Wagner P D, Vu N D
Laboratory of Biochemistry, NCI, National Institutes of Health, Bethesda, Maryland 20892-4255, USA.
J Biol Chem. 1995 Sep 15;270(37):21758-64. doi: 10.1074/jbc.270.37.21758.
Rat liver nucleoside diphosphate kinase (NDPK) and PC12 cell cytosol were used to determine whether NDPK could function as a protein kinase. NDPK was phosphorylated on its catalytic histidine using [gamma-32P]ATP, and the phosphorylated NDPK separated from [gamma-32P]ATP. The addition of phosphorylated NDPK to dialyzed PC12 cell cytosol resulted in the phosphorylation of a protein with a subunit molecular mass of about 120 kDa. This phosphorylation appeared to occur by a direct transfer of a phosphoryl group from the catalytic histidine of NDPK to a histidine on the 120-kDa protein. The 120-kDa protein was partially purified and shown by peptide sequencing to be ATP-citrate lyase. ATP-citrate lyase is the primary source of cytosolic acetyl-CoA. NDPK phosphorylated the histidine at the catalytic site of ATP-citrate lyase. This histidine can also be phosphorylated by ATP, and its phosphorylation is the first step in the conversion of citrate and CoA to oxaloacetate and acetyl-CoA by ATP-citrate lyase. The level of phosphorylation of PC12 cell ATP-citrate lyase by phosphorylated NDPK was comparable with that by ATP. Thus, in addition to its nucleoside diphosphate kinase activity, NDPK can function as a protein kinase.
使用大鼠肝脏核苷二磷酸激酶（NDPK）和PC12细胞胞质溶胶来确定NDPK是否可以作为蛋白激酶发挥作用。使用[γ-32P]ATP使NDPK的催化组氨酸磷酸化，并将磷酸化的NDPK与[γ-32P]ATP分离。向透析后的PC12细胞胞质溶胶中添加磷酸化的NDPK导致一种亚基分子量约为120 kDa的蛋白质发生磷酸化。这种磷酸化似乎是通过磷酸基团从NDPK的催化组氨酸直接转移到120 kDa蛋白质上的组氨酸而发生的。对120 kDa的蛋白质进行了部分纯化，并通过肽测序表明其为ATP-柠檬酸裂解酶。ATP-柠檬酸裂解酶是胞质乙酰辅酶A的主要来源。NDPK使ATP-柠檬酸裂解酶催化位点的组氨酸磷酸化。该组氨酸也可以被ATP磷酸化，并且其磷酸化是ATP-柠檬酸裂解酶将柠檬酸和辅酶A转化为草酰乙酸和乙酰辅酶A的第一步。磷酸化的NDPK对PC12细胞ATP-柠檬酸裂解酶的磷酸化水平与ATP相当。因此，除了其核苷二磷酸激酶活性外，NDPK还可以作为蛋白激酶发挥作用。
