Christiane R. Maroun

Distinct roles for CD4 and CD8 as co-receptors in antigen receptor signalling

The co-ordinated interactions of multiple membrane molecules with the T-cell receptor for antigen (the TCR-CD3) are prerequisite for T-cell activation. In this review we consider the involvement of CD4, CD8, and CD45 on the two lymphocyte lineages. Experiments from many laboratories have provided concordant results leading to the consensus that CD4 and CD8 are functional analogues, providing similar supplementary signals to those generated through the TCR-CD3 complex on MHC class-II- and MHC class-I-restricted T cells, respectively. However, recent results demonstrate striking differences in the coreceptor functions of CD4 and CD8. These differences reflect the distinct properties of the molecules themselves, which in turn are associated with CD45 involvement in the activation of CD4+ and CD8+ T cells.

The Tyrosine Phosphatase SHP-2 Is Required for Sustained Activation of Extracellular Signal-Regulated Kinase and Epithelial Morphogenesis Downstream from the Met Receptor Tyrosine Kinase

ABSTRACT Epithelial morphogenesis is critical during development and wound healing, and alterations in this program contribute to neoplasia. Met, the hepatocyte growth factor (HGF) receptor, promotes a morphogenic program in epithelial cell lines in matrix cultures. Previous studies have identified Gab1, the major phosphorylated protein following Met activation, as important for the morphogenic response. Gab1 is a docking protein that couples the Met receptor with multiple signaling proteins, including phosphatidylinositol-3 kinase, phospholipase Cγ, the adapter protein Crk, and the tyrosine specific phosphatase SHP-2. HGF induces sustained phosphorylation of Gab1 and sustained activation of extracellular signal-regulated kinase (Erk) in epithelial Madin-Darby canine kidney cells. In contrast, epidermal growth factor fails to promote a morphogenic program and induces transient Gab1 phosphorylation and Erk activation. To elucidate the Gab1-dependent signals required for epithelial morphogenesis, we undertook a structure-function approach and demonstrate that association of Gab1 with the tyrosine phosphatase SHP-2 is required for sustained Erk activation and for epithelial morphogenesis downstream from the Met receptor. Epithelial cells expressing a Gab1 mutant protein unable to recruit SHP-2 elicit a transient activation of Erk in response to HGF. Moreover, SHP-2 catalytic activity is required, since the expression of a catalytically inactive SHP-2 mutant, C/S, abrogates sustained activation of Erk and epithelial morphogenesis by the Met receptor. These data identify SHP-2 as a positive modulator of Erk activity and epithelial morphogenesis downstream from the Met receptor.

The Gab1 PH Domain Is Required for Localization of Gab1 at Sites of Cell-Cell Contact and Epithelial Morphogenesis Downstream from the Met Receptor Tyrosine Kinase

ABSTRACT Stimulation of the hepatocyte growth factor (HGF) receptor tyrosine kinase, Met, induces mitogenesis, motility, invasion, and branching tubulogenesis of epithelial and endothelial cell lines in culture. We have previously shown that Gab1 is the major phosphorylated protein following stimulation of the Met receptor in epithelial cells that undergo a morphogenic program in response to HGF. Gab1 is a member of the family of IRS-1-like multisubstrate docking proteins and, like IRS-1, contains an amino-terminal pleckstrin homology domain, in addition to multiple tyrosine residues that are potential binding sites for proteins that contain SH2 or PTB domains. Following stimulation of epithelial cells with HGF, Gab1 associates with phosphatidylinositol 3-kinase and the tyrosine phosphatase SHP2. Met receptor mutants that are impaired in their association with Gab1 fail to induce branching tubulogenesis. Overexpression of Gab1 rescues the Met-dependent tubulogenic response in these cell lines. The ability of Gab1 to promote tubulogenesis is dependent on its pleckstrin homology domain. Whereas the wild-type Gab1 protein is localized to areas of cell-cell contact, a Gab1 protein lacking the pleckstrin homology domain is localized predominantly in the cytoplasm. Localization of Gab1 to areas of cell-cell contact is inhibited by LY294002, demonstrating that phosphatidylinositol 3-kinase activity is required. These data show that Gab1 is an important mediator of branching tubulogenesis downstream from the Met receptor and identify phosphatidylinositol 3-kinase and the Gab1 pleckstrin homology domain as crucial for subcellular localization of Gab1 and biological responses.

