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1-Phosphatidylinositol 4-Phosphate 5-Kinase (EC 2.7.1.68): A Proliferation- and Malignancy-linked Signal Transduction Enzyme¹

Laboratory for Experimental Oncology, Indiana University School of Medicine, Indianapolis, Indiana 46202-5200

The activity of PIP kinase (1-phosphatidylinositol 4-phosphate 5-kinase; EC 2.7.1.68), the second ATP-utilizing enzyme of 1,4,5-trisphosphate and diacylglycerol biosynthesis, was determined in the rat in a spectrum of transplantable solid hepatomas of different growth rates and in normal tissues of high and low cell renewal rates. In a standard isotopic method developed for the assay, the enzyme activity was linear with time for 4 min and proportional with protein concentration over a range of 0.05 to 1 mg per 0.135-ml reaction mixture. The apparent K_m for the substrate PIP (phosphatidylinositol 4-phosphate) and for ATP and Mg²⁺ in normal liver were 0.06, 0.5, and 4.2 mM, respectively, and in rapidly growing hepatoma 3924A, 0.08, 0.7, and 7.1 mM. The kinase activity in adult Wistar rat liver was 0.046 ± 0.003 nmol/h/mg protein. In hepatomas of slow and intermediate growth rates, PIP kinase activity increased 3.3–9.7-fold, and in hepatoma 3924A, it was elevated 45-fold over that of normal liver. When hepatoma 3924A cells were plated and expressed their proliferative program, enzyme activity increased 4.3-fold in mid-log phase. To further clarify the linkage between PIP kinase activity and proliferation, enzyme activity was determined in rat organs of high and low cell renewal capacity. The PIP kinase activity in rat thymus, bone marrow, spleen, and testes was 5.4-, 6.3-, 4.8- and 4.3-fold higher, respectively, than in normal rat liver; in lung, brain, skeletal muscle, renal cortex, and heart, the activities were low. In all tissues examined, the activity of PIP kinase was 4.6 to 18% of that of phosphatidylinositol kinase.

Since enzymes of crucial significance frequently have short half-lives, the decay rates of PIP kinase were examined in liver, bone marrow, and hepatoma 3924A in rats injected with cycloheximide, which inhibits protein biosynthesis. In cycloheximide-treated animals, PIP kinase had the shortest decay rate (t = 0.12 h) in comparison with eight enzymes of purine and pyrimidine biosynthesis of rat bone marrow (t = 0.6 to 4.3 h). In liver and solid hepatoma 3924A, the activity of PIP kinase was degraded less rapidly (t = 5 h).

The relationship of PIP kinase activity with proliferation and transformation is apparent in the high activity in thymus, bone marrow, spleen, and testes and in the increased activities in the rat hepatomas of different growth rates. The coordinate increases in phosphatidylinositol and PIP kinase activities suggest that the capacity for signal transduction is heightened in cancer cells. The elevated inositol 1,4,5-trisphosphate concentrations observed in the hepatomas support this conclusion. Since both kinase activities are linked with malignancy, these may well be sensitive targets for designing new chemotherapeutic agents.

¹ This work was supported, in part, by USPHS Outstanding Investigator Grant CA-42510 (to G. W.) and by OTKA Grant 3024 (to N. P.).

² Current address: National Institute of Oncology, Rath Gy. utca, Budapest, Hungary.

³ To whom requests for reprints should be addressed, at Laboratory for Experimental Oncology, Indiana University School of Medicine, 702 Barnhill Drive, Indianapolis, IN 46202-5200.

Received 6/27/94. Accepted 8/30/94.

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