Phosphatidylinositol Kinase Activity Research Articles

Transforming protein of avian sarcoma virus UR2 is associated with phosphatidylinositol kinase activity: possible role in tumorigenesis.

The transforming protein of avian sarcoma virus UR2, p68v-ros, has an associated tyrosine-specific protein kinase activity similar to that of p60v-src and several other oncogene products. However, this activity has not been linked unequivocally to transformation, and the physiological action of these proteins remains in doubt. We now have found that immunoprecipitated p68v-ros also is associated with phosphatidylinositol (PtdIns) kinase (ATP:PtdIns 4-phosphotransferase, EC 2.7.1.67) activity. PtdIns 4,5-bisphosphate [PtdIns(4,5)P2] specifically inhibits both this activity and the autophosphorylation of p68v-ros. Moreover, cells transformed by UR2 showed significant increases in 32P-labeling of PtdIns 4-phosphate (PtdIns4P) and PtdIns(4,5)P2 and in the formation of their catabolites, inositol 1,4-bisphosphate and inositol 1,4,5-trisphosphate, as compared to uninfected cells. These results suggest that a physiologically relevant function of oncogene kinases might be the phosphorylation of PtdIns and that increased turnover of PtdIns4P and PtdIns(4,5)P2 might play a role in transformation by increasing the formation of diacylglycerol, a catabolite of polyphosphoinositides that activates kinase C. This protein copurifies with the phorbol ester receptor, and its activation is likely to be intimately linked with mitogenesis. This hypothesis suggests a mechanism whereby certain oncogene proteins might cause the unrestricted growth typical of transformed cells and could explain why tumor promoters mimic many of the effects of transformation.

Ca2+-sensitive phosphatidylinositol 4-phosphate metabolism in a rat beta-cell tumour.

Subcellular fractions were isolated from a rat beta-cell tumour by centrifugation of homogenates on Percoll and Urografin density gradients. Fractions were incubated with [gamma-32P]ATP, and labelling of phosphatidylinositol 4-phosphate and phosphatidylinositol 4,5-bisphosphate was used to measure phosphatidylinositol kinase and phosphatidylinositol 4-phosphate kinase activities, respectively. The distribution of enzyme markers in density gradients indicated that phosphatidylinositol kinase was located in both the plasma membrane and the secretory-granule membrane. Phosphatidylinositol 4-phosphate kinase activity was low in all fractions. Phosphatidylinositol kinase activity of secretory granules and plasma membranes was decreased to 10-20% of its initial value by raising the free [Ca2+] from 1 microM to 5 microM. The enzyme had a Km (apparent) for ATP of 110 microM (secretory granule) or 120 microM (plasma membrane) and a Ka for Mg2+ of 7 mM (secretory granule) or 6 mM (plasma membrane). Ca2+-sensitivity of phosphatidylinositol kinase in calmodulin-depleted secretory granules and plasma membranes was not affected by addition of exogenous calmodulin, although activity was stimulated by trifluoperazine in the presence of 0.1 microM or 40 microM-Ca2+. Trifluoperazine oxide had no effect on the enzyme activity of secretory granules. Plasma membranes had a phosphatidylinositol 4-phosphate phosphatase activity which was stimulated by raising the free [Ca2+] from 0.1 to 40 microM. The secretory granule showed no phosphatidylinositol 4-phosphate-degrading activity. These results suggest the presence in the tumour beta-cell of Ca2+-sensitive mechanisms responsible for the metabolism of polyphosphoinositides in the secretory granule and plasma membrane.

Phospholipid synthesis in the squid giant axon: enzymes of phosphatidylinositol metabolism.

We examined the properties of several enzymes of phospholipid metabolism in axoplasm extruded from squid giant axons. The following synthetic enzymes, CDP-diglyceride: inositol transferase (EC 2.7.8.11), ATP:diglyceride phosphotransferase, diglyceride kinase (EC 2.7.2.-), and phosphatidylinositol kinase (EC 2.7.1.67), were all present in axoplasm. Phospholipid exchange proteins, which catalyzed the transfer of phosphatidylinositol and phosphatidylcholine between membrane preparations and unilamellar lipid vesicles, were also found. However, we did not find conditions under which the synthesis of CDP-diglyceride, phosphatidylserine, and phosphatidylinositol-4,5-diphosphate could be measured. Subcellular fractionation by differential centrifugation showed that the axoplasmic inositol transferase and phosphatidylinositol kinase activities were largely "microsomal," while the diglyceride kinase and exchange protein activities were primarily "cytosolic."

