Extended Data Fig. 2: IP-MS/MS analysis identifies K228 as an acetylation site modulating LCK conformation.
From: SIRT2-mediated deacetylation of LCK governs the magnitude of T cell receptor signaling
a,b, CID spectra of LSRPCQTQKPQKPWWEDEWEVPR peptide derived from LCK carrying acetylation on K228 identified by LC–MS/MS from LCK-immunoprecipitated primary mouse T cell lysates (a) and human Jurkat cell lysates (b). c, Quantification of LCKK228 acetylation in NTC (Cas9–crRNA non-targeting control) and crSIRT2 (Cas9–crRNA targeting SIRT2) Jurkat cell lysates by LC–MS/MS following LCK immunoprecipitation. Acetylated peptide intensity was normalized to total peptide intensity. n = 3 biological replicates. Data are mean ± SEM. P values by two-tailed Student’s t-test. d, Quantification of LCKK228 acetylation in freshly isolated T cells (TN), OVA-activated OT-II T cells (TEFF-like), IL-15–differentiated OT-II TM-like cells and mouse TILs isolated from B16F10 subcutaneous by LC–MS/MS following LCK immunoprecipitation. Intensities of acetylated peptides shown relative to TN cells. TN, n = 1 (pooled from 10 mice); TEFF, n = 4 independent mice; TM, n = 4 independent mice; TILs, n = 1 (pooled from 10 mice). e, Schematic of LCK domains. Src homology 4 (SH4), unique region (UR), SH3, SH2, linker region (LR), kinase domain and negative-regulatory tail (NR) are depicted. f, Model of LCK conformational control by intramolecular interactions. Left, inactive closed form stabilized by: (i) phospho-Y505 (on the NR) binding to the SH2 domain and (ii) LR–SH3 interaction. Right, active open form achieved through: (i) Y505 dephosphorylation releasing the NR from SH2 and (ii) LR disengagement from SH3. g,h, Predicted binding mode of the LCK linker region to the SH3 domain modeled using AlphaFold3 and visualized in PyMOL. Left panels depict the non-acetylated form; right panels show the structural rearrangement upon K228 acetylation, which disrupts charge interaction and excludes the side chain from the pocket. g, Surface representation showing a shallow pocket on the SH3 domain (circled in blue) predicted to accommodate K228. The sequence of the LCK linker region (residues Q225–P232) was labeled. h, Stick model of the same complex showing the linker region (Q225–P232) and the three SH3 domain residues Y72, E73 and S75 defining the pocket predicted to accommodate K228. Data are representative of one (d), three (b,c) and four (a) independent experiments.