A Conserved Inositol Phospholipid Binding Site within the Pleckstrin Homology Domain of the Gab1 Docking Protein Is Required for Epithelial Morphogenesis

Stimulation of the hepatocyte growth factor receptor tyrosine kinase, Met, induces the inherent morphogenic program of epithelial cells. The multisubstrate binding protein Gab1 (Grb2-associated binder-1) is the major phosphorylated protein in epithelial cells following activation of Met. Gab1 contains a pleckstrin homology domain and multiple tyrosine residues that act to couple Met with multiple signaling proteins. Met receptor mutants that are impaired in their association with Gab1 fail to induce a morphogenic program in epithelial cells, which is rescued by overexpression of Gab1. The Gab1 pleckstrin homology domain binds to phosphatidylinositol 3,4,5-trisphosphate and contains conserved residues, shown from studies of other pleckstrin homology domains to be crucial for phospholipid binding. Mutation of conserved phospholipid binding residues tryptophan 26 and arginine 29, generates Gab1 proteins with decreased phosphatidylinositol 3,4,5-trisphosphate binding, decreased localization at sites of cell-cell contact, and reduced ability to rescue Met-dependent morphogenesis. We conclude that the ability of the Gab1 pleckstrin homology domain to bind phosphatidylinositol 3,4,5-trisphosphate is critical for subcellular localization of Gab1 and for efficient morphogenesis downstream from the Met receptor.

The Gab1 Docking Protein Links the B Cell Antigen Receptor to the Phosphatidylinositol 3-Kinase/Akt Signaling Pathway and to the SHP2 Tyrosine Phosphatase

B cell antigen receptor (BCR) signaling causes tyrosine phosphorylation of the Gab1 docking protein. This allows phosphatidylinositol 3-kinase (PI3K) and the SHP2 tyrosine phosphatase to bind to Gab1. In this report, we tested the hypothesis that Gab1 acts as an amplifier of PI3K- and SHP2-dependent signaling in B lymphocytes. By overexpressing Gab1 in the WEHI-231 B cell line, we found that Gab1 can potentiate BCR-induced phosphorylation of Akt, a PI3K-dependent response. Gab1 expression also increased BCR-induced tyrosine phosphorylation of SHP2 as well as the binding of Grb2 to SHP2. We show that the pleckstrin homology (PH) domain of Gab1 is required for BCR-induced phosphorylation of Gab1 and for Gab1 participation in BCR signaling. Moreover, using confocal microscopy, we show that BCR ligation can induce the translocation of Gab1 from the cytosol to the plasma membrane and that this requires the Gab1 PH domain as well as PI3K activity. These findings are consistent with a model in which the binding of the Gab1 PH domain to PI3K-derived lipids brings Gab1 to the plasma membrane, where it can be tyrosine-phosphorylated and then act as an amplifier of BCR signaling.

Enhanced Transformation by a Plasma Membrane-Associated Met Oncoprotein: Activation of a Phosphoinositide 3′-Kinase-Dependent Autocrine Loop Involving Hyaluronic Acid and CD44

ABSTRACT A Met-hepatocyte growth factor receptor oncoprotein, Tpr-Met, generated by chromosomal rearrangement, fuses a protein dimerization motif with the cytoplasmic domain of the Met receptor, producing a cytosolic, constitutively activated tyrosine kinase. Although both the Met receptor and the Tpr-Met oncoprotein associate with the same substrates, activating mutations of the Met receptor in hereditary papillary renal carcinomas have different signaling requirements for transformation than Tpr-Met. This suggests differential activation of membrane-localized pathways by oncogenic forms of the membrane-bound Met receptor but not by the cytoplasmic Tpr-Met oncoprotein. To establish which pathways might be differentially regulated, we have localized the constitutively activated Tpr-Met oncoprotein to the membrane using the c-src myristoylation signal. Membrane localization enhances cellular transformation, focus formation, and anchorage-independent growth and induces tumors with a distinct myxoid phenotype. This correlates with the induction of hyaluronic acid (HA) and the presence of a distinct form of its receptor, CD44. A pharmacological inhibitor of phosphoinositide 3′ kinase (PI3′K), inhibits the production of HA, and conversely, an activated, plasma membrane-targeted form of PI3′K is sufficient to enhance HA production. Furthermore, the multisubstrate adapter protein Gab-1, which couples the Met receptor with PI3′K, enhances Met receptor-dependent HA synthesis in a PI3′K-dependent manner. These results provide a positive link to a role for HA and CD44 in Met receptor-mediated oncogenesis and implicate PI3′K in these events.