Abstract
Rat-1 fibroblasts transformed with the v-src oncogene show a 6-fold increase in the apparent amount of an inositol polyphosphate which has a high performance liquid chromatography (HPLC) elution characteristic of the D/L-myo-inositol 1,4,5,6-tetrakisphosphate enantiomeric pair (Johnson, R.M., Wasilenko, W.J., Mattingly, R.R., Weber, M.J., and Garrison, J.C. (1989) Science 246, 121-124). By chemical and enzymatic analysis, the structure of this compound produced in both normal and v-src-transformed rat-1 fibroblasts has been determined to be principally D-myoinositol 1,4,5,6-tetrakisphosphate (D-Ins(1,4,5,6)P4). Chronic stimulation with endothelin-1 in the presence of Li+ significantly increased the amount of D/L-Ins(1,4,5,6)P4 only in the v-src-transformed rat-1 cells, suggesting that production of this compound may be remotely coupled to long term agonist-induced phosphatidylinositol turnover. Further evidence for such a link is provided by the progressive loss of D-Ins(1,4,5,6)P4 from the normal cells deprived of serum stimulation. To define a possible synthetic pathway for D-Ins(1,4,5,6)P4, cytosolic extracts of normal and v-src-transformed cells were incubated with [3H]inositol polyphosphates, and the reaction products were identified by HPLC elution and chemical analysis. Although inositol 1,3,4-trisphosphate 6-kinase activity was prominent in extracts of both normal and transformed cells, only the cytosol from v-src-transformed cells ultimately formed measurable amounts of D-Ins(1,4,5,6)P4 from [3H]inositol 1,3,4-trisphosphate. Approximately 6% of 0.1 microM inositol 1,3,4-trisphosphate was converted to D-Ins(1,4,5,6)P4 during a 2-h incubation at 37 degrees C. Inositol pentakisphosphate was identified as a likely intermediate in this conversion, and extracts of both normal and transformed cells converted [3H]inositol 1,3,4,5,6-pentakisphosphate to D-Ins(1,4,5,6)P4. The synthetic pathway described is consistent with the long term regulation of D/L-Ins(1,4,5,6)P4 levels in rat-1 fibroblasts seen in response to src transformation, serum withdrawal, and chronic endothelin treatment, and identifies several new potential interactions between the pathways of inositol polyphosphate metabolism and those of src transformation.
Highlights
From the $Departmentof Pharmacology and Cancer Research Center, Uniuersity of Virginia School of Medicine, Charlottesuille, Virginia22908 and the Wepartmentof Biochemistry, Institute of Animal Physiology, Agricultural andFood Research Council Babraham, Cambridge CB2 4AT, Great Britain
In rat-1 zymatic analysis, the structuroef this compound pro- fibroblasts,transformation with v-src is associated with a duced in both normal and v-src-transformed rat-1 fi- sustained increase in phosphatidylinositolmetabolism (8),an broblasts hasbeen determined to be principaDlly-my0- increase in the activity of Ins(1,4,5)P33-kinase' (9), and an inositol 1,4,5,6-tetrakisphosphate (~-Ins(1,4,5,6)P,). increase in the apparent formation of an InsP, which has an Chronic stimulation with endothelin-1 in the presenceHPLC elution characteristic of the enantiometric pair D/L
The results shown in Fig. 1are from experiments using a 60-min stimulation with endothelin-1 inthe presence of LiCl to trapcertain release of calcium from intracellularstoresin rat-1 fibroblasts: we hypothesized that chronic serum stimulation might be responsible for maintenance of the albeit low levels of D/
Summary
' the HPLC analysis used t o quantify the InsPl levels doesnotseparatethe D/L-Ins(1,4,5,6)P4 enantiomers,subsequent chemical analysis demonstrates tha D-Ins(1,4,5,6)P4 is the predominant form in these cells. Cytosolic extracts relatively long half-life of the Ins(1,3,4,5)P4produced in the were prepared andused to metabolize Ins(1,3,4)P:las described under incubationswith cytosolfrom v-src-transformed cells may "Experimental Procedures." Products of the metabolism were idenalso indicate a suppression of inositol polyphosphate 5-phos- tified by their elution from a strong anion exchange HPLC column. It was decided to repeat the assay conditions using [3H]by the appearanceof Ins(1,3,4,6)P4 Both Inspa and IannsP,, Ins(1,3,4)P3 as the substrate, beocthause it was further along which has the same strong anionexchange HPLC elution as the potential pathway (Scheme I), and because it is present the D/L-Ins(1,4,5,6)P4present in the intacct ells, were formed at high levels in the endothelin-stimulated transformed fibro-after 20 min,but only intheincubations using cytosolic blasts that show enhanced formation of D/L-Ins( 1,4,5,6)P4 extractsfromv-src-transformed cells(Fig. 3B).,the (Fig. 1). The mean efficiency of the conversion of Ins(1,3,4)P3 toD/L-1ns(1,4,5,6)P4was 17% for the low substrateconcentrationand 6% for[100] nM substrate
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