Lck phosphorylation status of Lck Tyr192 and Tyr192/Tyr505 mutantsLck phosphorylation status of Lck Tyr192 and Tyr192/Tyr505 mutants

Lymphocyte-specific protein tyrosine kinase (Lck) is crucial for signaling from the T cell receptor (TCR) and is controlled through tyrosine phosphorylations. Phosphorylated Tyr505 (pTyr505) promotes a closed, inactive conformation of Lck, while pTyr394 is critical for kinase activity. Additionally, pTyr192 has been suggested to regulate Lck activity by changing the specificity of the Lck Src-homology 2 (SH2) domain and/or by affecting Lck association with CD45 thus drastically increasing pTyr505. However, little is known about how pTyr192 affects endogenously expressed Lck. Here we used CRISPR/Cas9 genome editing to generate Jurkat cell lines expressing Lck Glu192 mimicking Lck pTyr192, or Lck Phe192 mimicking unphosphorylated Lck Tyr192. We confirmed that Lck Glu192 is hyperphosphorylated on Tyr505, possibly explaining reduced association of Lck Glu192 with prototypic Lck-SH2 ligands. To isolate the effect of Lck Tyr192 mutations from the effect on Lck pTyr505, we subsequently generated Jurkat cells doubly mutated on Lck Tyr192 and Lck Tyr505. Both Lck Phe192/Phe505 and Lck Glu192/Phe505 mutants co-precipitated similar amounts of binding partners. Moreover, both mutants displayed hyperphosphorylation of Tyr394. Our results indicate that CD45 is the main phosphatase controlling Lck pTyr394 in steady-state T cells. Additionally, our data demonstrate that the prototypic specificity of the Lck SH2 domain (owned by the Lck Phe192 mutants) promotes transphosphorylation of Tyr394. These observations pinpoint the fundamental role of Tyr192 in regulation of Lck activity and simultaneously reveal the most potent Lck mutants so far described

淋巴细胞特异性蛋白酪氨酸激酶（Lymphocyte-specific protein tyrosine kinase, Lck）是T细胞受体（T cell receptor, TCR）信号传导的关键调控分子，其活性通过酪氨酸磷酸化精准控制。磷酸化酪氨酸505（Phosphorylated Tyr505, pTyr505）可诱导Lck形成闭合的非活性构象，而磷酸化酪氨酸394（Phosphorylated Tyr394, pTyr394）则对维持激酶活性至关重要。另有研究指出，磷酸化酪氨酸192（Phosphorylated Tyr192, pTyr192）可通过两种途径调控Lck活性：一是改变Lck的Src同源2（Src-homology 2, SH2）结构域的底物特异性，二是影响Lck与CD45的结合，进而大幅提升pTyr505的修饰水平。然而，目前学界对pTyr192如何调控内源性表达的Lck仍缺乏深入认知。
本研究采用CRISPR/Cas9基因组编辑技术，构建了两种Jurkat细胞系：分别表达模拟磷酸化酪氨酸192的Lck谷氨酸192（Lck Glu192）突变体，以及模拟未磷酸化酪氨酸192的Lck苯丙氨酸192（Lck Phe192）突变体。实验证实，Lck Glu192在酪氨酸505（Tyrosine505, Tyr505）位点呈现过度磷酸化，这一现象可解释其与典型Lck-SH2配体的结合能力显著降低的原因。
为了单独解析酪氨酸192（Tyrosine192, Tyr192）突变的效应，排除pTyr505的干扰，我们进一步构建了同时携带酪氨酸192（Tyrosine192, Tyr192）与酪氨酸505（Tyrosine505, Tyr505）双突变的Jurkat细胞。结果显示，Lck Phe192/Phe505与Lck Glu192/Phe505两种双突变体的结合伴侣沉淀量并无显著差异；且二者均表现出酪氨酸394（Tyrosine394, Tyr394）位点的过度磷酸化。
本研究结果表明，CD45是稳态T细胞中调控Lck pTyr394的主要磷酸酶。同时，实验数据证实，Lck Phe192突变体所保留的典型Lck SH2结构域特异性，可促进酪氨酸394（Tyrosine394, Tyr394）的反式磷酸化。上述发现明确了酪氨酸192（Tyrosine192, Tyr192）在Lck活性调控中的核心功能，同时也揭示了目前已报道的活性最强的Lck突变体。