Properties of phosphatidylinositol kinase activities in rabbit erythrocyte membranes

Properties of phosphatidylinositol kinase activities in rabbit erythrocyte membranes were studied by measuring 32P incorporation into di- and triphosphoinositide from Mg-[γ- 32P]ATP. The K m 's for 32P incorporation into di- and triphosphoinositide were 110 and 48 μ m ATP, respectively. The optimal temperature for 32P incorporation into diphosphoinositide was at 32 °C, whereas the optimum for triphosphoinositide labeling occurred at 43 °C. Differences in the effects of pH on the rate of 32P incorporation into di- and triphosphoinositide were also found. At 37 °C but not at 25 °C 32P-labeled diphosphoinositide was phosphorylated to triphosphoinositide in the presence of Mg-ATP. Triton X-100 partially inhibited 32P incorporation into diphosphoinositide but completely inhibited the synthesis of triphosphoinositide. At physiological concentrations, 0.4 m m MgCl 2 half-maximally activated di- and triphosphoinositide synthesis. Higher concentrations of MgCl 2 (5 to 50 m m) decreased 32P incorporation into diphosphoinositide and greatly enhanced 32P incorporation into triphosphoinositide. NaCl or KCl (⩽100 m m) did not have any effects on polyphosphoinositide synthesis, whereas 150 to 300 m m NaCl or KCl decreased synthesis of diphosphoinositide and increased synthesis of triphosphoinositide. Further studies showed that 50 m m MgCl 2 and 200 m m NaCl or KCl stimulate kinase-mediated phosphorylation of diphosphoinositide to triphosphoinositide. Triton X-100 inhibited the ability of 50 m m MgCl 2 and neomycin to stimulate phosphorylation of diphosphoinositide to triphosphoinositide. The pathways for synthesis of di- and triphosphoinositides in erythrocyte membranes are discussed.

Identification of phosphatidylinositol kinase in rat liver lysosomal membranes.

Liver lysosomes from Triton-injected or normal rats were found to rapidly incorporate 32P from [gamma-32P]ATP into a lipid component of the membrane, in vitro. The lipid was identified as phosphatidylinositol 4-phosphate based on its chromatographic behavior on Silica Gel H thin layer plates as compared with standard phosphoinositides. The deacylation product, glyceryl-phosphorylinositol phosphate, was compared with standards in chromatographic and electrophoretic systems to further substantiate the identification of the radioactive material. A trace of phosphatidylinositol 4,5-bisphosphate was also found. The properties of the lysosomal membrane phosphatidylinositol kinase were examined using both endogenous lipid and exogenous phosphatidylinositol as substrate. The enzyme was active at neutral pH in the presence of 20 mM MgCl2. The addition of 0.4% Triton X-100 stimulated the enzyme activity toward endogenous substrate, and the highest activity was observed in the presence of detergent and 1 mM phosphatidylinositol. Degradation of the product was seen only in the presence of Triton X-100. The specific activity of the lysosomal phosphatidylinositol kinase is comparable to the detergent-stimulated activity of liver microsomes and plasma membrane, the previously recognized sources of this enzyme in the liver cell.

Detergent solubilization and hydrophobic chromatography of rat brain phosphatidylinositol kinase.

Rat brain microsomal phosphatidylinositol kinase activity was maximally activated in the presence of either 3 mM sodium deoxycholate, 2% Triton-X-100, or 30-40 mM octylglucoside. Among these detergents, 1% Triton-X-100 was most effective in solubilizing the enzyme, and after treatment with this agent, 100% of the activity was recovered in the high speed supernatant. Octylglucoside solubilized 40% of the enzyme at concentrations below its critical micelle concentration of 25 mM and up to 80% at higher levels. Solubilized phosphatidylinositol kinase failed to absorb to adenosine nucleotide affinity resins. However, when the Triton-X-100 extract was chromatographed on an uncharged hydrophobic resin, consisting of dodecyl chains attached to Sepharose 4B by ether bonds, nearly all the enzyme activity was retained, and from 44-85% could be eluted with 8 mM sodium deoxycholate. Solubilization followed by hydrophobic chromatography resulted in several-fold purification of phosphatidylinositol kinase and may have disrupted interactions of the enzyme with other hydrophobic proteins sufficiently to allow its substantial purification by conventional or affinity chromatography techniques.

