Sequential phosphorylation of SLP-76 at tyrosine 173 is required for activation of T and mast cells

Abstract

Cooperatively assembled signalling complexes, nucleated by adaptor proteins, integrate information from surface receptors to determine cellular outcomes. In T and mast cells, antigen receptor signalling is nucleated by three adaptors: SLP-76, Gads and LAT. Three well-characterized SLP-76 tyrosine phosphorylation sites recruit key components, including a Tec-family tyrosine kinase, Itk. We identified a fourth, evolutionarily conserved SLP-76 phosphorylation site, Y173, which was phosphorylated upon T-cell receptor stimulation in primary murine and Jurkat T cells. Y173 was required for antigen receptorinduced phosphorylation of phospholipase C-c1 (PLC-c1) in both T and mast cells, and for consequent downstream events, including activation of the IL-2 promoter in T cells, and degranulation and IL-6 production in mast cells. In intact cells, Y173 phosphorylation depended on three, ZAP-70-targeted tyrosines at the N-terminus of SLP-76 that recruit and activate Itk, a kinase that selectively phosphorylated Y173 in vitro. These data suggest a sequential mechanism whereby ZAP-70-dependent priming of SLP-76 at three N-terminal sites triggers reciprocal regulatory interactions between Itk and SLP-76, which are ultimately required to couple active Itk to its substrate, PLC-c1.

Modulation of TCR responsiveness by the Grb2-family adaptor, Gads

“…Phosphospecific anti-human SLP-76 pY128 was from BD Pharmingen (San Jose, CA). Polyclonal phospho-specific antibodies to human SLP-76 pY173 were previously described [21]. Anti-CD69-PE-Cy5 (FN50) was from Dako, an Agilent Technologies company (Glostrup, Denmark).…”

“…Jurkat-derived cell lines were transfected with plasmid vectors by electroporation or infected with IRES-GFP-tagged retroviral vectors as described [21]. Cells were sorted by FACS for homogeneous GFP expression, approximately one week following infection.…”

“…Itk subsequently phosphorylates SLP-76 at Y173, and PLC-γ1 at Y783, both sites required for PLC-γ1 activation [19][20][21]. Gads may facilitate these events, by bridging the interaction between LAT-bound PLC-γ1 and SLP-76-bound Itk.…”

Cutting Edge: Feed-Forward Activation of Phospholipase Cγ2 via C2 Domain–Mediated Binding to SLP65

“…The phosphorylation reaction is independent of other pTyr. By contrast, the corresponding site of SLP76 becomes phosphorylated by Btk-related Itk in a hierarchical manner only upon preceding phosphorylations on other sites by Syk-related ZAP70 (9). The independence of Y 119 phosphorylation of a "primer kinase" is most likely caused by the N-terminal glutamate, which is absent in SLP76.…”

“…A similar motif can be found in the T cell paralog SLP76 (3), albeit at a different position (i.e., Y 173 in murine SLP76) and without a negatively charged residue N terminal to the tyrosine. Phosphorylation of Y 173 in SLP76 by the Btk-related kinase Itk is pivotal for PLCg1 activation in T lymphocytes and mast cells in vitro and in vivo (9). The mechanism of phospho-Y173 signaling remained elusive.…”