Signaling in the Late Phase of T cell Activation

Abstract

Engagement of the T cell antigen receptor (TCR) induces multiple signaling pathways, including the activation of extracellular signal-regulated kinase (ERK), a mitogen-activated protein kinase (MAPK). We previously reported the importance of sustained ERK activation for interleukin-2 (IL-2) production. Inhibition of ERK activation from 2 to 6 hours after TCR stimulation significantly impaired IL-2 production and activation of the nuclear factor-kappaB (NF-KB) family transcription factor, c-Rel, whereas inhibition during the first 4 hours had no effect. Loss of the adaptor protein, She, results in impaired ERK activation during the late phase ofTCR stimulation, and leads to severely reduced IL-2 production and c-Rel activation. These data suggest a novel activation process following TCR stimulation that involves She and late ERK activation-dependent regulation of c-Rel activation and IL-2 production. To further understand the mechanisms underlying this pathway, we employed a two- dimensional differential in-gel electrophoresis/ mass spectrometry (2D-DIGE/MS)-based proteomics approach. This approach to identify members of a She-containing signaling complex revealed alpha tubulin and beta actin as She associated proteins. Furthermore, we identified proteins whose expression and modification are triggered by TCR stimulation and are under control of the ERK signaling pathway, by comparing TCR-stimulated samples with or without MEK (MAPK kinase) inhibitor treatment. Here we report heterogeneous nuclear l ribonucleoprotein K (hnRNP-K) as a novel downstream target of ERK in TCR signaling. Functional studies using small RNA interference showed that hnRNPK regulated IL-2 production at the transcriptional level. We also showed that knockdown ofhnRNP-K expression specifically impaired NF-KB activity, but caused a relatively minor effect on activating protein-I (AP-1) activity and expression of CD69 or CD25. Biochemical analysis showed that knockdown of hnRNP-K caused enhanced proteolysis of the protooncogene Vav. MEK inhibitor treatment during the late phase of stimulation also enhanced proteolysis of Vav. Moreover, knockdown of hnRNP-K impaired Vav-mediated transcriptional activation ofIL-2 gene. T!\ken together, these results indicate that ERK signaling modulates IL-2 production by regulating Vav activity through the function of hnRNP-K. We also examined changes in phosphoprotein profiles upon TCR stimulation and MEK inhibitor treatment. Results obtained from these three different approaches provide a further understanding of the mechanisms that regulate late phase T cell activation as well as the components required for full activation of T cells.