THE CATALYTIC SUBUNIT OF PHOSPHATIDYLINOSITOL 3-KINASE IS A SUBSTRATEFOR THE ACTIVATED PLATELET-DERIVED GROWTH-FACTOR RECEPTOR, BUT NOT FOR MIDDLE-T ANTIGEN PP60(C-SRC) COMPLEXES

The interaction of phosphatidylinositol 3-kinase (PI 3-K) with polyoma -virus middle-T antigen-pp60(c.src) (mT:cSrc complexes and with the pl atelet-derived growth factor (PDGF) receptor has been investigated. Fi rstly, we undertook reconstitution studies, using proteins derived fro m a baculovirus expression system. The p110 catalytic subunit of the P I 3-K associated with tyrosine kinases only when complexed with the p8 5 alpha regulatory subunit. Both p85 alpha and p110 were substrates of the PDGF receptor. In contrast, only the p85 alpha subunit was detect ably phosphorylated when PI 3-K was associated with mT:cSrc. Secondly, we studied PI 3-K in mammalian cells. In mT-antigen-transformed NIH-3 T3 cells neither p85 alpha nor p110 was phosphorylated on tyrosine res idues in vivo, even though p85 alpha. was a substrate in kinase assays in vitro. In quiescent NIH-3T3 cells, PI 3-K showed detectable activi ty in vitro; PDGF stimulation resulted in a rapid and transient associ ation of PI 3-K with the receptor, which was correlated with a transie nt increase in intrinsic PI 3-K activity (approx. 2-fold). The activat ed PDGF receptor phosphorylated p110 in vitro, at one major site. In v ivo, PDGF stimulation induced tyrosine phosphorylation of p110 that pe rsisted for at least 1 h after stimulation. Immunodepletion of the PDG F receptor from stimulated cell lysates showed that p110 was released from the receptor in a tyrosine-phosphorylated form. From these result s we conclude that (i) the mT:cSrc complex and the PDGF receptor diffe r in their association with PI 3-K activity, (ii) PDGF receptor appear s to activate PI 3-K in vivo both by relocation of the enzyme and by s timulation of its intrinsic activity, and (iii) tyrosine phosphorylati on of the p110 subunit by the PDGF receptor may play a role in PI 3-K regulation in some circumstances.