RESUMO
Voltage-gated proton currents regulate generation of reactive oxygen species (ROS) in phagocytic cells. In B cells, stimulation of the B cell antigen receptor (BCR) results in the production of ROS that participate in B cell activation, but the involvement of proton channels is unknown. We report here that the voltage-gated proton channel HVCN1 associated with the BCR complex and was internalized together with the BCR after activation. BCR-induced generation of ROS was lower in HVCN1-deficient B cells, which resulted in attenuated BCR signaling via impaired BCR-dependent oxidation of the tyrosine phosphatase SHP-1. This resulted in less activation of the kinases Syk and Akt, impaired mitochondrial respiration and glycolysis and diminished antibody responses in vivo. Our findings identify unanticipated functions for proton channels in B cells and demonstrate the importance of ROS in BCR signaling and downstream metabolism.
Assuntos
Linfócitos B/imunologia , Canais Iônicos/imunologia , Espécies Reativas de Oxigênio/imunologia , Receptores de Antígenos de Linfócitos B/imunologia , Animais , Linfócitos B/enzimologia , Ativação Enzimática/imunologia , Immunoblotting , Peptídeos e Proteínas de Sinalização Intracelular/imunologia , Camundongos , Camundongos Knockout , Microscopia Confocal , Mitocôndrias/imunologia , Proteína Oncogênica v-akt/imunologia , Proteínas Tirosina Quinases/imunologia , Transdução de Sinais , Quinase SykRESUMO
HVCN1 (Hydrogen voltage-gated channel 1) is the only mammalian voltage-gated proton channel. In human B lymphocytes, HVCN1 associates with the B-cell receptor (BCR) and is required for optimal BCR signaling and redox control. HVCN1 is expressed in malignant B cells that rely on BCR signaling, such as chronic lymphocytic leukemia (CLL) cells. However, little is known about its regulation in these cells. We found that HVCN1 was expressed in B cells as two protein isoforms. The shorter isoform (HVCN1S) was enriched in B cells from a cohort of 76 CLL patients. When overexpressed in a B-cell lymphoma line, HVCN1S responded more profoundly to protein kinase C-dependent phosphorylation. This more potent enhanced gating response was mediated by increased phosphorylation of the same residue responsible for enhanced gating in HVCN1L, Thr(29). Furthermore, the association of HVCN1S with the BCR was weaker, which resulted in its diminished internalization upon BCR stimulation. Finally, HVCN1S conferred a proliferative and migratory advantage as well as enhanced BCR-dependent signaling. Overall, our data show for the first time, to our knowledge, the existence of a shorter isoform of HVCN1 with enhanced gating that is specifically enriched in malignant B cells. The properties of HVCN1S suggest that it may contribute to the pathogenesis of BCR-dependent B-cell malignancies.
Assuntos
Linfócitos B/metabolismo , Neoplasias Hematológicas/imunologia , Canais Iônicos/metabolismo , Leucemia Linfocítica Crônica de Células B/imunologia , Animais , Linhagem Celular Tumoral , Células HEK293 , Humanos , Camundongos , Técnicas de Patch-Clamp , Fosforilação , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Proteína Quinase C/metabolismo , Espécies Reativas de Oxigênio/metabolismoRESUMO
Voltage-gated proton channels and NADPH oxidase function cooperatively in phagocytes during the respiratory burst, when reactive oxygen species are produced to kill microbial invaders. Agents that activate NADPH oxidase also enhance proton channel gating profoundly, facilitating its roles in charge compensation and pH(i) regulation. The "enhanced gating mode" appears to reflect protein kinase C (PKC) phosphorylation. Here we examine two candidates for PKC-delta phosphorylation sites in the human voltage-gated proton channel, H(V)1 (Hvcn1), Thr(29) and Ser(97), both in the intracellular N terminus. Channel phosphorylation was reduced in single mutants S97A or T29A, and further in the double mutant T29A/S97A, by an in vitro kinase assay with PKC-delta. Enhanced gating was evaluated by expressing wild-type (WT) or mutant H(V)1 channels in LK35.2 cells, a B cell hybridoma. Stimulation by phorbol myristate acetate enhanced WT channel gating, and this effect was reversed by treatment with the PKC inhibitor GF109203X. The single mutant T29A or double mutant T29A/S97A failed to respond to phorbol myristate acetate or GF109203X. In contrast, the S97A mutant responded like cells transfected with WT H(V)1. We conclude that under these conditions, direct phosphorylation of the proton channel molecule at Thr(29) is primarily responsible for the enhancement of proton channel gating. This phosphorylation is crucial to activation of the proton conductance during the respiratory burst in phagocytes.
Assuntos
Ativação do Canal Iônico/fisiologia , Canais Iônicos/metabolismo , Leucócitos/metabolismo , Explosão Respiratória/fisiologia , Treonina/metabolismo , Substituição de Aminoácidos , Carcinógenos/farmacologia , Linhagem Celular , Inibidores Enzimáticos/farmacologia , Humanos , Indóis/farmacologia , Ativação do Canal Iônico/efeitos dos fármacos , Canais Iônicos/genética , Maleimidas/farmacologia , Mutação de Sentido Incorreto , NADPH Oxidases/metabolismo , Fosforilação/efeitos dos fármacos , Fosforilação/fisiologia , Proteína Quinase C-delta/antagonistas & inibidores , Proteína Quinase C-delta/metabolismo , Explosão Respiratória/efeitos dos fármacos , Acetato de Tetradecanoilforbol/farmacologia , Treonina/genéticaRESUMO
Two forms of the growth hormone-releasing hormone (GHRH) receptor (GHRH-R) exist in terms of a polymorphism at codon 57. The most common allele possesses GCG, coding for Ala. This codon can also be ACG, replacing the Ala with Thr. The present study demonstrates that the latter occurs in about 20% of pituitary somatotrophinomas, removed from patients with acromegaly. Somatotrophinomas possessing the alternative allele respond, on average, more strongly to GHRH in terms of GH secretion in vitro than tumors which are homozygous for the more common allele. The distribution of the two allelic forms of the GHRH-R did not significantly differ between acromegalic and non-acromegalic subjects. Thus, while the alternative allelic forms may, at least partially, contribute to the variable response of serum GH levels to i.v. GHRH observed in acromegalic and normal subjects, it is unlikely that subjects possessing the rarer form containing Thr in place of Ala at residue 57 are at increased risk of developing acromegaly.