Jefferson A B, Majerus P W
Washington University School of Medicine, St. Louis, Missouri 63110, USA.
J Biol Chem. 1995 Apr 21;270(16):9370-7. doi: 10.1074/jbc.270.16.9370.
We have isolated additional cDNA clones encoding type II inositol polyphosphate 5-phosphatase (5-phosphatase II) resulting in a combined cDNA of 3076 nucleotides encoding a protein of 942 amino acids. The 5-phosphatase II hydrolyzed both Ins(1,4,5)P3 to Ins(1,4)P2 and the phospholipid PtdIns(4,5)P2 to PtdIns(4)P both in vitro and in vivo. There are two motifs highly conserved between types I and II 5-phosphatase and several other proteins presumed to be inositol phosphatases suggesting a possible role in catalysis. The type II 5-phosphatase also contains homology to several GTPase activating proteins although no such activity for 5-phosphatase II was found. The predicted protein ends with the sequence CNPL, suggesting that it is isoprenylated as a mechanism for membrane attachment. We found evidence for isoprenylation by demonstrating incorporation of [3H]mevalonate into native but not C939S mutant 5-phosphatase II expressed in Sf9 insect cells. Furthermore, we showed that membrane localization and the activity of 5-phosphatase II toward its lipid substrate PtdIns(4,5)P2 is reduced by eliminating 5-phosphatase II isoprenylation in the mutant C939S relative to the native enzyme.
我们分离得到了额外的编码II型肌醇多磷酸5-磷酸酶（5-磷酸酶II）的cDNA克隆，得到了一个3076个核苷酸的复合cDNA，其编码一个含942个氨基酸的蛋白质。5-磷酸酶II在体外和体内均可将Ins(1,4,5)P3水解为Ins(1,4)P2，并将磷脂PtdIns(4,5)P2水解为PtdIns(4)P。I型和II型5-磷酸酶之间有两个高度保守的基序，还有其他几种被认为是肌醇磷酸酶的蛋白质，这表明它们在催化过程中可能发挥作用。II型5-磷酸酶与几种GTP酶激活蛋白也存在同源性，尽管未发现5-磷酸酶II有此类活性。预测的蛋白质序列以CNPL结尾，表明它通过异戊二烯化作用附着于细胞膜。我们通过证明[3H]甲羟戊酸掺入在Sf9昆虫细胞中表达的天然5-磷酸酶II而非C939S突变体中，找到了异戊二烯化的证据。此外，我们还表明，相对于天然酶，通过消除突变体C939S中的5-磷酸酶II异戊二烯化作用，5-磷酸酶II对其脂质底物PtdIns(4,5)P2的膜定位和活性降低。
