Mieskes G, Kuduz J, Söling H D
Eur J Biochem. 1987 Sep 1;167(2):383-9. doi: 10.1111/j.1432-1033.1987.tb13349.x.
Changes in glycolytic flux have been observed in liver under conditions where effects of cAMP seem unlikely. We have, therefore, studied the phosphorylation of four enzymes involved in the regulation of glycolysis and gluconeogenesis (6-phosphofructo-1-kinase from rat liver and rabbit muscle; pyruvate kinase, 6-phosphofructo-2-kinase and fructose-1,6-bisphosphatase from rat liver) by defined concentrations of two cAMP-independent protein kinases: Ca2+/calmodulin-dependent protein kinase and Ca2+/phospholipid-dependent protein kinase (protein kinase C). The results were compared with those obtained with the catalytic subunit of cAMP-dependent protein kinase. The following results were obtained. 1. Ca2+/calmodulin-dependent protein kinase phosphorylates 6-phosphofructo-1-kinase and L-type pyruvate kinase at a slightly lower rate as compared to cAMP-dependent protein kinase. 2. 6-Phosphofructo-1-kinase is phosphorylated by the two kinases at a single identical position. There is no additive phosphorylation. The final stoichiometry is 2 mol phosphate/mol tetramer. The same holds for L-type pyruvate kinase except that the stoichiometry with either kinase or both kinases together is 4 mol phosphate/mol tetramer. 3. Rabbit muscle 6-phosphofructo-1-kinase is phosphorylated by cAMP-dependent protein kinase but not by Ca2+/calmodulin-dependent protein kinase. 4. Fructose-1,6-bisphosphatase from rat but not from rabbit liver is phosphorylated at the same position but at a markedly lower rate by Ca2+/calmodulin-dependent protein kinase when compared to the phosphorylation by cAMP-dependent protein kinase. 5. 6-Phosphofructo-2-kinase is phosphorylated by Ca2+/calmodulin-dependent protein kinase only at a negligible rate. 6. Protein kinase C does not seem to be involved in the regulation of the enzymes examined: only 6-phosphofructo-2-kinase became phosphorylated to a significant degree. In contrast to the phosphorylation by cAMP-dependent protein kinase, this phosphorylation is not associated with a change of enzyme activity. This agrees with our observation that the sites of phosphorylation by the two kinases are different. The results indicate that Ca2+/calmodulin-dependent protein kinase but not protein kinase C could be involved in the regulation of hepatic glycolytic flux under conditions where changes in the activity of cAMP-dependent protein kinase seem unlikely.
在一些情况下，即使环磷酸腺苷（cAMP）似乎不太可能发挥作用，肝脏中的糖酵解通量仍会发生变化。因此，我们研究了两种不依赖cAMP的蛋白激酶（Ca²⁺/钙调蛋白依赖性蛋白激酶和Ca²⁺/磷脂依赖性蛋白激酶（蛋白激酶C））在特定浓度下对参与糖酵解和糖异生调节的四种酶（大鼠肝脏和兔肌肉中的6-磷酸果糖-1-激酶；大鼠肝脏中的丙酮酸激酶、6-磷酸果糖-2-激酶和果糖-1,6-二磷酸酶）的磷酸化作用。将结果与用cAMP依赖性蛋白激酶催化亚基获得的结果进行了比较。得到了以下结果。1.与cAMP依赖性蛋白激酶相比，Ca²⁺/钙调蛋白依赖性蛋白激酶对6-磷酸果糖-1-激酶和L型丙酮酸激酶的磷酸化速率略低。2.两种激酶在单个相同位置对6-磷酸果糖-1-激酶进行磷酸化。不存在累加性磷酸化。最终化学计量比为2摩尔磷酸/摩尔四聚体。L型丙酮酸激酶也是如此，只是无论单独一种激酶还是两种激酶一起作用时，化学计量比都是4摩尔磷酸/摩尔四聚体。3.兔肌肉中的6-磷酸果糖-1-激酶可被cAMP依赖性蛋白激酶磷酸化，但不能被Ca²⁺/钙调蛋白依赖性蛋白激酶磷酸化。4.大鼠肝脏而非兔肝脏中的果糖-1,6-二磷酸酶在相同位置被磷酸化，但与cAMP依赖性蛋白激酶的磷酸化相比，Ca²⁺/钙调蛋白依赖性蛋白激酶的磷酸化速率明显较低。5.6-磷酸果糖-2-激酶仅以可忽略不计的速率被Ca²⁺/钙调蛋白依赖性蛋白激酶磷酸化。6.蛋白激酶C似乎不参与所检测酶的调节：只有6-磷酸果糖-2-激酶被显著磷酸化。与cAMP依赖性蛋白激酶的磷酸化不同，这种磷酸化与酶活性的变化无关。这与我们观察到的两种激酶的磷酸化位点不同一致。结果表明，在cAMP依赖性蛋白激酶活性变化似乎不太可能的情况下，Ca²⁺/钙调蛋白依赖性蛋白激酶而非蛋白激酶C可能参与肝脏糖酵解通量的调节。
