Phosphorylation of L-type pyruvate kinase by a Ca2+/calmodulin-dependent protein kinase.
Phosphorylation of L-type pyruvate kinase by a Ca2+/calmodulin-dependent protein kinase.
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Ca2/钙调蛋白依赖性蛋白激酶对 L 型丙酮酸激酶进行磷酸化。
DOI：
10.1016/s0021-9258(17)38831-2
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发表时间：
1985
期刊：
影响因子：
--
通讯作者：
T. Soderling
中科院分区：
文献类型：
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作者：
C. Schworer;M. El‐Maghrabi;S. Pilkis;T. Soderling
大鼠肝脏l型丙酮酸激酶被纯化自兔肝的Ca2+/钙调素依赖性蛋白激酶体外磷酸化。钙调蛋白（CaM）依赖性激酶催化了高达1.7 mol 32P/mol的丙酮酸激酶亚基的结合；最大磷酸化与p -烯醇丙酮酸的K0.5增加3.0倍有关。这与camp依赖性蛋白激酶催化的0.7至1.0 mol 32P/mol的掺入相比，p -烯醇丙酮酸的K0.5增加了2倍。当被cam依赖性蛋白激酶磷酸化的[32P]丙酮酸激酶随后与5 mM ADP和camp依赖性蛋白激酶（激酶逆转条件）孵育时，从丙酮酸激酶中去除了50-60%的32PO4，但p -烯醇丙酮酸的K0.5仅下降了20-30%。经部分酸水解鉴定的32p氨基酸表明，cam依赖性蛋白激酶磷酸化苏氨酸基和丝氨酸基残基（比例分别为1:2），而camp依赖性蛋白激酶仅磷酸化丝氨酸基。这两个磷酸化位点存在于位于酶亚基氨基末端的相同3-4-kDa CNBr片段中。这些结果表明，cam依赖性蛋白激酶在两个离散位点催化l型丙酮酸激酶的磷酸化。其中一个位点显然是被camp依赖性蛋白激酶磷酸化的同一丝氨酸。第二个位点是一个独特的苏氨酸残基，其磷酸化也通过提高对烯醇丙酮酸的K0.5而使丙酮酸激酶失活。这些结果可能解释了在分离的肝细胞中观察到的丙酮酸激酶的Ca2+依赖性磷酸化。
Rat liver L-type pyruvate kinase was phosphorylated in vitro by a Ca2+/calmodulin-dependent protein kinase purified from rabbit liver. The calmodulin (CaM)-dependent kinase catalyzed incorporation of up to 1.7 mol of 32P/mol of pyruvate kinase subunit; maximum phosphorylation was associated with a 3.0-fold increase in the K0.5 for P-enolpyruvate. This compares to incorporation of 0.7 to 1.0 mol of 32P/mol catalyzed by the cAMP-dependent protein kinase with a 2-fold increase in K0.5 for P-enolpyruvate. When [32P]pyruvate kinase, phosphorylated by the CaM-dependent protein kinase, was subsequently incubated with 5 mM ADP and cAMP-dependent protein kinase (kinase reversal conditions), 50-60% of the 32PO4 was removed from pyruvate kinase, but the K0.5 for P-enolpyruvate decreased only 20-30%. Identification of 32P-amino acids after partial acid hydrolysis showed that the CaM-dependent protein kinase phosphorylated both threonyl and seryl residues (ratio of 1:2, respectively) whereas the cAMP-dependent protein kinase phosphorylated only seryl groups. The two phosphorylation sites were present in the same 3-4-kDa CNBr fragment located near the amino terminus of the enzyme subunit. These results indicate that the CaM-dependent protein kinase catalyzed phosphorylation of L-type pyruvate kinase at two discrete sites. One site is apparently the same serine which is phosphorylated by the cAMP-dependent protein kinase. The second site is a unique threonine residue whose phosphorylation also inactivates pyruvate kinase by elevating the K0.5 for P-enolpyruvate. These results may account for the Ca2+-dependent phosphorylation of pyruvate kinase observed in isolated hepatocytes.