Endocytosis of Na(+),K(+)-ATPase molecules in response to G protein-coupled receptor stimulation requires activation of class I(A) phosphoinositide-3 kinase (PI3K-I(A)) in a protein kinase C-dependent manner. In this paper, we report that PI3K-I(A), through its p85alpha subunit-SH3 domain, binds to a proline-rich region in the Na(+),K(+)-ATPase catalytic alpha subunit. This interaction is enhanced by protein kinase C-dependent phosphorylation of a serine residue that flanks the proline-rich motif in the Na(+),K(+)-ATPase alpha subunit and results in increased PI3K-I(A) activity, an effect necessary for adaptor protein 2 binding and clathrin recruitment. Thus, Ser-phosphorylation of the Na(+),K(+)-ATPase catalytic subunit serves as an anchor signal for regulating the location of PI3K-I(A) and its activation during Na(+),K(+)-ATPase endocytosis in response to G protein-coupled receptor signals.