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首页> 外文期刊>Proceedings of the National Academy of Sciences of the United States of America >In situ detection of activated Bruton's tyrosine kinase in the Ig signaling complex by phosphopeptide-specific monoclonal antibodies
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In situ detection of activated Bruton's tyrosine kinase in the Ig signaling complex by phosphopeptide-specific monoclonal antibodies

机译：磷酸肽特异性单克隆抗体原位检测Ig信号复合物中活化的Bruton酪氨酸激酶

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摘要

Bruton's tyrosine kinase (Btk) is a critical transducer of signals originating from the B cell antigen receptor (BCR). Dosage, sequential phosphorylation, and protein interactions are interdependent mechanisms influencing Btk function. ;hosphopeptide-specific habs recogniz- ing two distinct phosphotyrosine modifications were used to quantify Btk activation by immunofluorescent techniques during B cell stimulation. In a population of cultured B cells stimulated by BCR crosslinking and analyzed by flow cytometry, transient phosphorylation of the regulatory Btk tyrosine residues (551Y and 223) was detected. The kinftics of phosphorylation of the residues were temporally distinct. Tyrosine 551, a transactivating substrate site for Src-family kinases, was maximally phosphorylated within approximaately equals 30 seconds of stimulation as monitored by flow cytometry. Tyrosine 223, an autophosphorylation site within the SH3 domain, was maximally phosphorylated at aproximately equals 5 minutes. Btk returned to a low tyrosine phosphorylation level within 30 minutes, despite persistent elevation of global tyrosine phosphorylation. Colocalization of activated Btk molecules with the crosslinked BCR signaling complex was observed to coincide with the period of maximal Btk tyrosine phosphorylation when stimulated B cells were analyzed with confocal microscopy. The results of these in sito temporal and spatial analyses imply that Btk signaling occurs in the region of the Ig receptor signaling complex, suggesting a similar location for downstream targets of its activity.
机译：布鲁顿酪氨酸激酶（Btk）是源自B细胞抗原受体（BCR）的信号的关键转导者。剂量，顺序磷酸化和蛋白质相互作用是影响Btk功能的相互依赖的机制。 ；识别两种截然不同的磷酸酪氨酸修饰的磷酸肽特异的嗜血杆菌，用于在B细胞刺激过程中通过免疫荧光技术对Btk活化进行定量。在通过BCR交联刺激并通过流式细胞仪分析的培养B细胞群体中，检测到调节性Btk酪氨酸残基（551Y和223）的瞬时磷酸化。残基的磷酸化的动力学在时间上是不同的。通过流式细胞术监测，酪氨酸551（一种Src家族激酶的反式激活底物位点）在大约等于30秒的刺激时间内被最大程度地磷酸化。 SH3结构域中的一个自磷酸化位点酪氨酸223在大约等于5分钟时被最大程度地磷酸化。尽管全球酪氨酸磷酸化水平持续升高，但Btk在30分钟内恢复到了较低的酪氨酸磷酸化水平。当用共聚焦显微镜分析刺激的B细胞时，观察到活化的Btk分子与交联的BCR信号复合物的共定位与最大Btk酪氨酸磷酸化的时间段一致。这些在时态和时空分析中的结果表明，Btk信号传导发生在Ig受体信号传导复合物的区域中，表明其活性下游靶标的位置相似。

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