Fast receptor-induced formation of glycerophosphoinositol-4-phosphate, a putative novel intracellular messenger in the Ras pathway.

Abstract

Glycerophosphoinositols are phosphoinositide metabolites whose levels are constitutively elevated in Ras-transformed cells. Here, we show that one of these compounds, glycerophosphoinositol-4-phosphate (GroPlns-4-P) responds acutely to the stimulation of the epidermal growth factor receptor, with a fast, massive and transient increase. The mechanism leading to GroPIns-4-P formation involves the activation of phosphoinositide-3 kinase and the small GTP-binding protein Rac, since GroPIns-4-P was neither formed in cells expressing the dominant negative form of Rac nor in cells treated with the phosphoinositide-3 kinase inhibitor wortmannin. GroPIns-4-P has been previously shown to inhibit adenylyl cyclase. Accordingly, epidermal growth factor also decreased the basal, cholera toxin-stimulated, and forskolin-stimulated cyclic AMP levels with kinetics similar to those of GroPIns4-P formation, suggesting that GroPIns-4-P mediates this inhibitory effect. The hormone-induced formation of GroPIns-4-P was detected in several cell lines of various origin, suggesting that GroPIns4-P is a novel intracellular messenger of the Ras pathway, possibly able to convey information from tyrosine kinase receptors to the cyclic AMP cascade.

Release of the mitogen lysophosphatidylinositol from H-Ras-transformed fibroblasts; a possible mechanism of autocrine control of cell proliferation

“…Notably, Table 2 suggests that not all of the increase of Ins-1-P and Ins-2-P can be explained as derived from Ins-1 : 2-cycP since these two metabolites remained elevated in Ras-transformed ®broblasts even after neutral extraction (four-and 40-fold higher, respectively). Indeed, it is possible that these metabolites derive from GroPIns via enzymatic hydrolysis, as previously suggested Falasca et al, 1996Falasca et al, , 1997. In contrast, inositol phosphates produced by phospholipase C activation such as Ins-1,4-P 2 and Ins-1,4,5-P 3 , in agreement with previous studies, were not signi®cantly higher in Rastransformed than in normal cells (Table 1) (Alonso et al, 1988;Valitutti et al, 1991;Iacovelli et al, 1993;Corda and Falasca, 1996;Falasca and Corda, 1994).…”

“…However, also LysoPtdIns and glycerophosphoinositol-4-phosphate (GroPlns-4-P) possess biological activity (Iacovelli et al, 1993;Falasca and Corda, 1994;Corda and Falasca, 1996). Exogenously added GroPlns-4-P inhibits adenylyl cyclase, probably by acting at the level of the Gs protein (Iacovelli et al, 1993), and a similar eect is observed when GroPlns-4-P is formed intracellularly by growth factor receptor activation (Falasca et al, 1997). More important in the context of the Ras-induced transformation, exogenously added LysoPtdIns stimulates cell proliferation in Rastransformed thyroid cells via the activation of phospholipases C and A 2 (Falasca and Corda, 1994;Falasca et al, 1995).…”

“…In particular, high levels of both lysophosphatidylinositol (LysoPtdIns), a molecule generated by a constitutively-active phosphoinositide (PtdIns)-speci®c phospholipase A 2 , and of glycerophosphoinositols (derived from LysoPtdIns through a second deacylation step) have been reported in cells of dierent origin transformed by the ras oncogene (Alonso et al, 1988;Valitutti et al, 1991;Iacovelli et al, 1993;Corda and Falasca, 1996;Falasca and Corda, 1994;Falasca et al, 1995). The increase in glycerophosphoinositols and LysoPtdIns in Ras-transformed cells is most likely due to the generation of these compounds directly by enzymes participating in the Ras signaling pathway, rather than being a generic consequence of transformation, as indicated by several lines of evidence: (a) glycerophosphoinositols, the direct metabolic products of LysoPtdIns deacylation, are very rapidly and transiently increased by the activation of membrane growth-factor receptors linked to a PtdIns-speci®c phospholipase A 2 through the RasRac pathway (Falasca et al, 1997); (b) cell lines expressing temperature-sensitive mutants of oncogenic Ras display high levels of LysoPtdIns and glycerophosphoinositols only at the permissive temperature (Alonso and Santos, 1990;Valitutti et al, 1991;Falasca and Corda, 1994); (c) high glycerophosphoinositol levels are associated with Ras activation in cellular responses dierent from transformation (hepatic and neuronal dierentiation; Falasca et al, 1996;Corda and Falasca, 1996).…”

Reorganization of Actin Cytoskeleton by the Phosphoinositide Metabolite Glycerophosphoinositol 4-Phosphate

“…Moreover, increases in cAMP levels result in the inhibition of RhoA, with respect to its ability to induce either morphological changes in neuroblastoma cells or cell migration in colon carcinoma and hepatoma cells (Dong et al, 1998;Mukai et al, 2000;O'Connor et al, 2000). Thus, a decrease in cAMP induced in Swiss 3T3 cells by GroPIns-4P (Falasca et al, 1997) might play at least an accessory role in Rac and Rho activation also in fibroblasts. Irrespective of the precise manner in which Rac is activated, an intriguing and potentially important mechanistic aspect emerges from the comparison between the stimulatory effects of GroPIns-4P and those of the other glycerophosphoinositols.…”

“…First, they define a new biological activity of an endogenous, freely diffusable inositol-containing molecule. Moreover, because GroPIns-4P is a natural metabolite of the phosphoinositides generated by the activation of the Ras cascade (Falasca et al, 1995(Falasca et al, , 1997, the actin remodeling it induces is likely to play a role in motility and metastatic invasion. Second, because GroPIns-4P is a small, water-soluble molecule that is active when added extracellularly, our observations suggest that it has the potential to serve as a prototype of a new class of pharmacologically active agents.…”