Receptor specificity of growth factor-stimulated synthesis of 3-phosphorylated inositol lipids in Swiss 3T3 cells.

T.R Jackson,L.R Stephens,P.T Hawkins

doi:10.1016/s0021-9258(18)42049-2

Abstract

We have investigated synthesis of 3-phosphorylated inositol lipids in growth factor-stimulated Swiss 3T3 cells. Those growth factors tested which act via tyrosine kinase-containing receptors (platelet-derived growth factor (PDGF), insulin growth factor I (IGF-I), epidermal growth factor (EGF), and basic fibroblast growth factor (bFGF)) caused the rapid synthesis of [32P]PtdIns(3,4)P2 and [32P]PtdIns(3,4,5)P3 (PtdIns is phosphatidylinositol) in [32P]P(i)-prelabeled cells and the appearance of an inositol lipid 3-OH kinase in antiphosphotyrosine immunoprecipates. In contrast, those growth factors tested which act via G-protein-coupled receptors (bombesin, vasopressin, prostaglandin E1) were unable to stimulate either of the above responses. Furthermore, while PDGF was able to increase the formation of PtdIns(3,4)P2 and PtdIns(3,4,5)P3 in streptolysin-permeabilized cells, guanosine 5'-3-(thio)triphosphate and guanyl-5'-yl imidodiphosphate were not. These results suggest that Swiss 3T3 cells possess the machinery for tyrosine kinase but not G-protein-mediated activation of PtdIns(4,5)P2 3-OH kinase; a situation which is the inverse to that recently described for human neutrophils. The tyrosine kinase-containing receptors differed markedly in their relative abilities to elevate the levels of [32P] PtdIns(3,4,5)P3 (ranked in the order PDGF greater than or equal to IGF-I greater than EGF greater than bFGF), [32P]Ptd-OH (PDGF greater than EGF greater than bFGF; undetectable for IGF-I), and [32P]PtdIns4P (EGF greater than bFGF greater than PDGF; undetectable for IGF-I) in [32P]P(i)-prelabeled cells. These differences are epitomized by IGF-I, which was the joint most powerful stimulus for [32P] PtdIns(3,4,5)P3 formation, but was unable to stimulate a measurable accumulation of [32P]Ptd-OH (and hence, by deduction, was unable to stimulate phospholipase C). These results indicate that there is a differential ability among the tyrosine kinase-containing receptors present in a single cell to recruit phospholipase C and PtdIns(4,5)P2 3-OH kinase into their signalling complexes and further emphasizes the notion that the rapid synthesis of PtdIns(3,4,5)P3 may be a signalling event.

Highlights

We have investigated synthesis of 3-phosphorylated insights into themechanisms by which receptors regulate this inositol lipids in growth factor-stimulated Swiss 3T3 response, the nature of the response itself, and the results of cells
(thi0)triphosphate andguanyl-5'-yl imidodiphosphate man et al, 1990; Varticovski et al, 1989; Reith et al, 1991; were not. These results suggest that Swiss 3T3 cells Remillard et al, 1991, Bjorge et al, 1990)
PtdIns(3,4,5)P3 (rankedin the orderPDGF 2 IGF-I > ation (Kaplan et al, 1987) and subsequent activation of the EGF > bFGF), [32P]Ptd-OH(PDGF > EGF > bFGF; undetectable forIGF-I),and [""P]PtdIns4P (EGF > bFGF > PDGF; undetectable for IGF-I) in [32P]Pi-prelabeled cells. These differences are epitomizedby IGFI, which was thejoint most powerful stimulus for [""PI PtdIns(3,4,5)P3formation, but was unable to stimulate a measurable accumulation of [""PIPtd-OH(and by deduction, was unable to stimulate phospholipase C). These results indicate that there is a differential lipid kinase, the activity of which is expressed in the intact cell as a PtdIns(4,5)P23-OH kinase (Stephens et al, 1991).' Incontrast, aseparate mechanism for receptor-stimulated synthesis of 3-phosphorylated inositol lipids has been demonstrated in human neutrophils, where a number of agonistsactivate a distinct PtdIns(4,5)P2-3-OHkinase via a G - p r ~ t e i n . ~ These two mechanisms for the activation of PtdIns(3,4,5)P3 ability among the tyrosinekinase-containing receptors synthesis, G-protein regulation, and tyrosine kinase regulapresent in a single cell to recruit phospholipase C and tion (Rhee et al, 1991; Boyer et al, 1989) are directly analo

Summary

RESULTS

Measurement of Hormone-stimulated Changes in theLevels of 32P-Labeled Inositol Lipidsand P*P]PtdOH in Intact,PzP]. Wedecided to investigate the ability of growth factors to cause the association of an inositol lipid 3-OH kinase with the levels of PtdIns(3,4,5)P3 in response to both PDGF and IGF-I were sustained for relatively long periods of time (at least 20 min in the case of PDGF), while the responses to an antiphosphotyrosine antibody in Swiss 3T3 cell lysates because (1)we wished to compare the relative magnitude of this effect, both with respect to time and the nature of the EGF andbFGF appeared quite transient, with PtdIns(3,4,5)P3 agonist, with the stimulated formation of PtdIns(3,4,5)P3in levels almost returning to those of the control by 5 min. The valuthees cfoonrtrol samples did not change significantly over the first 5 min of stimulation andhave been combined (see Figs. 1-4).Similar resultshave been obtained intwo other independent experiments performed with vasopressin and oontheer independent experimenwt ith bFGF whichwere performed withoutcorrection for [3H] glycerol incorporation

Control bFGF

Con PDGF IGF Bomb

GTPVS PDGF