Protein Interactions between CD2 and Lck Are Required for the Lipid Raft Distribution of CD2

Abstract

In T lymphocytes, lipid rafts are preferred sites for signal transduction initiation and amplification. Many cell membrane receptors, such as the TCR, coreceptors, and accessory molecules associate within these microdomains upon cell activation. However, it is still unclear in most cases whether these receptors interact with rafts through lipid-based amino acid modifications or whether raft insertion is driven by protein-protein interactions. In murine T cells, a significant fraction of CD2 associates with membrane lipid rafts. We have addressed the mechanisms that control the localization of rat CD2 at the plasma membrane, and its redistribution within lipid rafts induced upon activation. Following incubation of rat CD2-expressing cells with radioactive-labeled palmitic acid, or using CD2 mutants with Cys226 and Cys228 replaced by alanine residues, we found no evidence that rat CD2 was subjected to lipid modifications that could favor the translocation to lipid rafts, discarding palmitoylation as the principal mechanism for raft addressing. In contrast, using Jurkat cells expressing different CD2 and Lck mutants, we show that the association of CD2 with the rafts fully correlates with CD2 capacity to bind to Lck. As CD2 physically interacts with both Lck and Fyn, preferentially inside lipid rafts, and reflecting the increase of CD2 in lipid rafts following activation, CD2 can mediate the interaction between the two kinases and the consequent boost in kinase activity in lipid rafts.

Quantitative Interactomics of Lck-TurboID in Living Human T Cells Unveils T Cell Receptor Stimulation-Induced Proximal Lck Interactors

“…TCR engagement is followed by the formation of an immunological synapse, where TCR-associated molecules and selected cytosolic signaling effectors are clustered to allow for more efficient signal transduction and amplification . The formation of immunological synapse requires repositioning of membrane lipid rafts, during which CD2 exhibits inducible translocation to lipid rafts and becomes integral to the structure of the immunological synapse. , Notably, CD2 translocation to the immunological synapse of TCR leads to physical association with Lck, supporting our findings. An affinity purification proteomic study of Lck similarly identified CD2 as an Lck interactor in primary mouse CD4+ T cells .…”

CD6 attenuates early and late signaling events, setting thresholds for T‐cell activation

“…SHP‐1, or modulating the activity of signaling‐enhancing tyrosine kinases 28, 29. Alternatively, given that CD6 associates with several kinases, it might sequester these kinases away from the TCR apparatus or it could function analogously to the IgSF glycoprotein CD2, which transduces mitogenic signals via its association with lipid rafts and the tyrosine kinases Lck and Fyn 30, 31, and inhibitory signals via its association with CD5 32–34. Since CD5 and CD6 also associate at the surface of T cells and one major effect of CD6 stimulation is the phosphorylation of CD5 13, it is feasible that CD5 can integrate or transform the signals generated by the triggering of CD6.…”

“…Transient transfections of primary T lymphocytes and of Raji cells were performed as described previously 21. Jurkat E6.1 cells were stably transfected as described previously 31. K562 cells (5×10 6 ) were transfected with 50 μg of hCD166/pECFP‐N2 or rCD166/pECFP‐N2 and by electroporation at 500 μF and 875 V and cultured in complete RPMI supplemented with 5 mg/mL of G418 (Invitrogen) to select for positive cells.…”

A New Pathway of CD5 Glycoprotein-mediated T Cell Inhibition Dependent on Inhibitory Phosphorylation of Fyn Kinase

“…These membrane microdomains are heterogeneous (54) and highly mobile (55), and receptors such as CD5 may serve as scaffolds that allow distinct types of rafts and their components to interact (56). CD5 associates with CD2 and with the TCR (23, 57), and these two surface receptors signal via Src kinases in the context of lipid rafts (33, 58). As we show here that CD5 is present at the cell surface as a homodimer, this may promote further the establishment of interaction arrays with a variety of lipid raft and signaling receptors.…”

“…Sucrose gradient centrifugation was performed as described (33). Briefly, activated cells were washed twice with ice-cold PBS and lysed for 30 min on ice in 1 ml of MBS buffer (25 m m MES, pH 6.5, 150 m m NaCl) containing 1% Triton X-100, 1 m m PMSF, and protease inhibitors (1 m m 4-(2-aminoethyl)benzenesulfonyl fluoride, 0.8 μ m aprotinin, 50 μ m bestatin, 15 μ m E-64, 20 μ m leupeptin, 10 μ m pepstatin A; Calbiochem).…”