p38 molecule can be activated through phosphorylation on Thr¹⁸⁰-Tyr¹⁸² by upstream MAPK kinases and via an alternative pathway through phosphorylation on Tyr³²³. We undertook this study to quantify the phosphorylation of Tyr³²³ p38 and of Thr¹⁸⁰-Tyr¹⁸² p38 on T cells from healthy controls and patients with RA or ankylosing spondylitis (AS) to identify variables associated with p38 phosphorylation and disease activity.nnnMETHODSnWe measured p38 phosphorylation on Tyr³²³ and Thr¹⁸⁰-Tyr¹⁸² by flow cytometry and Western blotting on T cells from 30 control subjects, 33 AS patients, 30 patients with RA in remission, and 79 patients with active RA. We collected the clinical characteristics and analyzed correlations between clinical variables, the Disease Activity Score in 28 joints (DAS28), and p38 phosphorylation levels. Multivariate regression analysis was performed to identify variables associated with p38 phosphorylation on Tyr³²³ and Thr¹⁸⁰-Tyr¹⁸².nnnRESULTSnPhosphorylation of p38 on Tyr³²³ was higher in T cells from patients with active RA (P = 0.008 versus healthy controls) than in patients with RA in remission or in patients with AS. Tyr³²³ p38 phosphorylation was associated with disease activity determined by the DAS28 (P = 0.017). Enhanced p38 phosphorylation was linked to Lck-mediated activation of the Tyr³²³-dependent pathway in the absence of upstream MAPKK activation.nnnCONCLUSIONnOur results indicate that phosphorylation status on Tyr³²³ p38 correlates with RA disease activity and suggest that the Tyr³²³-dependent pathway is an attractive target for down-regulation of p38 activity in RA patients.

Gadd45a acts as a modifier locus for lymphoblastic lymphoma.

Gadd45a−/− and p53−/− mice and cells derived from them share similar phenotypes, most notably genomic instability. However, p53−/− mice rapidly develop a variety of neoplasms, while Gadd45a−/− mice do not. The two proteins are involved in a regulatory feedback loop, whereby each can increase the expression or activity of the other, suggesting that common phenotypes might result from similar molecular mechanisms. Mice lacking both genes were generated to address this issue. Gadd45a−/−p53−/− mice developed tumors with a latency similar to that of tumor-prone p53−/− mice. However, while p53−/− mice developed a variety of tumor types, nearly all Gadd45a−/−p53−/− mice developed lymphoblastic lymphoma (LBL), often accompanied by mediastinal masses as is common in human patients with this tumor type. Deletion of Gadd45a in leukemia/lymphoma-prone AKR mice decreased the latency for LBL. These results indicate that Gadd45a may act as modifier locus for T-cell LBL, whereby deletion of Gadd45a enhances development of this tumor type in susceptible mice. Gadd45a is localized to 1p31.1, and 1p abnormalities have been described in T-cell lymphomas. Related human tumor samples did not show Gadd45a deletion or mutation, although changes in expression could not be ruled out.

p38α regulates cytokine-induced IFNγ secretion via the Mnk1/eIF4E pathway in Th1 cells

The p38 mitogen‐activated protein kinase (MAPK) pathway is involved in the regulation of immune and inflammatory processes. We used p38α‐conditional, p38β‐deficient and p38α/β double‐null mouse models to address the role of these two p38 MAPK in CD4+ T cells, and found that p38α deficiency causes these cells to hyperproliferate. Our studies indicate that both p38α and p38β are dispensable for T helper cell type 1 (Th1) differentiation but, by controlling interferon (IFN)γ and tumor necrosis factor (TNF)α production, are critical for normal Th1 effector function. We found that both p38α and p38β modulate T‐cell receptor‐induced IFNγ and TNFα production, whereas only p38α regulates cytokine‐induced IFNγ production. The lack of p38α and p38β did not affect transcription and mRNA stability of Ifng. However, the absence of p38α in Th1 cells resulted in a decreased MNK1 phosphorylation after cytokine activation, and MNK1 inhibition blocked IFNγ production. Our results indicate that p38α regulates IFNγ secretion through the activation of the MNK1/eIF4E pathway of translation initiation and identify specific functions for p38α and p38β in T‐cell proliferation.