Activation of the Src family kinase Hck without SH3-linker release
Activation of the Src family kinase Hck without SH3-linker release
复制标题
DOI：
10.1074/jbc.m508782200
复制
发表时间：
2005-12-09
影响因子：
4.8
通讯作者：
Smithgall, TE
中科院分区：
文献类型：
--
作者：
Lerner, EC;Trible, RP;Smithgall, TE
Src家族蛋白酪氨酸激酶通过SH 2结构域与C-末端尾的分子内结合以及SH 3结构域与SH 2激酶-接头的缔合来调节。两种调节相互作用的存在提出了一个问题，即激酶激活是否需要破坏两者。为了解决这个问题，我们设计了Src家族成员Hck的高亲和力接头（HAL）突变体，其中最佳SH 3配体取代了天然接头。表面等离子体共振分析表明，紧密的分子内结合的修改后的HAL序列SH 3。然后将Hck-HAL与尾部酪氨酸突变（Y501 F）组合并在Rat-2成纤维细胞中表达。令人惊讶的是，Hck-HAL-Y501 F显示出强的转化和激酶活性，证明分子内SH 3-接头释放对于基于SH 2的激酶活化不是必需的。在酿酒酵母中，缺乏负调控尾激酶Csk，野生型HCK更强烈地激活的SH 3结合蛋白（人类免疫缺陷病毒-1 Nef）的存在下，表明持久性的天然SH 3-接头相互作用的活性HCK构象。总之，这些数据支持Src家族激酶的多种活性构象的存在，这些构象可能产生独特的下游信号。
Src family protein-tyrosine kinases are regulated by intramolecular binding of the SH2 domain to the C-terminal tail and association of the SH3 domain with the SH2 kinase-linker. The presence of two regulatory interactions raises the question of whether disruption of both is required for kinase activation. To address this question, we engineered a high affinity linker (HAL) mutant of the Src family member Hck in which an optimal SH3 ligand was substituted for the natural linker. Surface plasmon resonance analysis demonstrated tight intramolecular binding of the modified HAL sequence to SH3. Hck-HAL was then combined with a tail tyrosine mutation (Y501F) and expressed in Rat-2 fibroblasts. Surprisingly, Hck-HAL-Y501F showed strong transforming and kinase activities, demonstrating that intramolecular SH3-linker release is not required for SH2-based kinase activation. In Saccharomyces cerevisiae, which lacks the negative regulatory tail kinase Csk, wild-type Hck was more strongly activated in the presence of an SH3-binding protein ( human immunodeficiency virus-1 Nef), indicating persistence of native SH3-linker interaction in an active Hck conformation. Taken together, these data support the existence of multiple active conformations of Src family kinases that may generate unique downstream signals.