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. 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. 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. 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. 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.

中文翻译：

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Src 蛋白酪氨酸激酶 p56Lck 的 SH2 结构域内的酪氨酸 192 独立于 Lck/CD45 相互作用调节 T 细胞活化

在与 T 细胞受体 (TCR) 结合后，Src 家族蛋白酪氨酸激酶 p56Lck 使 TCR 的成分（例如 TCRζ 链）磷酸化，从而启动 T 细胞活化。Lck 的酶活性主要通过两个酪氨酸残基 Y394 和 Y505 的可逆和动态磷酸化进行调节。Lck 拥有一个额外的高度保守的酪氨酸 Y192，位于 SH2 域内，其在 T 细胞激活中的作用尚未完全了解。产生了表达 Lck 的磷酸化模拟 (Y192E) 形式的敲入小鼠。进行细胞和生化表征以阐明 Y192 在原代 T 细胞中的功能。HEK 293T 和 Jurkat T 细胞用于体外研究。使用来自 LckY192E 敲入小鼠的 T 细胞进行的免疫共沉淀研究和生化分析显示，LckY192E 与 CD45 的结合减弱，同时伴随着 Y505 的过度磷酸化，从而证实了之前在 Jurkat T 细胞中获得的数据。然而，令人惊讶的是，体外激酶测定表明 LckY192E 在人和鼠 T 细胞中具有正常的酶活性。采用 LckY192E 生物传感器的 FLIM/FRET 测量进一步表明 LckY192E 突变体的稳态构象与 Lckwt 相似。这些数据表明 Y192 可能也独立于 Lck/CD45 关联调节 Lck 功能。事实上，当 LckY192E 在 CD45-/-/Csk-/- 非 T 细胞（HEK 293T 细胞）中表达时，Y505 的磷酸化与 Lckwt 相似，但 LckY192E 仍无法最佳磷酸化和激活 Lck 下游底物 ZAP70。此外，在 TCR 刺激后，LckY19E 被招募到 CD3 中较少。总之，Y192 的磷酸化通过阻止 Lck 与 CD45 结合和通过调节配体诱导的 Lck 募集到 TCR 来至少双重调节 T 细胞中的 Lck 功能。我们的数据改变了目前对 Y192 功能的看法，并表明 Y192 还以独立于 Y505 磷酸化的方式调节 Lck 活性。