Abstract 15645: Phosphoinositde 3-Kinase Regulates β2-Adrenergic Receptor Stimulated Epidermal Growth Factor Receptor Transactivation
A mechanism by which β2-adrenergic receptors (β2ARs) stimulate signaling is transactivation of the epidermal growth factor receptor (EGFR), a cardioprotective signaling pathway that requires the formation of a β2AR-EGFR complex and the activation of Src. We have shown that β2AR internalization requires phosphoinositide 3-kinase (PI3K) through both its lipid kinase and protein kinase activities. We therefore tested the hypothesis that the lipid kinase activity of PI3K mediates β2AR-EGFR complex formation, while the protein kinase activity of PI3K phosphorylates Src. PI3K kinase mutants with lipid kinase and/or protein kinase activity, and PI3K inhibitors were used in co-immunoprecipitation and FRET experiments to study the role of PI3K on β2AR-EGFR complex formation. To identify putative Src phosphorylation sites, HEK-293 cells expressing hemagglutinin (HA)-tagged Src and β2ARs were stimulated with isoproterenol (ISO) in the presence or absence of the selective PI3K inhibitor LY−294002. Immunoprecipitated HA-Src was analyzed by mass spectrometry for PI3K-dependent Src phosphorylation. In co-immunoprecipitation and FRET experiments, PI3K kinase-dead mutants and inhibitors of PI3K activity blocked ISO-mediated complex formation. Moreover, a PI3K mutant with only lipid kinase activity allowed β2AR-EGFR complexes to form, whereas a PI3K mutant containing only protein kinase activity did not. Complex formation in the protein kinase only mutant is rescued by adding the phospholipid product of PI3K lipid kinase activity. HA-Src immunoprecipitated from ISO stimulated cells with and without LY-294002 pretreatment were collected, purified, and analyzed for Src phosphopeptides by mass spectrometry. Analysis reveals a number of potential PI3K-dependent Src phosphorylation sites in the regulatory SH2 and SH3 domains of Src. In conclusion, both lipid and protein kinase activities of PI3K appear to play a role in β2AR-mediated EGFR transactivation by generating phosphatidylinositols needed for β2AR-EGFR complex formation, and with the phosphorylation of Src. Ongoing site-directed mutagenesis studies will determine whether the potential PI3K phosphorylation sites affect Src activity.
- © 2010 by American Heart Association, Inc.