R Di Stasio, Anna Rita Migliaccio, Concetta Cuozzo, M. Di Domenico, Mariarosaria Santillo, Savina Agnese, Enrico V. Avvedimento, Antonio Porcellini, Antonio Feliciello, C Cosentino, G De Gregorio, C., Cosentino, M., DI DOMENICO, Porcellini, Antonio, C., Cuozzo, G., DE GREGORIO, Santillo, Mariarosaria, S., Agnese, R., DI STASIO, Feliciello, Antonio, A., Migliaccio, Avvedimento, VITTORIO ENRICO, Cosentino, C, DI DOMENICO, Marina, Porcellini, A, Cuozzo, C, DE GREGORIO, G, Santillo, Mr, Agnese, S, DI STASIO, R, Feliciello, A, Migliaccio, Antimo, and Avvedimento, Ev
Cyclic adenosine 3'5' monophosphate (cAMP) and protein kinase A (PKA) cooperate with phosphatidylinositol 3' kinase (PI3K) signals in the control of growth and survival. To determine the molecular mechanism(s) involved, we identified and mutagenized a specific serine (residue 83) in p85alpha(PI3K), which is phosphorylated in vivo and in vitro by PKA. Expression of p85alpha(PI3K) mutants (alanine or aspartic substitutions) significantly altered the biological responses of the cells to cAMP. cAMP protection from anoikis was reduced in cells expressing the alanine version p85alpha(PI3K). These cells did not arrest in G1 in the presence of cAMP, whereas cells expressing the aspartic mutant p85D accumulated in G1 even in the absence of cAMP. S phase was still efficiently inhibited by cAMP in cells expressing both mutants. The binding of PI3K to Ras p21 was greatly reduced in cells expressing p85A in the presence or absence of cAMP. Conversely, expression of the aspartic mutant stimulated robustly the binding of PI3K to p21 Ras in the presence of cAMP. Mutation in the Ser 83 inhibited cAMP, but not PDGF stimulation of PI3K. Conversely, the p85D aspartic mutant amplified cAMP stimulation of PI3K activity. Phosphorylation of Ser 83 by cAMP-PKA in p85alpha(PI3K) was also necessary for estrogen signaling as expression of p85A or p85D mutants inhibited or amplified, respectively, the binding of estrogen receptor to p85alpha and AKT phosphorylation induced by estrogens. The data presented indicate that: (1) phosphorylation of Ser 83 in p85alpha(PI3K) is critical for cAMP-PKA induced G1 arrest and survival in mouse 3T3 fibroblasts; (2) this site is necessary for amplification of estrogen signals by cAMP-PKA and related receptors. Finally, these data suggest a general mechanism of PI3K regulation by cAMP, operating in various cell types and under different conditions.