PHYSIOLOGICAL INHIBITORS OF THE CATALYTIC SUBUNIT OF CAMP-DEPENDENT PROTEIN-KINASE - EFFECT OF MGATP ON PROTEIN-PROTEIN INTERACTIONS
PHYSIOLOGICAL INHIBITORS OF THE CATALYTIC SUBUNIT OF CAMP-DEPENDENT PROTEIN-KINASE - EFFECT OF MGATP ON PROTEIN-PROTEIN INTERACTIONS
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DOI：
10.1021/bi00213a035
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发表时间：
1993-12-21
期刊：
影响因子：
2.9
通讯作者：
TAYLOR, SS
中科院分区：
文献类型：
--
作者：
HERBERG, FW;TAYLOR, SS
cAMP依赖性蛋白激酶的催化（C）亚基与两类抑制剂相互作用。调节（R）亚单位，I型和II型，结合形成一个无活性的全酶复合物，该复合物响应cAMP被激活。C亚基也被小的热稳定蛋白激酶抑制剂（PKI）抑制。PKI和R（I）-亚基的抑制需要MgATP的协同高亲和力结合。ATP的稳定作用通过使用分析凝胶色谱法定量。在MgATP存在下，I型全酶和C.PKI复合物均显示亚基缔合的表观K（d）低于0.1 nM，而在MgATP不存在下，两种复合物的表观K（d）分别为125 nM和2.3 μ M。在没有MgATP的情况下，这两种复合物也可以容易地解离，因此，通过盐诱导的解离活化。在生理盐浓度下，盐诱导的解离在MgATP的高亲和力结合的情况下将是实质性的。在这两种复合物中，游离C亚基的ATP酶活性被消除。MgATP的解离速率也表明I型全酶比C-PKI复合物更稳定。MgATP从C.PKI复合物的解离速率（t1/2）为17 min，而I型全酶的解离速率为11.7 h。当C-PKI复合物与R（I）-亚基在MgATP存在或不存在下孵育时，C-亚基优先与R（I）-亚基重新结合，形成全酶。 相比之下，游离的PKI不能竞争的C-亚基时，它是一个全酶复合物的一部分。这些体外结果表明，C.PKI复合物的形成可能不是死端途径。相反，PKI可以作为一种穿梭机制，最终将C亚基返回全酶复合物。
The catalytic (C) subunit of cAMP-dependent protein kinase interacts with two classes of inhibitors. The regulatory (R) subunits, types I and II, associate to form an inactive holoenzyme complex that is activated in response to cAMP. The C-subunit is also inhibited by small heat-stable protein kinase inhibitors (PKI's). Inhibition by both PKI and R(I)-subunit requires the synergistic high-affinity binding of MgATP. The stabilizing effect of ATP was quantitated by using analytical gel chromatography. Both the type I holoenzyme and the C.PKI complex in the presence of MgATP show apparent K(d)'s for subunit association that are below 0.1 nM, while in the absence of MgATP the apparent K(d)'s are 125 nM and 2.3 muM, respectively, for the two complexes. In the absence of MgATP both complexes also can be dissociated readily and, hence, activated by salt-induced dissociation. Under physiological salt concentrations, salt-induced dissociation would be substantial in the absence of the high-affinity binding of MgATP. In both complexes, the ATPase activity of the free C-subunit is abolished. The off rates for MgATP also indicate that the type I holoenzyme is more stable than the C-PKI complex. The off rate (t1/2) for MgATP from the C.PKI complex is 17 min, while the off rate for the type I holoenzyme is 11.7 h. When the C-PKI complex is incubated with R(I)-subunit in the presence or absence of MgATP, the C-subunit preferentially reassociates with the R(I)-subunit, forming holoenzyme. In contrast, free PKI cannot compete for the C-subunit when it is part of a holoenzyme complex. These in vitro results suggest that the formation of the C.PKI complex may not be a dead-end pathway. Instead, PKI could function as a shuttle mechanism for eventually returning the C-subunit to a holoenzyme complex.