Dataset Information

Tyrosine 192 within the SH2 domain of the Src-protein tyrosine kinase p56^Lck 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 Lck^Y192E knock-in mice revealed a diminished binding of Lck^Y192E 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 Lck^Y192E possesses a normal enzymatic activity in human and murine T cells. FLIM/FRET measurements employing an Lck^Y192E biosensor further indicated that the steady state conformation of the Lck^Y192E mutant is similar to Lck^wt. These data suggest that Y192 might regulate Lck functions also independently from the Lck/CD45-association. Indeed, when Lck^Y192E was expressed in CD45^-/-/Csk^-/- non-T cells (HEK 293T cells), phosphorylation of Y505 was similar to Lck^wt, but Lck^Y192E still failed to optimally phosphorylate and activate the Lck downstream substrate ZAP70. Furthermore, Lck^Y19E 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. Video Abstract.

SUBMITTER: Kastle M

PROVIDER: S-EPMC7682018 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Gleevec, a well-known cancer therapeutic agent, is an effective inhibitor of several tyrosine kinases, including Abl and c-Kit, but displays less potency to inhibit closely homologous tyrosine kinases, such as Lck and c-Src. Because many structural features of the binding site are highly conserved in these homologous kinases, the molecular determinants responsible for the binding specificity of Gleevec remain poorly understood. To address this issue, free energy perturbation molecular dynamics (FEP/MD) simulations with explicit solvent was used to compute the binding affinity of Gleevec to Abl, c-Kit, Lck, and c-Src. The results of the FEP/MD calculations are in good agreement with experiments, enabling a detailed and quantitative dissection of the absolute binding free energy in terms of various thermodynamic contributions affecting the binding specificity of Gleevec to the kinases. Dominant binding free energy contributions arises from the van der Waals dispersive interaction, compensating about two-thirds of the unfavorable free energy penalty associated with the loss of translational, rotational, and conformational freedom of the ligand upon binding. In contrast, the contributions from electrostatic and repulsive interactions nearly cancel out due to solvent effects. Furthermore, the calculations show the importance of the conformation of the kinase activation loop. Among the kinases examined, Abl provides the most favorable binding environment for Gleevec via optimal protein-ligand interactions and a small free energy cost for loss of the translational, rotational, and conformational freedom upon ligand binding. The FEP/MD calculations additionally reveal that Lck and c-Src provide similar nonbinding interactions with the bound-Gleevec, but the former pays less entropic penalty for the ligand losing its translational, rotational, and conformational motions to bind, examining the empirically observed differential binding affinities of Gleevec between the two Src-family kinases.

Dataset Information

Tyrosine 192 within the SH2 domain of the Src-protein tyrosine kinase p56^Lck regulates T-cell activation independently of Lck/CD45 interactions.

Background

Methods

Results

Conclusions

Major findings

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets

Dataset Information

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

Background

Methods

Results

Conclusions

Major findings

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets

Tyrosine 192 within the SH2 domain of the Src-protein tyrosine kinase p56^Lck regulates T-cell activation independently of Lck/CD45 interactions.