RESUMEN
Synaptic transmission is mediated by a complex set of molecular events that must be coordinated in time and space. While many proteins that function at the synapse have been identified, the signaling pathways regulating these molecules are poorly understood. Pak5 (p21-activated kinase 5) is a brain-specific isoform of the group II Pak kinases whose substrates and roles within the central nervous system are largely unknown. To gain insight into the physiological roles of Pak5, we engineered a Pak5 mutant to selectively radiolabel its substrates in murine brain extract. Using this approach, we identified two novel Pak5 substrates, Pacsin1 and Synaptojanin1, proteins that directly interact with one another to regulate synaptic vesicle endocytosis and recycling. Pacsin1 and Synaptojanin1 were phosphorylated by Pak5 and the other group II Paks in vitro, and Pak5 phosphorylation promoted Pacsin1-Synaptojanin1 binding both in vitro and in vivo. These results implicate Pak5 in Pacsin1- and Synaptojanin1-mediated synaptic vesicle trafficking and may partially account for the cognitive and behavioral deficits observed in group II Pak-deficient mice.
Asunto(s)
Proteínas del Tejido Nervioso/metabolismo , Neuronas/enzimología , Neuropéptidos/metabolismo , Fosfoproteínas/metabolismo , Monoéster Fosfórico Hidrolasas/metabolismo , Vesículas Sinápticas/enzimología , Quinasas p21 Activadas/metabolismo , Proteínas Adaptadoras Transductoras de Señales , Animales , Transporte Biológico , Encéfalo/enzimología , Péptidos y Proteínas de Señalización Intracelular , Ratones , Modelos Biológicos , Fosforilación , Unión Proteica , Especificidad por SustratoRESUMEN
The Tec kinases ITK (interleukin-2-inducible T-cell kinase) and RLK (resting lymphocyte kinase) are critical components of the proximal TCR/CD3 signal transduction machinery, and data in mice suggest that ITK negatively modulates regulatory T cell (TREG) differentiation. However, whether Tec kinases modulate TREG development and/or function in human T cells remains unknown. Using a novel self-delivery siRNA platform (sdRNA), we found that ITK knockdown in human primary naïve peripheral blood CD4 T cells increased Foxp3+ expression under both TREG and T helper priming conditions. TREG differentiated under ITK knockdown conditions exhibited enhanced expression of the co-inhibitory receptor PD-1 and were suppressive in a T cell proliferation assay. ITK knockdown decreased IL-17A production in T cells primed under Th17 conditions and promoted Th1 differentiation. Lastly, a dual ITK/RLK Tec kinase inhibitor did not induce Foxp3 in CD4 T cells, but conversely abrogated Foxp3 expression induced by ITK knockdown. Our data suggest that targeting ITK in human T cells may be an effective approach to boost TREG in the context of autoimmune diseases, but concomitant inhibition of other Tec family kinases may negate this effect.
Asunto(s)
Diferenciación Celular , Factores de Transcripción Forkhead/metabolismo , Proteínas Tirosina Quinasas/metabolismo , Linfocitos T Reguladores/citología , Linfocitos T Reguladores/enzimología , Polaridad Celular , Humanos , Activación de Linfocitos/inmunología , Receptor de Muerte Celular Programada 1/metabolismo , Inhibidores de Proteínas Quinasas/farmacología , Células TH1/citología , Células Th17/citología , Regulación hacia Arriba/efectos de los fármacosRESUMEN
The earliest events leading to autoimmune type 1 diabetes (T1D) are not known in any species. A T-cell receptor (TCR)-variable region, TCR-Vß13, is required for susceptibility to autoimmune diabetes in rats, and selective depletion of Vß13(+) T cells with an allele-specific monoclonal antibody prevents disease in multiple rat strains. To investigate the role of Vß13 early in diabetes, we examined islet T-cell transcripts in susceptible (LEW.1WR1) and resistant (LEW.1W and Wistar Furth) strains induced with polyinosinic:polycytidylic acid. Vß13(+) T cells displayed antigenic focusing in LEW.1WR1 islets 5 days postinduction and were characterized by a substantial decrease in complementarity determining region 3 diversity. This occurred prior to significant islet T-cell accumulation (day 7) or frank diabetes (days 10-14). Vß13(+) transcripts increased in LEW.1WR1 islets during diabetes progression, but not in resistant rats. We also analyzed transcript clonality of rat TCR-Vα5, an ortholog of the dominant TCR-Vα chain found on insulin B:9-23-reactive T cells in nonobese diabetic rat islets. We observed clonal expansion of Vα5(+) transcripts in prediabetic LEW.1WR1 islets, suggesting that rat Vα5 is also an important component of islet autoantigen recognition. These data provide additional evidence that genome-encoded TCR sequences are important determinants of genetic susceptibility to T1D.