The activation kinetics of MAPK Erk are critical for T cell development and activation. In particular, sustained Erk signaling is required for T cell activation and effector functions, such as IL-2 production. Although Raf-1 triggers transient Erk activation, B-Raf is implicated in sustained Erk signaling after TCR stimulation. In this study, we show that B-Raf is dephosphorylated on its inhibitory serine 365 upon TCR triggering. However, it is unknown how B-Raf activation is coupled to the TCR. Using mass spectrometry, we identified protein kinase D–interacting substrate of 220 kDa (Kidins220)/ankyrin repeat-rich membrane spanning protein, mammalian target of rapamycin, Rictor, Dock2, and GM130 as novel B-Raf interaction partners. We focused on Kidins220, a protein that has been studied in neuronal cells and found that it associated with the pre-TCR, αβTCR, and γδTCR. Upon prolonged TCR stimulation, the Kidins220–TCR interaction was reduced, as demonstrated by immunoprecipitation and proximity ligation assays. We show that Kidins220 is required for TCR-induced sustained, but not transient, Erk activation. Consequently, induction of the immediate early gene products and transcription factors c-Fos and Erg-1 was blocked, and upregulation of the activation markers CD69, IL-2, and IFN-γ was reduced. Further, Kidins220 was required for optimal calcium signaling. In conclusion, we describe Kidins220 as a novel TCR-interacting protein that couples B-Raf to the TCR. Kidins220 is mandatory for sustained Erk signaling; thus, it is crucial for TCR-mediated T cell activation.
BackgroundCanine mast cell tumour proliferation depends to a large extent on the activity of KIT, a tyrosine kinase receptor. Inhibitors of the KIT tyrosine kinase have recently been introduced and successfully applied as a therapeutic agent for this tumour type. However, little is known on the downstream target genes of this signaling pathway and molecular changes after inhibition.ResultsTranscriptome analysis of the canine mast cell tumour cell line C2 treated for up to 72 hours with the tyrosine kinase inhibitor masitinib identified significant changes in the expression levels of approximately 3500 genes or 16% of the canine genome. Approximately 40% of these genes had increased mRNA expression levels including genes associated with the pro-proliferative pathways of B- and T-cell receptors, chemokine receptors, steroid hormone receptors and EPO-, RAS and MAP kinase signaling. Proteome analysis of C2 cells treated for 72 hours identified 24 proteins with changed expression levels, most of which being involved in gene transcription, e.g. EIA3, EIA4, TARDBP, protein folding, e.g. HSP90, UCHL3, PDIA3 and protection from oxidative stress, GSTT3, SELENBP1.ConclusionsTranscriptome and proteome analysis of neoplastic canine mast cells treated with masitinib confirmed the strong important and complex role of KIT in these cells. Approximately 16% of the total canine genome and thus the majority of the active genes were significantly transcriptionally regulated. Most of these changes were associated with reduced proliferation and metabolism of treated cells. Interestingly, several pro-proliferative pathways were up-regulated, which may represent attempts of masitinib treated cells to activate alternative pro-proliferative pathways. These pathways may contain hypothetical targets for a combination therapy with masitinib to further improve its therapeutic effect.
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