Meggio F, Donella Deana A, Ruzzene M, Brunati A M, Cesaro L, Guerra B, Meyer T, Mett H, Fabbro D, Furet P
Dipartimento di Chimica Biologica, Università di Padova, Italy.
Eur J Biochem. 1995 Nov 15;234(1):317-22. doi: 10.1111/j.1432-1033.1995.317_c.x.
A systematic analysis reveals that out of 20 protein kinases examined, specific for either Ser/Thr or Tyr, the majority are extremely sensitive to staurosporine, with IC50 values in the low nanomolar range. A few of them however, notably protein kinases CK1 and CK2, mitogen-activated protein (MAP) kinase and protein-tyrosine kinase CSK, are relatively refractory to staurosporine inhibition, exhibiting IC50 values in the micromolar range. With all protein kinases tested, namely PKA, CK1, CK2, MAP kinase (ERK-1), c-Fgr, Lyn, CSK and TPK-IIB/p38Syk, staurosporine inhibition was competitive with respect to ATP, regardless of its inhibitory power. In contrast, either uncompetitive or noncompetitive kinetics of inhibition with respect to the phosphoacceptor substrate were exhibited by Ser/Thr and Tyr-specific protein kinases, respectively, consistent with a different mechanism of catalysis by these two sub-families of kinases. Computer modeling based on PKA crystal structure in conjunction with sequence analysis suggest that the low sensitivity to staurosporine of CK2 may be accounted for by the bulky nature of three residues, Val66, Phe113 and Ile174 which are homologous to PKA Ala70, Met120 and Thr183, respectively. In contrast these PKA residues are either conserved or replaced by smaller ones in protein kinases highly sensitive to staurosporine inhibition. On the other hand, His160 which is homologous to PKA Glu170, appears to be responsible for the unique behaviour of CK2 with respect to a staurosporine derivative (CGP44171A) bearing a negatively charged benzoyl substituent: while CGP44171A is 10- 100-fold less effective than staurosporine against PKA and most of the other protein kinases tested, it is actually more effective than staurosporine for CK2 inhibition, but it looses part of its efficacy if it is tested on a CK2 mutant (H160D) in which His160 has been replaced by Asp. It can be concluded from these data that the catalytic sites of protein kinases are divergent enough as to allow a competitive inhibitor like staurosporine to be fairly selective, a feature that can be enhanced by suitable modifications designed based on the structure of the catalytic site of the kinase.
一项系统分析表明，在所检测的20种特异性作用于丝氨酸/苏氨酸或酪氨酸的蛋白激酶中，大多数对星形孢菌素极为敏感，其半数抑制浓度（IC50）值处于低纳摩尔范围。然而，其中少数激酶，特别是酪蛋白激酶1（CK1）和酪蛋白激酶2（CK2）、丝裂原活化蛋白（MAP）激酶以及蛋白酪氨酸激酶CSK，对星形孢菌素抑制作用相对不敏感，其IC50值处于微摩尔范围。对于所有测试的蛋白激酶，即蛋白激酶A（PKA）、CK1、CK2、MAP激酶（细胞外信号调节激酶1，ERK-1）、原癌基因酪氨酸蛋白激酶c-Fgr、淋巴细胞特异性蛋白酪氨酸激酶Lyn、CSK以及酪氨酸蛋白激酶IIB/p38Syk，无论星形孢菌素的抑制能力如何，其抑制作用相对于ATP均具有竞争性。相比之下，丝氨酸/苏氨酸特异性和酪氨酸特异性蛋白激酶分别对磷酸受体底物表现出非竞争性或反竞争性抑制动力学，这与这两个激酶亚家族不同的催化机制一致。基于PKA晶体结构结合序列分析的计算机模拟表明，CK2对星形孢菌素低敏感性可能是由于三个残基（缬氨酸66、苯丙氨酸113和异亮氨酸174）体积较大所致，它们分别与PKA的丙氨酸70、甲硫氨酸120和苏氨酸183同源。相反，在对星形孢菌素抑制高度敏感的蛋白激酶中，这些PKA残基要么保守，要么被较小的残基取代。另一方面，与PKA的谷氨酸170同源的组氨酸160，似乎是CK2对于带有带负电荷苯甲酰取代基的星形孢菌素衍生物（CGP44171A）表现出独特行为的原因：虽然CGP44171A对PKA和大多数其他测试蛋白激酶的抑制效果比对星形孢菌素低10 - 100倍，但实际上它对CK2的抑制作用比对星形孢菌素更有效，但如果在组氨酸160被天冬氨酸取代的CK2突变体（H160D）上进行测试，其部分效能会丧失。从这些数据可以得出结论，蛋白激酶的催化位点差异足够大，以至于像星形孢菌素这样的竞争性抑制剂具有相当的选择性，基于激酶催化位点结构设计的适当修饰可以增强这一特性。
