RESUMEN

Tumor progression locus 2 (TPL-2) kinase is essential for Toll-like receptor 4 activation of the mitogen-activated protein kinase extracellular signal-regulated kinase (ERK) and for upregulation of the inflammatory cytokine tumor necrosis factor (TNF) in lipopolysaccharide (LPS)-stimulated macrophages. LPS activation of ERK requires TPL-2 release from associated NF-kappaB1 p105, which blocks TPL-2 access to its substrate, the ERK kinase MEK. Here we demonstrate that TPL-2 activity is also regulated independently of p105, since LPS stimulation was still needed for TPL-2-dependent activation of ERK in Nfkb1(-/-) macrophages. In wild-type macrophages, LPS induced the rapid phosphorylation of serine (S) 400 in the TPL-2 C-terminal tail. Mutation of this conserved residue to alanine (A) blocked the ability of retrovirally expressed TPL-2 to induce the activation of ERK in LPS-stimulated Nfkb1(-/-) macrophages. TPL-2(S400A) expression also failed to reconstitute LPS activation of ERK and induction of TNF in Map3k8(-/-) macrophages, which lack endogenous TPL-2. Consistently, the S400A mutation was found to block LPS stimulation of TPL-2 MEK kinase activity. Thus, induction of TPL-2 MEK kinase activity by LPS stimulation of macrophages requires TPL-2 phosphorylation on S400, in addition to its release from NF-kappaB1 p105. Oncogenic C-terminal truncations of TPL-2 that remove S400 could promote its transforming potential by eliminating this critical control step.

Asunto(s)

Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Lipopolisacáridos/farmacología , Quinasas Quinasa Quinasa PAM/metabolismo , Macrófagos/efectos de los fármacos , Macrófagos/enzimología , Proteínas Proto-Oncogénicas/metabolismo , Serina/metabolismo , Secuencia de Aminoácidos , Animales , Catálisis/efectos de los fármacos , Línea Celular , Proteína 1 de la Respuesta de Crecimiento Precoz/genética , Activación Enzimática/efectos de los fármacos , Humanos , Quinasa I-kappa B/metabolismo , Quinasas Quinasa Quinasa PAM/química , Activación de Macrófagos/efectos de los fármacos , Ratones , Datos de Secuencia Molecular , Subunidad p50 de NF-kappa B/metabolismo , Fosforilación/efectos de los fármacos , Unión Proteica/efectos de los fármacos , Proteínas Proto-Oncogénicas/química , Proteínas Proto-Oncogénicas c-akt/metabolismo , Factores de Necrosis Tumoral/genética , Regulación hacia Arriba/efectos de los fármacos

RESUMEN

The MEK kinase TPL-2 (also known as Cot) is required for lipopolysaccharide (LPS) activation of the extracellular signal-regulated kinase (ERK) mitogen-activated protein (MAP) kinase cascade in macrophages and consequent upregulation of genes involved in innate immune responses. In resting cells, TPL-2 forms a stoichiometric complex with NF-kappaB1 p105, which negatively regulates its MEK kinase activity. Here, it is shown that lipopolysaccharide (LPS) stimulation of primary macrophages causes the release of both long and short forms of TPL-2 from p105 and that TPL-2 MEK kinase activity is restricted to this p105-free pool. Activation of TPL-2, MEK, and ERK by LPS is also demonstrated to require proteasome-mediated proteolysis. p105 is known to be proteolysed by the proteasome following stimulus-induced phosphorylation of two serines in its PEST region by the IkappaB kinase (IKK) complex. Expression of a p105 point mutant, which is not susceptible to signal-induced proteolysis, in RAW264.7 macrophages impairs LPS-induced release of TPL-2 from p105 and its subsequent activation of MEK. Furthermore, expression of wild-type but not mutant p105 reconstitutes LPS stimulation of MEK and ERK phosphorylation in primary NF-kappaB1-deficient macrophages. Consistently, pharmacological blockade of IKK inhibits LPS-induced release of TPL-2 from p105 and TPL-2 activation. These data show that IKK-induced p105 proteolysis is essential for LPS activation of TPL-2, thus revealing a novel function of IKK in the regulation of the ERK MAP kinase cascade.

