Differential effects of substrate on type I and type II PKA holoenzyme dissociation.
Differential effects of substrate on type I and type II PKA holoenzyme dissociation.
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DOI：
10.1021/bi0499157
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发表时间：
2004-04
期刊：
影响因子：
2.9
通讯作者：
D. Vigil;D. Blumenthal;Simon H. J. Brown;Susan S. Taylor;J. Trewhella
中科院分区：
文献类型：
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作者：
D. Vigil;D. Blumenthal;Simon H. J. Brown;Susan S. Taylor;J. Trewhella
cAMP激活蛋白激酶A（PKA）全酶是通过解离调节亚基和催化亚基，从而释放催化亚基使其靶点磷酸化而实现的。然而，最近的实验表明，cAMP不能完全解离全酶。在这里，我们进一步研究这一机制，通过使用小角X-射线散射研究，在生理酶浓度下，在平衡溶液条件下，没有任何标记的蛋白质亚基的Ialpha和II beta型全酶结构。我们观察到，虽然加入摩尔过量的cAMP的I型α PKA全酶引起部分解离，它是只有在加入PKA肽底物与cAMP，完全解离发生。类似地，向II型β全酶中加入过量的cAMP仅引起部分解离。然而，虽然添加肽底物以及过量的cAMP会导致更多的解离，但仍有相当大比例的完整II β型全酶。这些结果证实，在cAMP存在下，I α型和II β型全酶比以前认为的更稳定。他们还表明，底物在I型与II型全酶的激活中起着不同的作用，这可以解释PKA亚型之间的一些重要功能差异。在这些数据和其他最近发表的数据的基础上，我们提出了一个结构模型的I型全酶激活cAMP。
It has been widely accepted that cAMP activates the protein kinase A (PKA) holoenzyme by dissociating the regulatory and catalytic subunits, thus freeing the catalytic subunit to phosphorylate its targets. However, recent experiments suggest that cAMP does not fully dissociate the holoenzyme. Here, we investigate this mechanism further by using small-angle X-ray scattering to study, at physiological enzyme concentrations, the type Ialpha and type IIbeta holoenzyme structures under equilibrium solution conditions without any labeling of the protein subunits. We observe that while the addition of a molar excess of cAMP to the type Ialpha PKA holoenzyme causes partial dissociation, it is only upon addition of a PKA peptide substrate together with cAMP that full dissociation occurs. Similarly, addition of excess cAMP to the type IIbeta holoenzyme causes only a partial dissociation. However, while the addition of peptide substrate as well as excess cAMP causes somewhat more dissociation, a significant percentage of intact type IIbeta holoenzyme remains. These results confirm that both the type Ialpha and the type IIbeta holoenzymes are more stable in the presence of cAMP than previously thought. They also demonstrate that substrate plays a differential role in the activation of type I versus type II holoenzymes, which could explain some important functional differences between PKA isoforms. On the basis of these data and other recently published data, we propose a structural model of type I holoenzyme activation by cAMP.