The Src Homology 2 Domain Containing Inositol 5-Phosphatase SHIP2 Is Recruited to the Epidermal Growth Factor (EGF) Receptor and Dephosphorylates Phosphatidylinositol 3,4,5-Trisphosphate in EGF-stimulated COS-7 Cells

Abstract

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...

Signal propagation from membrane messengers to nuclear effectors revealed by reporters of phosphoinositide dynamics and Akt activity

“…However, the observed difference in responses stimulated by various growth factors cannot be accounted for solely by the difference in the total receptor numbers, because the numbers of PDGFR and IGF-IR per cell were similar (1-2 ϫ 10 5 ) (25, 38). Instead, the different dynamics may result from differential coupling of the activated receptors to PI3K, involving, for instance, PI3K isoform specific activation by different receptor signaling pathways (23) or their regulation of lipid phosphatases (39,40). Furthermore, as a distinct mode of negative regulation, the p85 regulatory subunit of PI3K was shown to translocate to discrete foci after the initial production of 3Ј PI, in response to IGF-1 but not PDGF signaling (24).…”

In-depth Qualitative and Quantitative Profiling of Tyrosine Phosphorylation Using a Combination of Phosphopeptide Immunoaffinity Purification and Stable Isotope Dimethyl Labeling

“…The EGFR autophosphorylation sites Tyr(P)-1172 and Tyr(P)-1197 showed an increase in phosphorylation after 10 min of EGF stimulation whereby this phosphorylation decreased again after 30 min, consistent with previously published data (5,45). Furthermore, phosphopeptides of Gab1 (Tyr(P)-659), STAM2 (Tyr(P)-192 and Tyr(P)-371), RBCK1 (Tyr(P)-288), Cbl (Tyr(P)-674), SHIP-2 (Tyr(P)-986 and Tyr(P)-1135), and Epsin (Tyr(P)-17) showed an extensive increase in phosphorylation and have shown to be involved in EGF signaling and internalization (5,12,15,46,47). Some of the tyrosine phosphorylated peptides that showed an increase in abundance after EGF stimulation have not been reported before, or the phosphosite has not been described to be profoundly involved in EGFR signaling.…”

The PI3K effector Arap3 interacts with the PI(3,4,5)P3 phosphatase SHIP2 in a SAM domain-dependent manner

“…As shown in Fig. 3B, the SAM domain of Arap3 indeed pulls down full-length SHIP2, but not a mutant of SHIP2 that lacks the proline-rich and SAM domain-containing C-terminus (t-SHIP2 [20]). Similarly, the SAM domain of SHIP2 only interacts with full-length Arap3, and not with the mutant lacking the SAM domain (Fig.…”

“…FlagHis-ΔSAMArap3 (residues 71-1544) was also made using mutagenesis with FlagHis-Arap3 as a template. His-tagged SHIP2 and His-t-SHIP2 have been described before [20]. His-SHIP2-ΔSAM (residues 1-1192) was made using mutagenesis PCR.…”

“…Like Arap3, SHIP2 has several protein-protein interaction domains. Besides its SH2 domain that mediates the recruitment of SHIP2 to activated receptor tyrosine kinases [20,21] and its catalytic phosphatase domain, SHIP2 has a prolinerich region followed by a C-terminal SAM domain. Several interaction partners are known for SHIP2, including the HGF receptor c-Met [21], the E3 ubiquitin ligase Cbl and Cblassociated protein (CAP) [22].…”