Cornelius Flemming, Mahmmoud Yasser A
Department of Biophysics, University of Aarhus, Denmark.
Biophys J. 2003 Mar;84(3):1690-700. doi: 10.1016/S0006-3495(03)74977-7.
In this study we compared the protein kinase dependent regulation of gastric H,K-ATPase and Na,K-ATPase. The protein kinase A/protein kinase C (PKA/PKC) phosphorylation profile of H,K-ATPase was very similar to the one found in the Na,K-ATPase. PKC phosphorylation was taking place in the N-terminal part of the alpha-subunit with a stoichiometry of approximately 0.6 mol Pi/mole alpha-subunit. PKA phosphorylation was in the C-terminal part and required detergent, as is also found for the Na,K-ATPase. The stoichiometry of PKA-induced phosphorylation was approximately 0.7 mol Pi/mole alpha-subunit. Controlled proteolysis of the N-terminus abolished PKC phosphorylation of native H,K-ATPase. However, after detergent treatment additional C-terminal PKC sites became exposed located at the beginning of the M5M6 hairpin and at the cytoplasmic L89 loop close to the inner face of the plasma membrane. N-terminal PKC phosphorylation of native H,K-ATPase alpha-subunit was found to stimulate the maximal enzyme activity by 40-80% at saturating ATP, depending on pH. Thus, a direct modulation of enzyme activity by PKC phosphorylation could be demonstrated that may be additional to the well-known regulation of acid secretion by recruitment of H,K-ATPase to the apical membranes of the parietal cells. Moreover, a distinct difference in the regulation of H,K-ATPase and Na,K-ATPase is the apparent absence of any small regulatory proteins associated with the H,K-ATPase.
在本研究中，我们比较了蛋白激酶对胃H⁺，K⁺-ATP酶和Na⁺，K⁺-ATP酶的依赖性调节。H⁺，K⁺-ATP酶的蛋白激酶A/蛋白激酶C（PKA/PKC）磷酸化谱与在Na⁺，K⁺-ATP酶中发现的非常相似。PKC磷酸化发生在α亚基的N端部分，化学计量比约为0.6摩尔磷酸根/摩尔α亚基。PKA磷酸化发生在C端部分，并且需要去污剂，这在Na⁺，K⁺-ATP酶中也有发现。PKA诱导的磷酸化化学计量比约为0.7摩尔磷酸根/摩尔α亚基。对N端进行可控的蛋白水解消除了天然H⁺，K⁺-ATP酶的PKC磷酸化。然而，在去污剂处理后，额外的C端PKC位点暴露出来，位于M5M6发夹结构的起始处以及靠近质膜内表面的细胞质L89环处。发现天然H⁺，K⁺-ATP酶α亚基的N端PKC磷酸化在ATP饱和时可将最大酶活性提高40 - 80%，具体取决于pH值。因此，可以证明PKC磷酸化对酶活性有直接调节作用，这可能是壁细胞顶端膜募集H⁺，K⁺-ATP酶从而调节胃酸分泌之外的另一种调节方式。此外，H⁺，K⁺-ATP酶和Na⁺，K⁺-ATP酶调节的一个明显差异是，H⁺，K⁺-ATP酶明显没有任何与之相关的小调节蛋白。
