Y192 within the SH2 Domain of Lck Regulates TCR Signaling Downstream of PLC-γ1 and Thymic Selection

Abstract

Signaling via the TCR, which is initiated by the Src-family tyrosine kinase Lck, is crucial for the determination of cell fates in the thymus. Because of its pivotal role, ablation of Lck results in a profound block of T-cell development. Here, we show that, in addition to its well-known function in the initiation of TCR signaling, Lck also acts at a more downstream level. This novel function of Lck is determined by the tyrosine residue (Y192) located in its SH2 domain. Thymocytes from knock-in mice expressing a phosphomimetic Y192E mutant of Lck initiate TCR signaling upon CD3 cross-linking up to the level of PLC-γ1 phosphorylation. However, the activation of downstream pathways including Ca2+ influx and phosphorylation of Erk1/2 are impaired. Accordingly, positive and negative selections are blocked in LckY192E knock-in mice. Collectively, our data indicate that Lck has a novel function downstream of PLCγ-1 in the regulation of thymocyte differentiation and selection.

Combined Immunodeficiency Caused by a Novel Nonsense Mutation in LCK

“…Phosphorylation of Y192, Y394 (as detected by the anti-pSrcY416 antibody) and Y505 were undetectable in Jk.LCKKO cells expressing LCKmut after anti-CD3 stimulation (Fig. 5 a) [ 33 ]. As expected, global tyrosine phosphorylation was strongly reduced in control Jk.LCKKO cells and cells expressing LCKmut, but reconstituted after re-expression of LCKwt as detected by developing with the 4G10 antibody (Fig.…”

“…This potential interaction with ZAP70 was however not associated with a detectable anti-CD3-induced phosphorylation at Y192 [ 26 ] in LCKmut(S377KTer14) in our transfection system. Phosphorylation of Y192 is relevant for the co-localization of LCK with CD45 and the TCR [ 26 , 33 ]; thus, the lack of phosphorylation may contribute to the observed aberrant subcellular localization of the truncated protein despite an intact N-terminus domain anchoring LCK to the plasma membrane in the truncated LCKmut [ 37 ].…”

A Cysteine Residue within the Kinase Domain of Zap70 Regulates Lck Activity and Proximal TCR Signaling

“…It has been proposed that the phosphorylation of Y192 diminishes the interaction between Lck and its positive regulatory phosphatase CD45, thus resulting in the hyper-phosphorylation of Y505 in Lck and in its inactivation [ 14 ]. Conversely, we have shown that a phosphomimetic Lck Y192E mutant still retains its enzymatic activity and is able to initiate signaling in thymocytes [ 15 , 22 ], highlighting that the initial steps of TCR activation and signal propagation are not fully understood. In this study, we revealed a new feedback loop involving Lck and Zap70.…”

A Story of Kinases and Adaptors: The Role of Lck, ZAP-70 and LAT in Switch Panel Governing T-Cell Development and Activation

“…This is an unusual regulatory mechanism by which a phosphorylation event triggers the uncoupling of two proteins, and this should be the reason why the mutation to phenylalanine of tyrosine 192 does not have a negative impact on TCR signaling: Phe residues could be structurally not so different from Tyr residues, and are still able to bind to CD45, while Tyr to Ala or Glu induces a strong modification of the tertiary structure of the SH2 domain, preventing binding to CD45 and thus leading to a high basal level of Tyr505 phosphorylation, which sharply reduces the pool of active Lck. Accordingly, knockin mice expressing a Tyr to Glu (Y192E) form of Lck showed a strong decrease in the total thymocyte numbers, with reduced numbers of DP and SP populations and defective positive and negative selection [ 99 , 100 ]. Interestingly, T cells from LckY192E knockin mice showed a diminished binding to CD45 and a concomitant hyperphosphorylation of Y505, thus corroborating previous data obtained from Jurkat T cells.…”