Asunto(s)

Lipopolisacáridos/farmacología , Quinasas Quinasa Quinasa PAM/metabolismo , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , FN-kappa B/metabolismo , Precursores de Proteínas/metabolismo , Procesamiento Proteico-Postraduccional , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Animales , Células Cultivadas , Activación Enzimática/efectos de los fármacos , Inhibidores Enzimáticos/farmacología , Quinasa I-kappa B , Quinasas Quinasa Quinasa PAM/genética , Macrófagos/efectos de los fármacos , Macrófagos/metabolismo , Ratones , Ratones Noqueados , Mutación/genética , FN-kappa B/genética , Subunidad p50 de NF-kappa B , Fosforilación/efectos de los fármacos , Fosfoserina/metabolismo , Complejo de la Endopetidasa Proteasomal/metabolismo , Precursores de Proteínas/genética , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Proteínas Proto-Oncogénicas/genética

RESUMEN

NF-kappa B1 p105 forms a high-affinity, stoichiometric interaction with TPL-2, a MEK kinase essential for TLR4 activation of the ERK mitogen-activated protein kinase cascade in lipopolysaccharide (LPS)-stimulated macrophages. Interaction with p105 is required to maintain TPL-2 metabolic stability and also negatively regulates TPL-2 MEK kinase activity. Here, affinity purification identified A20-binding inhibitor of NF-kappa B 2 (ABIN-2) as a novel p105-associated protein. Cotransfection experiments demonstrated that ABIN-2 could interact with TPL-2 in addition to p105 but preferentially formed a ternary complex with both proteins. Consistently, in unstimulated bone marrow-derived macrophages (BMDMs), a substantial fraction of endogenous ABIN-2 was associated with both p105 and TPL-2. Although the majority of TPL-2 in these cells was complexed with ABIN-2, the pool of TPL-2 which could activate MEK after LPS stimulation was not, and LPS activation of TPL-2 was found to correlate with its release from ABIN-2. Depletion of ABIN-2 by RNA interference dramatically reduced steady-state levels of TPL-2 protein without affecting levels of TPL-2 mRNA or p105 protein. In addition, ABIN-2 increased the half-life of cotransfected TPL-2. Thus, optimal TPL-2 stability in vivo requires interaction with ABIN-2 as well as p105. Together, these data raise the possibility that ABIN-2 functions in the TLR4 signaling pathway which regulates TPL-2 activation.

Asunto(s)

Proteínas Adaptadoras Transductoras de Señales , Proteínas Portadoras/metabolismo , Quinasas Quinasa Quinasa PAM/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Factores de Transcripción/metabolismo , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Proteínas Portadoras/química , Proteínas Portadoras/genética , Línea Celular , ADN Complementario/genética , Activación Enzimática , Células HeLa , Humanos , Técnicas In Vitro , Quinasas Quinasa Quinasa PAM/química , Quinasas Quinasa Quinasa PAM/genética , Sustancias Macromoleculares , Macrófagos/metabolismo , Glicoproteínas de Membrana/metabolismo , Ratones , Ratones Endogámicos BALB C , Ratones Noqueados , Datos de Secuencia Molecular , Subunidad p50 de NF-kappa B , Proteínas Proto-Oncogénicas/química , Proteínas Proto-Oncogénicas/genética , ARN Interferente Pequeño/genética , Receptores de Superficie Celular/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Transducción de Señal , Solubilidad , Receptor Toll-Like 4 , Receptores Toll-Like , Factores de Transcripción/química , Factores de Transcripción/deficiencia , Factores de Transcripción/genética , Transfección

RESUMEN

Activation of the oncogenic potential of the MEK kinase TPL-2 (Cot) requires deletion of its C terminus. This mutation also weakens the interaction of TPL-2 with NF-kappaB1 p105 in vitro, although it is unclear whether this is important for the activation of TPL-2 oncogenicity. It is demonstrated here that TPL-2 stability in vivo relies on its high-affinity, stoichiometric association with NF-kappaB1 p105. Formation of this complex occurs as a result of two distinct interactions. The TPL-2 C terminus binds to a region encompassing residues 497 to 534 of p105, whereas the TPL-2 kinase domain interacts with the p105 death domain. Binding to the p105 death domain inhibits TPL-2 MEK kinase activity in vitro, and this inhibition is significantly augmented by concomitant interaction of the TPL-2 C terminus with p105. In cotransfected cells, both interactions are required for inhibition of TPL-2 MEK kinase activity and, consequently, the catalytic activity of a C-terminally truncated oncogenic mutant of TPL-2 is not affected by p105. Thus, in addition to its role as a precursor for p50 and cytoplasmic inhibitor of NF-kappaB, p105 is a negative regulator of TPL-2. Insensitivity of C-terminally truncated TPL-2 to this regulatory mechanism is likely to contribute to its ability to transform cells.

