Shc Interaction with Src Homology 2 Domain Containing Inositol Phosphatase (SHIP) in VivoRequires the Shc-Phosphotyrosine Binding Domain and Two Specific Phosphotyrosines on SHIP

Abstract

The adapter protein Shc has been implicated in mitogenic signaling via growth factor receptors, cytokine receptors, and antigen receptors on lymphocytes. Besides the well characterized interaction of Shc with molecules involved in Ras activation, Shc also associates with a 145-kDa tyrosine-phosphorylated protein upon triggering via antigen receptors and many cytokine receptors. This 145-kDa protein has been recently identified as an SH2 domain containing 5-inositol phosphatase (SHIP) and has been implicated in the regulation of growth and differentiation in hematopoietic cells. In this report, we have addressed the molecular details of the interaction between Shc and SHIP in vivo. During T cell receptor signaling, tyrosine phosphorylation of SHIP and its association with Shc occurred only upon activation. We demonstrate that the phosphotyrosine binding domain of Shc is necessary and sufficient for its association with tyrosine-phosphorylated SHIP. Through site-directed mutagenesis, we have identified two tyrosines on SHIP, Tyr-917, and Tyr-1020, as the principal contact sites for the Shc-phosphotyrosine binding domain. Our data also suggest a role for the tyrosine kinase Lck in phosphorylation of SHIP. We also show that the SH2 domain of SHIP is dispensable for the Shc-SHIP interaction in vivo. These data have implications for the localization of the Shc⅐SHIP complex and regulation of SHIP function during T cell receptor signaling.The adapter protein Shc is a key regulator of intracellular signaling events that lead to such varied biological processes as neuronal differentiation, lymphocyte proliferation, and cellular transformation via polyoma virus middle T antigen (1-6). Shc mediates these effects, at least in part, through the activation of Ras proteins following stimulation of many receptors, including the receptors for growth factors (2, 7-10), antigens (11, 12), and cytokines (13-18), as well as G protein-coupled receptors (19). Shc contains an amino-terminal phosphotyrosine binding (PTB) 1 domain, a central collagen homology (CH) region, and a carboxyl-terminal Src homology 2 (SH2) domain but no apparent catalytic domain (7,20,21). In hematopoietic cells, Shc exists in two isoforms of 46 and 52 kDa. Upon activation of many receptors, Shc is tyrosine-phosphorylated and subsequently interacts with the SH2 domain of Grb2 (22,23). Grb2, in turn, interacts with the Ras GTP/GDP exchange factor, mSOS (24 -26). The complex of Shc⅐Grb2⅐mSOS becomes localized to the membrane through the association of Shc with the activated, tyrosine-phosphorylated receptors, where it leads to Ras activation (27). Shc interaction with activated receptors can occur either via the SH2 or the PTB domain. While both domains bind phosphotyrosine-containing sequences, their specificities of recognition are different and require residues either COOH-terminal (for SH2) or NH 2 -terminal (for PTB) to the Tyr(P) (28 -33). In hematopoietic cells, the Shc SH2 domain binds to components of the T cell receptor (TCR) (6, 11) and B cell receptor ...

SHC and SHIP phosphorylation and interaction in response to activation of the FLT3 receptor

“…18 SHC phosphorylation is neither necessary nor sufficient for this interaction, as previously proposed by Lamkin et al, 18 in contrast to the conclusions of Liu et al 45 Moreover, we were not able to precipitate SHC in a GST pull down assay using the isolated SH2 domain of SHIP under conditions that permit efficient precipitation of SHC by a GRB2 GST fusion protein (not shown). The lack of SHIP inducible phosphorylation by the FLT3 TM mutant suggests that SRClike kinases are not involved in this process.…”

“…A tyrosine-phosphorylated band that comigrates with SHIP was coprecipitated only with SHC proteins harboring an intact PTB domain, in agreement with a previous study. 18 In contrast, GRB2 binding was not affected by these two point mutations, indicating that the SH2 and PTB domains are dispensable for SHC phosphorylation and subsequent GRB2 binding.…”

Signaling via Shc family adapter proteins

“…A competition model, where SHIP would compete with Grb2 for binding to phosphorylated Shc and thereby downmodulate the positive signaling via Shc, has also been proposed (Liu et al, 1997a;Tridandapani et al, 1997). However, most of these studies were done in vitro with phosphopeptides, and we and others have not been able to reproduce these results in vivo (Harmer and DeFranco, 1999;Lamkin et al, 1997). Moreover, in Shc-de®cient DT40 B cells, the FcgRIIB1-mediated inhibition of B cell signaling, which is absolutely dependent on SHIP, occurs normally (Aman et al, 2000).…”

PTEN, but not SHIP and SHIP2, suppresses the PI3K/Akt pathway and induces growth inhibition and apoptosis of myeloma cells

“…Thus, we expressed the SHIPWT, its catalytic inactive mutant (D675A), and NPXY motif mutant (Y917F/Y1020F) in NIH3T3 cells and determined their expression by a Western blot analysis ( Figure 3, bottom panel). The Y917F/Y1020F mutant has been known to abolish its association with Shc through Shc phosphotyrosine binding domain (Lamkin et al, 1997). Expression of SHIPWT did not significantly affect the Akt phosphorylation on Ser 473 ( Figure 3, top panel) or Akt activity on a substrate (data not shown) stimulated by PDGF and IGF-I, when compared to that of D675A mutant expressing line.…”