Research Article: Serine Phosphorylation of SLP76 Is Dispensable for T Cell Development but Modulates Helper T Cell Function

Date Published: January 20, 2017

Publisher: Public Library of Science

Author(s): Victor H. Navas, Céline Cuche, Andres Alcover, Vincenzo Di Bartolo, Jean Kanellopoulos.

http://doi.org/10.1371/journal.pone.0170396

Abstract

The adapter protein SLP76 is a key orchestrator of T cell receptor (TCR) signal transduction. We previously identified a negative feedback loop that modulates T cell activation, involving phosphorylation of Ser376 of SLP76 by the hematopoietic progenitor kinase 1 (HPK1). However, the physiological relevance of this regulatory mechanism was still unknown. To address this question, we generated a SLP76-S376A-expressing knock-in mouse strain and investigated the effects of Ser376 mutation on T cell development and function. We report here that SLP76-S376A-expressing mice exhibit normal thymocyte development and no detectable phenotypic alterations in mature T cell subsets or other lymphoid and myeloid cell lineages. Biochemical analyses revealed that mutant T cells were hypersensitive to TCR stimulation. Indeed, phosphorylation of several signaling proteins, including SLP76 itself, phospholipase Cγ1 and the protein kinases AKT and ERK1/2, was increased. These modifications correlated with increased Th1-type and decreased Th2-type cytokine production by SLP76-S376A T cells, but did not result in significant changes of proliferative capacity nor activation-induced cell death susceptibility. Hence, our results reveal that SLP76-Ser376 phosphorylation does not mediate all HPK1-dependent regulatory effects in T cells but it fine-tunes helper T cell responses.

Partial Text

Adaptive immune responses are initiated upon recognition by the T cell receptor (TCR) of peptide antigen–major histocompatibility complex (MHC) complexes, displayed on the surface of antigen-presenting cells. TCR engagement results in a rapid activation of protein tyrosine kinases e.g. Lck and ZAP-70 [1] that, in turn, phosphorylate two key scaffold proteins, LAT [2] and SLP76 [3]. Phosphorylated LAT recruits SLP76 via the GRB2-related adaptor GADS [4], thus nucleating a central hub that gathers a wide array of effectors to activate downstream signaling pathways. Hence, assembly and stability of this proximal signaling platform critically affect the outcome of immune responses. For instance, both LAT and SLP76 have been implicated in the control of T cell cytoskeleton reorganization, generation of second messengers and activation of transcription factors [5–9], thus driving T cell proliferation, differentiation and specific effector functions.

In this article, we present a characterization of a knock-in mouse strain expressing the mutant adaptor protein SLP76-S376A. Based on our previous work in human T cell lines and primary T cells, we expected this mutation to impair the negative feedback loop driven by HPK1-dependent phosphorylation of SLP76 at Ser376 and recruitment of 14-3-3 proteins. This, in turn, would result in increased TCR-dependent signaling and activation. Data presented herein confirm that this regulatory loop is relevant in TCR-stimulated murine T cells. Indeed, tyrosine phosphorylation of key signaling effectors, e.g. SLP76 (Tyr128) and PLCγ1, is increased in SLP76-S376A T cells, similarly to what was observed in SLP76-S376A-expressing or HPK1-silenced Jurkat-derived T cells [13]. Moreover, phosphorylation of AKT and ERK1/2 kinases is increased in CD4+ T cells from SLP76-S376A mice, unveiling the effects of Ser376 mutation on PI3K-dependent and MAPK pathways.

 

Source:

http://doi.org/10.1371/journal.pone.0170396