Asunto(s)

Quinasas Quinasa Quinasa PAM/metabolismo , FN-kappa B/metabolismo , Precursores de Proteínas/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Células 3T3 , Animales , Sitios de Unión , Estabilidad de Enzimas , MAP Quinasa Quinasa 1 , Quinasas Quinasa Quinasa PAM/genética , Ratones , Quinasas de Proteína Quinasa Activadas por Mitógenos/genética , Quinasas de Proteína Quinasa Activadas por Mitógenos/metabolismo , FN-kappa B/genética , Subunidad p50 de NF-kappa B , Fragmentos de Péptidos/metabolismo , Unión Proteica , Precursores de Proteínas/genética , Proteínas Serina-Treonina Quinasas/genética , Proteínas Serina-Treonina Quinasas/metabolismo , Estructura Terciaria de Proteína , Proteínas Proto-Oncogénicas/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo

RESUMEN

To identify novel genes induced in the early stage of T-cell activation, mRNA expression in alloactivated human lymphocytes was examined. Differential display-reverse transcription PCR analysis revealed a 207-bp cDNA fragment which was upregulated 24 h after allostimulation of a human T-cell line. The corresponding complete 1396 bp cDNA, named TGAM77, encodes a predicted 134 amino acid protein which shares 63% homology with the cornichon (cni) protein of Drosophila melanogaster. Upregulation of TGAM77 mRNA in the early phase of T-cell activation was confirmed by Northern blot and RT-PCR analysis of activated human lymphocytes. TGAM77 mRNA is expressed in a variety of human tissues with various expression levels. In analogy to cni which is involved in an epidermal growth factor-like signaling pathway inducing cellular asymmetry in Drosophila oogenesis, TGAM77 might function in similar signaling establishing vectorial re-localization and concentration of signaling events in T-cell activation.

Asunto(s)

ADN Complementario/química , Proteínas de Drosophila , Proteínas del Huevo/genética , Proteínas de la Membrana , Linfocitos T/metabolismo , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Drosophila melanogaster/genética , Proteínas del Huevo/química , Humanos , Activación de Linfocitos/genética , Ratones , Datos de Secuencia Molecular , ARN Mensajero/análisis , Homología de Secuencia de Ácido Nucleico , Transducción de Señal/genética

RESUMEN

A novel 75 kDa membrane protein, TIRC7, is described that exhibits a central role in T cell activation in vitro and in vivo. Modulation of TIRC7-mediated signals with specific anti-TIRC7 antibodies in vitro efficiently prevents human T cell proliferation and IL-2 secretion. Moreover, anti-TIRC7 antibodies specifically inhibit type 1 subset specific IFN-gamma expression but spare the type 2 cytokine IL-4. Diminished proliferation but not IFN-gamma secretion is reversible by exogenous rIL-2. An anti-TIRC7 antibody that cross-reacts with the 75 kDa rat homolog exhibits inhibition of rat alloimmune response in vitro and significantly prolongs kidney allograft survival in vivo. Targeting of TIRC7 may provide a novel therapeutic approach for modulation of the immune response.

Asunto(s)

Rechazo de Injerto/prevención & control , Proteínas de la Membrana/inmunología , Subunidades de Proteína , Linfocitos T/inmunología , ATPasas de Translocación de Protón Vacuolares , Enfermedad Aguda , Secuencia de Aminoácidos , Animales , Anticuerpos , Secuencia de Bases , Cartilla de ADN/genética , ADN Complementario/genética , Rechazo de Injerto/inmunología , Rechazo de Injerto/patología , Humanos , Técnicas In Vitro , Interleucina-2/biosíntesis , Trasplante de Riñón/inmunología , Trasplante de Riñón/patología , Activación de Linfocitos , Masculino , Proteínas de la Membrana/química , Proteínas de la Membrana/genética , Datos de Secuencia Molecular , Peso Molecular , ARN Mensajero/genética , ARN Mensajero/metabolismo , Ratas , Ratas Endogámicas Lew , Ratas Endogámicas WF , Transducción de Señal , Linfocitos T/metabolismo , Trasplante Homólogo