Activation loop sequences confer substrate specificity to phosphoinositide3-kinase alpha (PI3K alpha) - Functions of lipid kinase-deficient PI3K alpha in signaling

Phosphoinositide 3-kinases (PI3Ks) are dual specificity lipid and protein k inases. While the lipid-dependent PI3K downstream signaling is well charact erized, little is known about PI3K protein kinase signaling and structural determinants of lipid substrate specificity across the various PI3K classes . Here we show that sequences C-terminal to the PI3K ATP-binding site deter mine the lipid substrate specificity of the class IA PI3K alpha (p85/p110 a lpha). Transfer of such activation loop sequences from class II PI3Ks, clas s III PI3Ks, and a related mammalian target of rapamycin (FRaP) into p110 a lpha turns the lipid substrate specificity of the resulting hybrid protein into that of the donor protein, while leaving the protein kinase activity u naffected. All resulting hybrids lacked the ability to produce phosphatidyl inositol 3,4,5-trisphosphate in intact cells. Amino acid substitutions and structure modeling showed that two conserved positively charged (Lys and Ar g) residues in the activation loop are crucial for the functionality of cla ss I PI3Ks as phosphatidylinositol 4,5-bisphosphate kinases. By transient t ransfecion of 293 cells, we show that p110 alpha hybrids, although unable t o support lipid-dependent PI3K signaling, such as activation of protein kin ase B/Akt and p70(S6k), retain the capability to associate with and phospho rylate insulin receptor substrate-1, with the same specificity and higher e fficacy than wild type PI3K alpha. Our data lay the basis for the understan ding of the class I PI3K substrate selectivity and for the use of PI3K alph a hybrids to dissect PI3K alpha function as lipid and protein kinase.