Feschenko M S, Sweadner K J
Laboratory of Membrane Biology, Massachusetts General Hospital, Charlestown 02129, USA.
J Biol Chem. 1995 Jun 9;270(23):14072-7. doi: 10.1074/jbc.270.23.14072.
There is considerable evidence that protein kinases play a role in regulation of the activity of the Na,K-ATPase, but the characteristics of direct kinase phosphorylation of Na,K-ATPase subunits are still not well understood. There are 36 sites that could qualify as protein kinase C motifs in rat alpha 1. Here we have used protein fragmentation with trypsin to localize the site of phosphorylation of the rat Na,K-ATPase alpha 1 subunit to within the first 32 amino acids of the N terminus and then used direct sequencing of the phosphorylated protein to determine which of two candidate serine residues was modified. The result was that at most 25% of the 32P was found on Ser-11, a site that is well conserved in Na,K-ATPase alpha 1 subunits. The remaining 75% or more of the 32P was found on Ser-18, a site that is absent in many Na,K-ATPase alpha subunit sequences. This accounts for the observation that dog and pig alpha 1 subunits can be phosphorylated by protein kinase C only to much lower levels than can rat alpha 1. It is also likely to be relevant to other known species-specific effects of protein kinase C on Na,K-ATPase.
有大量证据表明蛋白激酶在钠钾 - ATP酶活性调节中发挥作用，但钠钾 - ATP酶亚基直接激酶磷酸化的特征仍未得到很好的理解。在大鼠α1亚基中有36个位点可能符合蛋白激酶C基序。在这里，我们使用胰蛋白酶进行蛋白片段化，将大鼠钠钾 - ATP酶α1亚基的磷酸化位点定位在N端的前32个氨基酸内，然后对磷酸化蛋白进行直接测序，以确定两个候选丝氨酸残基中哪一个被修饰。结果是，在Ser - 11上发现的32P最多占25%，该位点在钠钾 - ATP酶α1亚基中高度保守。其余75%或更多的32P在Ser - 18上被发现，该位点在许多钠钾 - ATP酶α亚基序列中不存在。这就解释了为什么犬和猪的α1亚基被蛋白激酶C磷酸化的水平比大鼠α1亚基低得多。这也可能与蛋白激酶C对钠钾 - ATP酶的其他已知物种特异性效应有关。
