PHOSPHORYLATION OF CALMODULIN BY THE CATALYTIC SUBUNIT OF CASEIN KINASE-II IS INHIBITED BY THE REGULATORY SUBUNIT
PHOSPHORYLATION OF CALMODULIN BY THE CATALYTIC SUBUNIT OF CASEIN KINASE-II IS INHIBITED BY THE REGULATORY SUBUNIT
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DOI：
10.1006/abbi.1993.1037
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发表时间：
1993-01-01
影响因子：
3.9
通讯作者：
GLOVER, CVC
中科院分区：
文献类型：
--
作者：
BIDWAI, AP;REED, JC;GLOVER, CVC
酪蛋白激酶 II (CKII) 由催化亚基 (α) 和调节亚基 (β) 组成，二者结合形成 α2β2 全酶。 α-亚基单体具有酶活性，尽管相对于全酶在动力学上减弱，并且纯化的 β 亚基的添加刺激其针对酪蛋白的活性（C. Cochet 和 E. M. Chambaz, 1983, J. Biol. Chem.258, 1403-1406）。在这里，我们报告了果蝇CKII的α亚基和霍尔酶对各种蛋白质和肽底物的磷酸化的动力学分析。我们证明，α 亚基与全酶一样，能够磷酸化典型的生理底物，例如 cAMP 依赖性蛋白激酶 (cAMPdPK) 的调节 (Rh) 亚基，以及人工底物，例如 α-酪蛋白和合成肽 RRREEETEEE。在每种情况下，α亚基的 Km 与全酶相似，而 Vmax 低 5 至 60 倍。相反，钙调蛋白是一种仅在多元化合物存在下才被全酶显着磷酸化的蛋白质，很容易被单独的α亚基磷酸化。虽然钙调蛋白α亚基和全酶的Km值相似，但α亚基的Vmax至少比全酶高10倍。这些结果表明，虽然 α 亚基包含 CKII 底物特异性所需的决定因素，但 β 亚基可以以底物依赖性方式抑制或激活它。最后，我们还证明多元化合物不仅刺激全酶，而且在较小程度上刺激α亚基。
Casein kinase II (CKII) is composed of a catalytic subunit (α) and a regulatory subunit (β) that combine to form an α2β2holoenzyme. The α-subunit monomer is enzymatically active, albeit kinetically attenuated relative to the holoenzyme, and the addition of purified β subunit stimulates its activity against casein (C. Cochet and E. M. Chambaz, 1983,J. Biol. Chem.258, 1403-1406). Here we report a kinetic analysis of the phosphorylation of various protein and peptide substrates by the a subunit and the holneazyme ofDrosophila melanogasterCKII. We demonstrate that the α subunit, like the holoenzyme, is competent to phosphorylate typical physiological substrates such as the regulatory (Rh) subunit of cAMP-dependent protein kinase (cAMPdPK), as well as artificial substrates such as α-casein and the synthetic peptide RRREEETEEE. TheKmof the α subunit in each case is similar to that of the holoenzyme, whereas theVmaxis 5- to 60-fold lower. In contrast, calmodulin, a protein that is significantly phosphorylated by the holoenzyme only in the presence of polybasic compounds, is readily phosphorylated by the α subunit alone. While theKmvalues of the α subunit and the holoenzyme for calmodulin are similar, theVmaxof the α subunit is at least 10-fold higher than that of the holoenzyme. These results suggest that while the α subunit contains the necessary determinants for CKII substrate specificity, the β subunit can either inhibit or activate it, in a substrate-dependent manner. Finally, we also demonstrate that polybasic compounds stimulate not only the holoenzyme but, to a lesser extent, the a subunit as well.