We recently identified a novel adaptor protein, termed dual adaptor for phosphotyrosine and 3-phosphoinositides (DAPP1), that possesses a Src homology (SH2) domain and a pleckstrin homology (PH) domain. DAPP1 exhibits a high-affinity interaction with PtdIns(3,4,5) P 3 and PtdIns(3,4) P 2 , which bind to the PH domain. In the present study we show that when DAPP1 is expressed in HEK-293 cells, the agonists insulin, insulin-like growth factor-1 and epidermal growth factor induce the phosphorylation of DAPP1 at Tyr 139 . Treatment of cells with phosphoinositide 3-kinase (PI 3-kinase) inhibitors or expression of a dominant-negative PI 3-kinase prevent phosphorylation of DAPP1 at Tyr 139 , and a PH-domain mutant of DAPP1, which does not interact with PtdIns(3,4,5) P 3 or PtdIns(3,4) P 2 , is not phosphorylated at Tyr 139 following agonist stimulation of cells. Overexpression of a constitutively active form of PI 3-kinase induced the phosphorylation of DAPP1 in unstimulated cells. We demonstrated that Tyr 139 of DAPP1 is likely to be phosphorylated in vivo by a Src-family tyrosine kinase, since the specific Src-family inhibitor, PP2, but not an inactive variant of this drug, PP3, prevented the agonist-induced tyrosine phosphorylation of DAPP1. Src, Lyn and Lck tyrosine kinases phosphorylate DAPP1 at Tyr 139 in vitro at similar rates in the presence or absence of PtdIns(3,4,5) P 3 , and overexpression of these kinases in HEK-293 cells induces the phosphorylation of Tyr 139 . These findings indicate that, following activation of PI 3-kinases, PtdIns(3,4,5) P 3 or PtdIns(3,4) P 2 bind to DAPP1, recruiting it to the plasma membrane where it becomes phosphorylated at Tyr 139 by a Src-family tyrosine kinase.