Kraybill Brian C, Elkin Lisa L, Blethrow Justin D, Morgan David O, Shokat Kevan M
Department of Cellular and Molecular Pharmacology, Box 0450, University of California- San Francisco, San Francisco, California 94143, USA.
J Am Chem Soc. 2002 Oct 16;124(41):12118-28. doi: 10.1021/ja0264798.
The elucidation of protein kinase signaling networks is challenging due to the large size of the protein kinase superfamily (>500 human kinases). Here we describe a new class of orthogonal triphosphate substrate analogues for the direct labeling of analogue-specific kinase protein targets. These analogues were constructed as derivatives of the Src family kinase inhibitor PP1 and were designed based on the crystal structures of PP1 bound to HCK and N(6)-(benzyl)-ADP bound to c-Src (T338G). 3-Benzylpyrazolopyrimidine triphosphate (3-benzyl-PPTP) proved to be a substrate for a mutant of the MAP kinase p38 (p38-T106G/A157L/L167A). 3-Benzyl-PPTP was preferred by v-Src (T338G) (k(cat)/K(M) = 3.2 x 10(6) min(-)(1) M(-)(1)) over ATP or the previously described ATP analogue, N(6) (benzyl) ATP. For the kinase CDK2 (F80G)/cyclin E, 3-benzyl-PPTP demonstrated catalytic efficiency (k(cat)/K(M) = 2.6 x 10(4) min(-)(1) M(-)(1)) comparable to ATP (k(cat)/K(M) = 5.0 x 10(4) min(-)(1) M(-)(1)) largely due to a significantly better K(M) (6.4 microM vs 530 microM). In kinase protein substrate labeling experiments both 3-benzyl-PPTP and 3-phenyl-PPTP prove to be over 4 times more orthogonal than N(6)-(benzyl)-ATP with respect to the wild-type kinases found in murine spleenocyte cell lysates. These experiments also demonstrate that [gamma-(32)P]-3-benzyl-PPTP is an excellent phosphodonor for labeling the direct protein substrates of CDK2 (F80G)/E in murine spleenocyte cell lysates, even while competing with cellular levels (4 mM) of unlabeled ATP. The fact that this new more highly orthogonal nucleotide is accepted by three widely divergent kinases studied here suggests that it is likely to be generalizable across the entire kinase superfamily.
由于蛋白激酶超家族规模庞大（人类激酶超过500种），阐明蛋白激酶信号网络颇具挑战性。在此，我们描述了一类新型的正交三磷酸底物类似物，用于直接标记类似物特异性激酶蛋白靶点。这些类似物构建为Src家族激酶抑制剂PP1的衍生物，并基于与HCK结合的PP1以及与c-Src（T338G）结合的N(6)-(苄基)-ADP的晶体结构进行设计。3-苄基吡唑并嘧啶三磷酸（3-苄基-PPTP）被证明是丝裂原活化蛋白激酶p38（p38-T106G/A157L/L167A）突变体的一种底物。相较于ATP或先前描述的ATP类似物N(6)（苄基）ATP，v-Src（T338G）对3-苄基-PPTP表现出更高的偏好性（催化常数/米氏常数=3.2×10⁶ min⁻¹ M⁻¹）。对于激酶CDK2（F80G）/细胞周期蛋白E，3-苄基-PPTP表现出与ATP相当的催化效率（催化常数/米氏常数=2.6×10⁴ min⁻¹ M⁻¹，而ATP的催化常数/米氏常数=5.0×10⁴ min⁻¹ M⁻¹），这主要归因于其显著更低的米氏常数（6.4微摩尔/升对530微摩尔/升）。在激酶蛋白底物标记实验中，就小鼠脾细胞裂解物中发现的野生型激酶而言，3-苄基-PPTP和3-苯基-PPTP的正交性均比N(6)-(苄基)-ATP高出4倍以上。这些实验还表明，[γ-(³²)P]-3-苄基-PPTP是在小鼠脾细胞裂解物中标记CDK2（F80G）/E直接蛋白底物的优良磷供体，即便与细胞内未标记ATP的水平（4毫摩尔/升）存在竞争。本文研究的三种差异很大的激酶都能接受这种新型的、正交性更高的核苷酸，这一事实表明它可能适用于整个激酶超家族。
