Liu Y, Shah K, Yang F, Witucki L, Shokat K M
Department of Chemistry, Princeton University, NJ 08544, USA.
Chem Biol. 1998 Feb;5(2):91-101. doi: 10.1016/s1074-5521(98)90143-0.
Protein kinases play a central role in controlling diverse signal transduction pathways in all cells. The identification of the direct cellular substrates of individual protein kinases remains the key challenge in the field.
We describe the protein engineering of v-Src to produce a kinase which preferentially uses an ATP analog, N6-(benzyl) ATP, as a substrate, rather than the natural v-Src substrate, ATP. The sidechain of a single residue (Ile338) controls specificity for N6-substituted ATP analogs in the binding pocket of v-Src. Elimination of this sidechain by mutation to glycine produces a v-Src kinase which preferentially utilizes N6-(benzyl) ATP as a phosphodonor substrate. Our engineering strategy is generally applicable to the Src family kinases: mutation of the corresponding residue (Thr339 to glycine) in the Fyn kinase confers specificity for N6-(benzyl) ATP on Fyn.
The v-Src tyrosine kinase has been engineered to exhibit specificity for an unnatural ATP analog, N6-(benzyl) ATP, even in a cellular context where high concentrations of natural ATP are present (1-5 mM), where preferential use of the ATP analog by the mutant kinase is essential. The mutant v-Src transfers phosphate more efficiently with the designed unnatural analog than with ATP. As the identical mutation in the Src-family kinase Fyn confers on Fyn the ability to recognize the same unnatural ATP analog, our strategy is likely to be generally applicable to other protein kinases and may help to identify the direct targets of specific kinases.
蛋白激酶在控制所有细胞中的多种信号转导途径中发挥核心作用。确定单个蛋白激酶的直接细胞底物仍然是该领域的关键挑战。
我们描述了v-Src的蛋白质工程改造，以产生一种激酶，该激酶优先使用ATP类似物N6-（苄基）ATP作为底物，而不是天然的v-Src底物ATP。单个残基（Ile338）的侧链控制v-Src结合口袋中对N6-取代的ATP类似物的特异性。通过突变为甘氨酸消除该侧链会产生一种v-Src激酶，其优先利用N6-（苄基）ATP作为磷酸供体底物。我们的工程策略通常适用于Src家族激酶：Fyn激酶中相应残基（Thr339突变为甘氨酸）的突变赋予Fyn对N6-（苄基）ATP的特异性。
v-Src酪氨酸激酶经过工程改造后，即使在存在高浓度天然ATP（1-5 mM）的细胞环境中，也能对非天然ATP类似物N6-（苄基）ATP表现出特异性，在这种环境中突变激酶优先使用该ATP类似物至关重要。与ATP相比，突变的v-Src与设计的非天然类似物转移磷酸的效率更高。由于Src家族激酶Fyn中的相同突变赋予Fyn识别相同非天然ATP类似物的能力，我们的策略可能普遍适用于其他蛋白激酶，并可能有助于确定特定激酶的直接靶点。
