INVOLVEMENT OF PHOSPHATIDYLINOSITOL 3-KINASE IN NFAT ACTIVATION IN T-CELLS

Phosphatidylinositol 3-kinase (PI3-K) has been implicated in the regul ation of cell proliferation in many cell types, We have previously sho wn that in T cells the PI3-K inhibitor, wortmannin, interferes with ac tivation of the mitogen-activated kinase, Erk2, after T cell receptor (TcR) stimulation, To further explore the involvement of PI3-K in T ce ll activation, we created a set of potentially dominant negative PI3-K constructs comprising individual or tandem domains of the regulatory p85 subunit and tested their effect on downstream signaling events lik e Erk2 activation and transcription from an NFAT (nuclear factor of ac tivated T cells) element taken from the interleukin-2 promoter, Follow ing TcR stimulation, activation of Erk2 was only inhibited by a previo usly described truncated form of p85 that cannot bind the catalytic su bunit, but not by other constructs of p85, In contrast, several mutant p85 alleles had dramatic effects on NFAT activation. Most interesting ly, the N-terminal SH2 domain had an inhibitory effect, whereas a muta nt p85 containing only the two SH2 domains enhanced basal NFAT activit y in a Ras-dependent manner, Ionomycin induced synergistic activation of NFAT in cells expressing p85 mutants that contained the C-terminal SH2 domain. Analysis of phosphotyrosine-containing proteins bound to t runcated p85 constructs revealed cooperative binding of the two SH2 do mains but no apparent differences between the N- and C-terminal SH2 do mains, Wortmannin did not interfere with NFAT activation, although it inhibited PI3-K and Erk2 activation in the same experiment, These resu lts suggest that PI3-K is involved in NFAT activation through a comple x adaptor function of its regulatory subunit and that its lipid kinase activity is dispensable for this effect.