三种Tyrphostin对重组人蛋白激酶CK2全酶的影响

Effects of 3 Tyrphostins on Recombinant Human Protein Kinase CK2 Holoenzyme

目的 :观察三种 Tyrphostins AG110 9、AG5 5 5和 AG1394对重组人蛋白激酶 CK2全酶的直接作用。方法 :利用基因工程克隆、表达和纯化获得重组人蛋白激酶 CK2α和β亚基 ,在体外等摩尔数混合构成有最大生物活性的重组 CK2全酶 ,在不同条件下测定 CK2的活性。 CK2活性通过测定转移到 CK2底物上的 [γ-3 2 P] ATP或 [γ-3 2 P] GTP的 [3 2 P]放射活度来检测。结果 :重组人 CK2是一种 Ca2 +、c AMP和 c GMP等第二信使非依赖性蛋白激酶 ,与天然 CK2的性质一致。 AG110 9对重组人CK2全酶具有较强的抑制作用 ,IC50 为 9.7μmol/L ,抑制作用稍大于已知 CK2抑制剂 DRB和 A3。 AG5 5 5对重组人 CK2全酶具有一定的抑制作用 ,而 AG1394则对人 CK2全酶基本无影响。 AG110 9对重组人 CK2的动力学研究表明 :它与 GTP呈现以竞争性为主的混合型抑制作用 ,与酪蛋白呈非竞争性抑制作用。结论 :AG110 9不仅是酪氨酸蛋白激酶的抑制剂 ,而且是一种十分有效的蛋白激酶 CK2的抑制剂。重组人蛋白激酶 CK2可作为一种较为简便筛选和开发有效的 CK2抑制剂的分子靶点。

AIM The purpose is to study the direct effects of tyrphostins AG1109, AG555 and AG1394 on recombinant human protein kinase CK2 holoenzyme and its kinetics. METHODS Recombinant human protein kinase CK2 α and β subunits were cloned and expressed by gene engineering, and purified to being homogeneous. The two subunits were mixed at the same molar ratio and reconstituted CK2 holoenzyme, which displayed the maximum biological activity. The CK2 activity was assayed by detecting incorporation of 32 P of [γ- 32 P]ATP or [γ- 32 P]GTP into the substrate under the various conditions. RESULTS The recombinant human CK2 was a second messenger (Ca 2+ , cAMP and cGMP) independent protein kinase, the characterization and function of the reconstituted holoenzyme were consistent with those of native CK2. AG1109 strongly inhibited the holoenzyme activity of recombinant human protein kinase CK2 with an IC 50 of 9.7 μmol/L, which was more slightly by effective than DRB and A3, known CK2 special inhibitors. AG555 had a little inhibitory effect on recombinant human CK2 holoenzyme, while AG1394 had no suppressive effect on this enzyme. Kinetic studies of AG1109 on recombinant human CK2 showed that the inhibition was mixed (competitive is dominant) with GTP and noncompetitive with casein. CONCLUSIONS AG1109 not only was an effective inhibitor of protein tyrosine kinases, but also was a novel potent inhibitor of protein kinase CK2. The recombinant human protein kinase CK2 may be used as a molecular target for simpler screening and development of more effective inhibitors of CK2.