The membrane adaptor LAT is proteolytically cleaved following Fas engagement in a tyrosine phosphorylation-dependent fashion

Abstract

Engagement of the TCR (T-cell receptor) induces tyrosine phosphorylation of the LAT (linker for the activation of T-cells) adaptor, and thereby it recruits several cytosolic mediators for downstream signalling pathways. The Fas protein is essential for T-lymphocyte apoptosis, and following Fas engagement, many proteins are proteolytically cleaved, including several molecules that are important for the transduction of TCR intracellular signals. In the present study, we demonstrate that the adaptor LAT is also subject to a proteolytic cleavage in mature T-lymphocytes and thymocytes in response to Fas engagement, and also on TCR stimulation, and we identify three aspartic acid residues at which LAT is cleaved. Interestingly, these aspartic acid residues are located in proximity to several functionally important tyrosine residues of LAT, raising the possibility that their phosphorylation could modulate LAT cleavage. Consistent with that hypothesis, we show that induction of phosphorylation by pervanadate or H2O2 in Jurkat cells and thymocytes inhibits Fas-mediated cleavage of LAT. Moreover, we show that LAT proteolysis is also enhanced during anergy induction of primary human T-cells, suggesting that LAT cleavage may act as a regulator of TCR-mediated activation of T-cells and not only as a transducer of cell death promoting stimuli.

Mutations in linker for activation of T cells (LAT) lead to a novel form of severe combined immunodeficiency

“…In the absence of LAT, there is reduced apoptosis, which leads to lymphoproliferation in murine LAT mutant knock-in models. 5,[20][21][22] In the absence of primary material (other than a limited source of PBMCs) from our patients who had either died or undergone hematopoietic stem cell transplantation, we chose to demonstrate the nonfunctional effect of the patient mutation in specific cell lines. We used 2 LAT mutant Jurkat T-cell lines, ANJ3 3 and J.CaM2, 12,13 which are defective in TCR-mediated signal transduction.…”

“…On TCR cross-linking, a number of studies have shown that LAT can be a negative regulator of TCR signaling, including mutant LAT knock-in models, which show a T-lymphoproliferative disease. 5,[20][21][22] We stimulated JE6.1 and LAT-deficient ANJ3 and J.CaM2 cells with immobilized OKT3 and showed that there was significantly reduced apoptosis in mutant lines than in JE6.1 control cells (Fig 4, A, upper panel, and B). In reconstituted lines introduction of wild-type LAT in ANJ3/wtLAT-GFP-lent and J.CaM2/wtLAT-GFP-lent led to a significant increase in apoptosis after TCR stimulation, whereas LAT-deficient lines transduced with mutant LAT, ANJ3/mutLAT-GFP-lent, and J.CaM2/ mutLAT-GFP-lent did not show any increase in levels of apoptosis (Fig 4, A, middle and lower panels, C and D).…”

Identification of functional, short-lived isoform of linker for activation of T cells (LAT)

“…Both LAT isoforms were found predominantly in lipid raft-enriched fractions but were absent from cytoplasmic fractions. As the partial proteolytic degradation of LAT in response to pro-apoptotic stimuli was previously recognized as a mechanism for attenuation of T-cell signaling, 31,32 we wished to examine whether JCam2.5 cells transduced with LAT i6 cDNA are prone to Fas-mediated degradation. In Figure 4a it is shown that upon anti-Fas crosslinking the LAT i6 was degraded similarly to the LAT isoform, indicating that an additional string of amino acids present in the LAT i6 does not change its sensitivity to Fas-induced degradation.…”

“…The cDNA encoding human LAT i6 or LAT isoforms in fusions with C-terminally 6-histidine tags or Zs-Green fluorescent protein cDNA were cloned into the self-inactivating (SIN) lentiviral transfer plasmid pHR'SINcPPT-Blast as previously described. 31 Lentiviral particles containing supernatants were used to transduce JCam2.5 cells. Stably transduced cells were selected using 10 mg ml À 1 of blasticidin.…”

“…[24][25][26] The activity of LAT undergoes post-translational modulation, mainly by phosphorylation of tyrosine and serine residues 27,28 and by palmitoylation of cysteine residues. 29 In addition, ubiquitylation 30 and partial proteolytic degradation 31,32 of LAT have been recognized as strategies for attenuation of T-cell signaling. In this report we identified a signaling-competent short-lived isoform of LAT that mediated increased long-term interleukin-2 (IL-2) secretion upon CD3/CD28 crosslinking.…”