The serine-threonine kinase Akt/PKB is activated downstream of phosphatidylinositol 3-kinase in response to several growth factor stimuli and has been implicated in the promotion of cell survival. Although both phosphatidylinositol 3,4,5-trisphosphate (PIP 3 ) and phosphatidylinositol 3,4-bisphosphate (PI 3,4-P 2 ) have been implicated in the regulation of Akt activity in vitro, the relative roles of these two phospholipids in vivo are not well understood. Co-ligation of the B cell receptor (BCR) and the inhibitory F c ␥RIIB1 on B cells results in the recruitment of the 5-inositol phosphatase SHIP to the signaling complex. Since SHIP is known to cleave PIP 3 to generate PI 3,4-P 2 both in vivo and in vitro, and Akt activity has been reported to be regulated by either PIP 3 or PI 3,4-P 2 , we hypothesized that recruitment of SHIP through F c ␥RIIB1 co-cross-linking to the BCR in B cells might regulate Akt activity. The nature of this regulation, positive or negative, might also reveal the relative contribution of PIP 3 and PI 3,4-P 2 to Akt activation in vivo. Here we report that Akt is activated by stimulation through the BCR in a phosphatidylinositol 3-kinase-dependent manner and that this activation is inhibited by co-cross-linking of the BCR to F c ␥RIIB1. Using mutants of F c ␥RIIB1 and SHIP-deficient B cells, we demonstrate that inhibition of Akt activity is mediated by the immune cell tyrosine-based inhibitory motif within F c ␥RIIB1 as well as SHIP. The SHIP-dependent inhibition of Akt activation also suggests that PIP 3 plays a greater role in Akt activation than PI 3,4-P 2 in vivo.Akt (also called protein kinase B) is a serine/threonine kinase that is activated upon ligation of several cell surface receptors, including the receptors for insulin and platelet-derived growth factor (1-4). The biological significance of Akt has been demonstrated by its ability to protect a variety of cell types from apoptosis (5-8). Akt, in addition to its kinase domain, contains an amino-terminal pleckstrin homology (PH) 1 domain, which can bind the phospholipids phosphatidylinositol 3,4,5-trisphosphate (PIP 3 ) or phosphatidylinositol 3,4-bisphosphate (PI 3,4-P 2 ) (9). Phosphatidylinositol-3 kinase (PI3K), which phosphorylates the 3Ј-position of the inositol ring in phosphatidylinositol 4,5-bisphosphate to generate PIP 3 , has been shown to function upstream of Akt (10), since the specific PI3K inhibitor wortmannin can block Akt activation, and platelet-derived growth factor receptor mutants that fail to activate PI3K also fail to activate Akt (1-3).Recent studies have established the importance of both 3Ј-phosphorylated inositol phosphates (PI 3,4-P 2 and PIP 3 ) and Akt phosphorylation in activation of Akt. PI 3,4-P 2 has been shown to directly activate Akt in vitro via interaction with the Akt PH domain, while PIP 3 was inhibitory in this experimental system (11-13). Additionally, Akt enzymatic activity was shown to be dependent on phosphorylation of Akt on a specific serine (Ser 473 ) and a specific threonine (Thr 30...

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 ...

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