PHOSPHOINOSITIDE KINASES IN CHICK BRAIN AND SCIATIC NERVE, A DEVELOPMENTAL STUDY

Abstract—Phosphatidylinositol kinase and diphosphoinositide kinase activities were measured in homogenates of brain and sciatic nerve of developing chick embryos and chicks. Characteristics of the chick nervous system enzymes were similar to those reported for rat brain. Diphosphoinositide kinase was inhibited by high concentrations of ATP and by low concentrations of triphosphoinositide. Both activities were greatly enhanced by the non‐ionic detergent, Cutscum, and the ratio of detergent to protein in the reaction mixture was important. Optimum phosphatidylinositol kinase activity required a ratio of 7 : 1 for both tissues. The optimum ratio for diphosphoinositide kinase was 3:1 for nerve homogenates and 0.6:1 for brain. Cutscum increased the concentration of diphosphoinositide that is required for maximum diphosphoinositide kinase activity.Developmental changes were the same for both kinase activities, which were low in unmyelinated brain and sciatic nerve. The activities correlated with the concentration of polyphosphoinositides in chick brain where they increased 4‐5 fold during the period of active myelination and remained high in the mature brain. The kinase activities correlated with the rate of triphosphoinositide deposition in sciatic nerve. Following a 2‐3 fold increase during the initial phase of myelination the activities declined to values as low as those of embryonic nerve.

Enzymes of phosphoinositide metabolism during rat brain development.

Abstract—The activities of four enzymes concerned with inositol lipid metabolism have been determined in homogenates of rat brains of different ages. The enzymes are CDP‐diglyceride inositol phosphatidate transferase, phosphatidylinositol kinase, diphosphoinositide kinase and triphosphoinositide phosphomonoesterase. The activities of all the enzymes increased with age. Phosphatidylinositol kinase activity rose most sharply well before myelination, reaching a maximum at about 6 days of age. Diphosphoinositide kinase and triphosphoinositide phosphomonoesterase activities increased most rapidly during myelination. The increase in CDP‐diglyceride inositol phosphatidate transferase showed no definite association with any period of development. It is concluded that triphosphoinositide metabolism is associated with myelin or a closely related structure.

The diphosphoinositide kinase of rat brain.

1. The supernatant fraction of adult rat brain contains a diphosphoinositide kinase. 2. Formation of triphosphoinositide by the enzyme in the presence of ATP and Mg(2+) ions was shown with labelled ATP or labelled diphosphoinositide. 3. The kinase was also activated by Ca(2+), Mn(2+) and Co(2+) ions, but to a smaller extent than by Mg(2+) ions. 4. In the presence of optimum Mg(2+) ion concentration the enzyme was inhibited by Ca(2+) ions. 5. Activity did not depend on thiol groups and the pH optimum was 7.3. 6. The dialysed supernatant fraction had no diglyceride kinase activity and negligible phosphatidylinositol kinase activity. 7. Triphosphoinositide phosphomonoesterase was present but showed little activity under the conditions used to assay the kinase. 8. Diphosphoinositide kinase was purified by ammonium sulphate fractionation, ethanol treatment and chromatography on Sephadex G-200. 9. This purification removed much of the triphosphoinositide phosphomonoesterase.

Characteristics of rat liver phosphatidylinositol kinase and its presence in the plasma membrane

1. 1. Rat liver contains a phosphatidylinositol kinase. 2. 2. The enzyme requires phosphatidylinositol, ATP and Mg 2+ or Mn 2+ for activity. 3. 3. The subcellular distribution of the enzyme does not parallel that of nuclei, mitochondria, lysosomes or endoplasmic reticulum. 4. 4. The kinase and 5'-nucleotidase (a plasma membrane enzyme) are similary distributed in subcellular fractions. 5. 5. The activity of phosphatidylinositol kinase is at least twice as high in purified plasma membrane as in nuclei prepared from the same crude nuclear fraction. 6. 6. The role of the kinase at the cell surface is discussed.