Involvement of phosphatidylinositol 3-kinase in NFAT activation in T cells.

Phosphatidylinositol 3-kinase (PI3-K) has been implicated in the regulation of cell proliferation in many cell types. We have previously shown that in T cells the PI3-K inhibitor, wortmannin, interferes with activation of the mitogen-activated kinase, Erk2, after T cell receptor (TcR) stimulation. To further explore the involvement of PI3-K in T cell 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 like Erk2 activation and transcription from an NFAT (nuclear factor of activated T cells) element taken from the interleukin-2 promoter. Following TcR stimulation, activation of Erk2 was only inhibited by a previously described truncated form of p85 that cannot bind the catalytic subunit, but not by other constructs of p85. In contrast, several mutant p85 alleles had dramatic effects on NFAT activation. Most interestingly, the N-terminal SH2 domain had an inhibitory effect, whereas a mutant p85 containing only the two SH2 domains enhanced basal NFAT activity 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 truncated p85 constructs revealed cooperative binding of the two SH2 domains but no apparent differences between the N- and C-terminal SH2 domains. Wortmannin did not interfere with NFAT activation, although it inhibited PI3-K and Erk2 activation in the same experiment. These results suggest that PI3-K is involved in NFAT activation through a complex adaptor function of its regulatory subunit and that its lipid kinase activity is dispensable for this effect.