The Tribbles 2 (TRB2) pseudokinase binds to ATP and autophosphorylates in a metal-independent manner.
The Tribbles 2 (TRB2) pseudokinase binds to ATP and autophosphorylates in a metal-independent manner.
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DOI：
10.1042/bj20141441
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发表时间：
2015-04-01
期刊：
影响因子：
--
通讯作者：
Eyers PA
中科院分区：
文献类型：
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作者：
Bailey FP;Byrne DP;Oruganty K;Eyers CE;Novotny CJ;Shokat KM;Kannan N;Eyers PA
人类 Tribbles (TRB) 相关假激酶是 CAMK（钙/钙调蛋白依赖性蛋白激酶）相关家族成员，在“假催化”结构域中进化出一系列高度不寻常的基序。在经典激酶中，保守氨基酸与二价金属离子结合并在有效磷酰基转移至底物之前排列 ATP。然而，在假激酶中，非典型残基会产生多种且常常未经研究的生化和结构特征，这些特征被认为是细胞功能的核心。 TRB 蛋白在多个信号网络中发挥着至关重要的作用，过度表达会赋予人类细胞癌症表型，使 TRB 假激酶成为一类新型药物靶点。在本文中，我们报道人假激酶 TRB2 在体外保留了弱结合和水解 ATP 的能力。激酶活性不依赖于金属，并且涉及催化赖氨酸残基，该残基在整个进化过程中与活性位点中的几个独特氨基酸一起在 TRB 蛋白中保守。密切相关的人类 TRB3 中也保留了类似的低水平自磷酸化。通过采用化学遗传学，我们确定“类似物敏感”（AS）TRB2突变体的核苷酸结合位点可以用吡唑并嘧啶（PP）化学型的特定大配体靶向。我们的分析证实，TRB2 保留了低水平的小分子可靶向的 ATP 结合和/或催化作用。鉴于用小分子抑制剂靶向癌症相关激酶相关的重大临床成功，类似的方法可能有助于进一步评估 TRB 假激酶，而这些信息的转化可能为药物发现提供新的线索。
The human Tribbles (TRB)-related pseudokinases are CAMK (calcium/calmodulin-dependent protein kinase)-related family members that have evolved a series of highly unusual motifs in the ‘pseudocatalytic’ domain. In canonical kinases, conserved amino acids bind to divalent metal ions and align ATP prior to efficient phosphoryl-transfer to substrates. However, in pseudokinases, atypical residues give rise to diverse and often unstudied biochemical and structural features that are thought to be central to cellular functions. TRB proteins play a crucial role in multiple signalling networks and overexpression confers cancer phenotypes on human cells, marking TRB pseudokinases out as a novel class of drug target. In the present paper, we report that the human pseudokinase TRB2 retains the ability to both bind and hydrolyse ATP weakly in vitro. Kinase activity is metal-independent and involves a catalytic lysine residue, which is conserved in TRB proteins throughout evolution alongside several unique amino acids in the active site. A similar low level of autophosphorylation is also preserved in the closely related human TRB3. By employing chemical genetics, we establish that the nucleotide-binding site of an ‘analogue-sensitive’ (AS) TRB2 mutant can be targeted with specific bulky ligands of the pyrazolo-pyrimidine (PP) chemotype. Our analysis confirms that TRB2 retains low levels of ATP binding and/or catalysis that is targetable with small molecules. Given the significant clinical successes associated with targeting of cancer-associated kinases with small molecule inhibitors, it is likely that similar approaches will be useful for further evaluating the TRB pseudokinases, with the translation of this information likely to furnish new leads for drug discovery.