INOSITOL POLYPHOSPHATES ARE NOT INCREASED BY OVEREXPRESSION OF INS(1,4,5)P-3 3-KINASE BUT SHOW CELL-CYCLE DEPENDENT CHANGES IN GROWTH FACTOR-STIMULATED FIBROBLASTS

NIH 3T3 fibroblasts were stably transfected with rat brain inositol 1,4,5-trisphosphate (Ins(1,4,5)P-3) 3-kinase to explore the relationship between increased production of Ins(1,3,4,5)P-4 and the formation of InsP(5) and InsP(6). Mass measurements of InsP(5) and InsP(6) revealed no significant difference between kinase- and vector-transfected fibroblasts. However, such 3-kinase-transfected cells, when labeled with [H-3]inositol for 48-72 h, showed lower levels of [H-3]InsP(5) and [H-3]InsP(6), as well as [H-3]Ins(1,3,4,6)P-4 and D/L[H-3]Ins(1,4,5,6)P-4, than their vector-transfected counterparts. Because Ins(1,4,5)P-3 3-kinase-transfected cells grew less rapidly than vector-transfected controls, we determined whether the synthesis of InsP(5) and InsP(6) was related to a specific phase of the cell cycle. When NIH 3T3 cells prelabeled with [H-3]inositol were synchronized by serum deprivation followed by stimulation with platelet-derived growth factor (PDGF), the amounts of labeled InsP(5) and InsP(6) began to increase only after 12 h of stimulation, when cells entered the S-phase as indicated by increased [H-3]thymidine incorporation. The enhanced synthesis of these inositol polyphosphates was preceded by an early increase in Ins(1,4,5)P-3 and its metabolites that was no longer evident by the fifth hour of PDGF action. There was also a prominent and biphasic increase in the level of D/L-Ins(1,4,5,6)P-4 with an early peak at similar to 3 h and a second rise that paralleled the increases in InsP(5) and InsP(6). These results indicate that the formation of highly phosphorylated inositols is not tightly coupled to the receptor-mediated formation of Ins(1,4,5)P-3 and its metabolites but is mainly determined by other factors that operate at specific points of the cell cycle.