Abstract
Amphioxus (e.g., Branchiostoma belcheri, Bb) has recently emerged as a new model for studying the origin and evolution of vertebrate immunity. Mammalian lymphocyte-specific tyrosine kinase (Lck) plays crucial roles in T cell activation, differentiation and homeostasis, and is reported to phosphorylate both the ITIM and ITSM of PD-1 to induce the recruitment of phosphatases and thus the inhibitory function of PD-1. Here, we identified and cloned the amphioxus homolog of human Lck. By generating and using an antibody against BbLck, we found that BbLck is expressed in the amphioxus gut and gill. Through overexpression of BbLck in Jurkat T cells, we found that upon TCR stimulation, BbLck was subjected to tyrosine phosphorylation and could partially rescue Lck-dependent tyrosine phosphorylation in Lck-knockdown T cells. Mass spectrometric analysis of BbLck immunoprecipitates from immunostimulants-treated amphioxus, revealed a BbLck-associated membrane-bound receptor LRR (BbLcLRR). By overexpressing BbLcLRR in Jurkat T cells, we demonstrated that BbLcLRR was tyrosine phosphorylated upon TCR stimulation, which was inhibited by Lck knockdown and was rescued by overexpression of BbLck. By mutating single tyrosine to phenylalanine (Y-F), we identified three tyrosine residues (Y539, Y655, and Y690) (3Y) of BbLcLRR as the major Lck phosphorylation sites. Reporter gene assays showed that overexpression of BbLcLRR but not the BbLcLRR-3YF mutant inhibited TCR-induced NF-κB activation. In Lck-knockdown T cells, the decline of TCR-induced IL-2 production was reversed by overexpression of BbLck, and this reversion was inhibited by co-expression of BbLcLRR but not the BbLcLRR-3YF mutant. Sequence analysis showed that the three tyrosine-containing sequences were conserved with the tyrosine-based inhibition motifs (ITIMs) or ITIM-like motifs. And TCR stimulation induced the association of BbLcLRR with tyrosine phosphatases SHIP1 and to a lesser extent with SHP1/2. Moreover, overexpression of wild-type BbLcLRR but not its 3YF mutant inhibited TCR-induced tyrosine phosphorylation of multiple signaling proteins probably via recruiting SHIP1. Thus, we identified a novel immunoreceptor BbLcLRR, which is phosphorylated by Lck and then exerts a phosphorylation-dependent inhibitory role in TCR-mediated T-cell activation, implying a mechanism for the maintenance of self-tolerance and homeostasis of amphioxus immune system and the evolutionary conservatism of Lck-regulated inhibitory receptor pathway.
Highlights
The amphioxus, the basal chordate, occupies the evolutionary position between invertebrates and vertebrates and serves as an excellent animal model to unveil the evolutionary enigma of the vertebrate adaptive immune system [1,2,3]
To determine whether the Lymphocyte-specific kinase (Lck) gene is present in the amphioxus, NCBI, JGI, and LanceletDB databases were used for BbLck sequence retrieval
Because BbLcLRR was identified in the BbLck coimmunoprecipitates, we determined whether BbLcLRR directly interacts with and is phosphorylated by BbLck. We found that both BbLck and human Lck associated with BbLcLRR when coexpressed in HEK293T cells (Figure 5A and Supplementary Figure 3)
Summary
The amphioxus, the basal chordate, occupies the evolutionary position between invertebrates and vertebrates and serves as an excellent animal model to unveil the evolutionary enigma of the vertebrate adaptive immune system [1,2,3]. After TCR and coreceptors engagement with cognate antigens, Lck is activated and phosphorylates the tyrosine residues on the immunoreceptor tyrosine-based activation motifs (ITAMs) on the CD3z and CD3g, d, and e subunits of the TCR complex. The phosphorylated ITAM tyrosine residues of CD3z serve as docking sites to recruit and activate the tyrosine kinase Zap, which phosphorylates the adaptor protein LAT (linker for activation of T cells) at multiple tyrosine residues. Phosphorylated LAT nucleates multiprotein signaling complexes, leading to T-cell activation [10,11,12,13]. In addition to phosphorylate ITAMs, Lck can phosphorylate the ITIMs of HLA-specific killer cell inhibitory receptors (KIR) in NK and T cells [14], and phosphorylate both the ITIM and immune receptor tyrosine–based switch motif (ITSM) of programmed cell death–1 (PD-1) to activate its inhibitory function in T cells [15]
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
