Characterization of Ly108 in the Thymus: Evidence for Distinct Properties of a Novel Form of Ly108

Abstract

Ly108 (CD352) is a member of the Signaling Lymphocyte Activation Molecule (SLAM) family of receptors that signals through SLAM-associated protein (SAP), an SH2 domain protein that can function by the recruitment of Src family kinases or by competition with phosphatases. Ly108 is expressed on a variety of hematopoietic cells and with especially high levels on developing thymocytes. We find that Ly108 is constitutively tyrosine phosphorylated in murine thymi in a SAP- and Fyn kinase-dependent manner. Phosphorylation of Ly108 is rapidly lost after thymocyte disaggregation, suggesting dynamic contact-mediated regulation of Ly108. Similar to recent reports, we find at least 3 isoforms of Ly108 mRNA and protein in the thymus, which are differentially expressed in the thymi of C57Bl/6 and 129S6 mice that express the lupus-resistant and lupus-prone haplotypes of Ly108, respectively. Notably, the recently described novel isoform, Ly108-H1, is not expressed in mice having the lupus-prone haplotype of Ly108, but is expressed in C57Bl/6 mice. We further provide evidence for differential phosphorylation of these isoforms; the novel Ly108-H1 does not undergo tyrosine phosphorylation, suggesting it functions as a decoy isoform that contributes to the reduced overall phosphorylation of Ly108 seen in C57Bl/6 mice. Our studies suggest that Ly108 is dynamically regulated in the thymus and shed light on Ly108 isoform expression and phosphorylation.

SAP Facilitates Recruitment and Activation of LCK at NTB-A Receptors during Restimulation-Induced Cell Death

“…FYN is considered the key Src-family kinase required for SLAM-R signaling, based on its SAP-dependent association with several other SLAM-Rs (13)(14)(15)(16)(17)(18)(19)(20). Although LCK was previously implicated in phosphorylation of both SLAM and CD84 (17,24), no direct association between LCK and a SLAM-R had been detected to date.…”

“…Previous studies either relied on overexpression constructs for biochemical analysis of NTB-A signaling (19) or focused on thymocytes, NK cells, or NK cell lines (13)(14)(15)(16)(17)(18)(19)(20). In the latter case, it is possible that cell type-based differences in the expression of FYN, LCK, SAP, or the SLAM family receptor examined could account for differences in the associations detected.…”

“…Two Srcfamily kinases are primarily involved in initiating TCR signaling: LCK and FYN (12). FYN is the major Src-family kinase shown to mediate signal transduction of SLAM-Rs (including NTB-A) in thymocytes and NK cells (13)(14)(15)(16)(17)(18)(19)(20). To examine the role of both FYN and LCK in RICD, we first used highly selective inhibitors for FYN (SU6656) (21) or LCK [LCK inhibitor I (LCKi)] (22).…”

“…The cytoplasmic tail of NTB-A contains three tyrosinebased motifs that regulate signal transduction: Y273 is part of a putative ITIM, whereas Y284 and Y308 are each part of an ITSM (8). Because SAP binding to NTB-A is reported to be dependent on phosphorylation of one or more of these tyrosines (19,20), we next asked which tyrosines in the NTB-A cytoplasmic tail are important for increased SAP-dependent LCK recruitment and phosphorylation during restimulation. To address this question, we stably expressed wild-type (WT) NTB-A or single tyrosine-tophenylalanine (Y→F) NTB-A cytoplasmic tail mutants (Y273F, Y284F, or Y308F) in the NTB-A-negative T cell line PEER (10).…”

The Receptor Ly108 Functions as a SAP Adaptor-Dependent On-Off Switch for T Cell Help to B Cells and NKT Cell Development

“…The neomycin resistance gene cassette remains. Ly108 protein expression is completely absent (Dutta and Schwartzberg, 2012). Slamf6 −/− mice have no gross B cell, CD4 + T cell, or CD8 T cell defects.…”