The lipid phosphatase SHIP2 (Src homology 2 domain containing inositol 5-phosphatase 2) has been shown to be expressed in nonhemopoietic and hemopoietic cells. It has been implicated in signaling events initiated by several extracellular signals, such as epidermal growth factor (EGF) and insulin. In COS-7 cells, SHIP2 was tyrosine-phosphorylated at least at two separated tyrosine phosphorylation sites in response to EGF. SHIP2 was coimmunoprecipitated with the EGF receptor (EGFR) and also with the adaptor protein Shc. A C-terminal truncated form of SHIP2 that lacks the 366 last amino acids, referred to as tSHIP2, was also precipitated with the EGFR when transfected in COS-7 cells. The Src homology 2 domain of SHIP2 was unable to precipitate the EGFR in EGF-stimulated cells. Moreover, when transfected in COS-7 cells, it could not be detected in immunoprecipitates of the EGFR. When the His-tagged fulllength enzyme was expressed in COS-7 cells and stained with anti-His 6 monoclonal antibody, a signal was observed at plasma membranes in EGF-stimulated cells that colocalize with the EGFR by double staining. Upon stimulation by EGF, phosphatidylinositol 3,4,5-trisphosphate and protein kinase B activity were decreased in SHIP2-transfected COS-7 cells as compared with the vector alone. SHIP2 appears therefore in a tyrosine-phosphorylated complex with at least two other proteins, the EGFR and Shc.Protein tyrosine phosphorylation plays a central role in the regulation of protein-protein interactions and modulation of enzyme activities (1). Key events in receptor signaling are the interactions of proteins such as the adaptor protein Shc with other phosphorylated proteins. In epidermal growth factor (EGF) 1 signaling, the activation of the EGF receptor (EGFR)could mediate the phosphorylation of a series of proteins, such as the p85 subunit of the phosphoinositide 3-kinase (PI 3-kinase) (2). This phosphorylation has been linked to the activation of PI 3-kinase, which plays an important role in signaling. All mammalian cell types express at least class IA PI 3-kinase isoform, and stimulation of almost every receptor that induces Tyr kinase activity also leads to class IA PI 3-kinase activation (3, 4). PI 3-kinase class IA substrates in vitro are phosphatidylinositol, phosphatidylinositol 4-phosphate, and phosphatidylinositol 4,5-bisphosphate, whereas the preferred substrate in vivo is phosphatidylinositol 4,5-bisphosphate (2). Phosphatidylinositol 3,4,5-trisphosphate (PIP3) and/or phosphatidylinositol 3,4-bisphosphate (PI 3,4-P 2 ) could activate protein kinase B (PKB) through the binding to the PH domain of 3Ј-phosphoinositide-dependent kinase 1 phosphorylating protein kinase B (5-7). The control of the levels of the second messenger PIP3 depends on the activity of both PI 3-kinase and PIP3 phosphatases (which are members of inositol and phosphatidylinositol 5-phosphatase family) and 3-phosphatases, such as PTEN (phosphatase and tensin deleted from chromosome 10) (8). SHIP1 and SHIP2 (SH2 domain containing inositol 5-phospha...