Tyrosine 192 within the SH2 domain of the Src-protein tyrosine kinase p56Lck regulates T-cell activation independently of Lck/CD45 interactions

Abstract

Background Upon engagement of the T-cell receptor (TCR), the Src-family protein tyrosine kinase p56Lck phosphorylates components of the TCR (e.g. the TCRζ chains), thereby initiating T-cell activation. The enzymatic activity of Lck is primarily regulated via reversible and dynamic phosphorylation of two tyrosine residues, Y394 and Y505. Lck possesses an additional highly conserved tyrosine Y192, located within the SH2 domain, whose role in T-cell activation is not fully understood. Methods Knock-in mice expressing a phospho-mimetic (Y192E) form of Lck were generated. Cellular and biochemical characterization was performed to elucidate the function of Y192 in primary T cells. HEK 293T and Jurkat T cells were used for in vitro studies. Results Co-immunoprecipitation studies and biochemical analyses using T cells from LckY192E knock-in mice revealed a diminished binding of LckY192E to CD45 and a concomitant hyperphosphorylation of Y505, thus corroborating previous data obtained in Jurkat T cells. Surprisingly however, in vitro kinase assays showed that LckY192E possesses a normal enzymatic activity in human and murine T cells. FLIM/FRET measurements employing an LckY192E biosensor further indicated that the steady state conformation of the LckY192E mutant is similar to Lckwt. These data suggest that Y192 might regulate Lck functions also independently from the Lck/CD45-association. Indeed, when LckY192E was expressed in CD45−/−/Csk−/− non-T cells (HEK 293T cells), phosphorylation of Y505 was similar to Lckwt, but LckY192E still failed to optimally phosphorylate and activate the Lck downstream substrate ZAP70. Furthermore, LckY19E was recruited less to CD3 after TCR stimulation. Conclusions Taken together, phosphorylation of Y192 regulates Lck functions in T cells at least twofold, by preventing Lck association to CD45 and by modulating ligand-induced recruitment of Lck to the TCR. Major findings Our data change the current view on the function of Y192 and suggest that Y192 also regulates Lck activity in a manner independent of Y505 phosphorylation.

LCK Regulates Homologous Recombination DNA Repair Identifying a New Target for Sensitizing PARP Inhibitors in HR Proficient Ovarian Cancer

“…For full activation of LCK kinase, presence of tyrosine at position 394 and lysine at position 273 is essential for autophosphorylation, conformational change and phosphate group transfer (29). Further phosphorylation at Y192 inhibits the function of LCK and leads to negative regulation (30). Thus, C_ LCK OE and C_ LCK Y192F mutant retain kinase activity while C_ LCK K273R and C_ LCK Y394F mutants lose kinase activity.…”

“…LCK interacts with BRCA1 and RAD51 in response to DNA damage, so we tested whether kinase activity and autophosphorylation of LCK is necessary for activity and DNA repair. We generated LCK mutants at lysine 273, which is necessary for kinase activity (K273R); tyrosine 394, an autophosphorylation and activation site (Y394F) 23 ; and tyrosine 192, a SH2 adaptor protein binding site (Y192F) 24 and transduced them into LCK KO CP70 cells (Fig. 6A).…”

Regulating the discriminatory response to antigen by T-cell receptor

“…Studies using phosphomimetic mutant of Lck suggest that residue Y192 in the SH2 domain is essential in determining the equilibrium between an active and inactive conformation [ 131 , 142 ]. Phosphorylation of Y192 prevents Lck interaction with the CD45, thus altering the enzymatic activity of Lck and shifting the equilibrium toward the closed-autoinhibited state [ 143 ].…”

Virtual Screening of Drug Proteins Based on Imbalance Data Mining

“…SRC kinase is widely present in tissue cells and can react with important molecules in signal transduction pathways, participating in cellular metabolic processes and regulating cell growth, development, and differentiation. is kinase has been implicated in the development of several cancers [14]. Activation of the SRC pathway has been detected in approximately more than 50% of various cancers, so the treatment of cancer can be achieved by inhibiting the activity of SRC [12].…”