Joanne C. Pratt

Essential role for Gab2 in the allergic response

Dos/Gab family scaffolding adapters (Dos, Gab1, Gab2) bind several signal relay molecules, including the protein-tyrosine phosphatase Shp-2 and phosphatidylinositol-3-OH kinase (PI(3)K); they are also implicated in growth factor, cytokine and antigen receptor signal transduction. Mice lacking Gab1 die during embryogenesis and show defective responses to several stimuli. Here we report that Gab2-/- mice are viable and generally healthy; however, the response (for example, degranulation and cytokine gene expression) of Gab2-/- mast cells to stimulation of the high affinity immunoglobulin-ε (IgE) receptor FcεRI is defective. Accordingly, allergic reactions such as passive cutaneous and systemic anaphylaxis are markedly impaired in Gab2-/- mice. Biochemical analyses reveal that signalling pathways dependent on PI(3)K, a critical component of FcεRI signalling, are defective in Gab2-/- mast cells. Our data identify Gab2 as the principal activator of PI(3)K in response to FcεRI activation, thereby providing genetic evidence that Dos/Gab family scaffolds regulate the PI(3)K pathway in vivo. Gab2 and/or its associated signalling molecules may be new targets for developing drugs to treat allergy.

Cloning of p97/Gab2, the major SHP2-binding protein in hematopoietic cells, reveals a novel pathway for cytokine-induced gene activation.

Several components in cytokine signaling remain unidentified. We report the cloning and initial characterization of one such component, p97, a widely expressed scaffolding protein distantly related to Drosophila DOS and mammalian Gab1. Upon cytokine, growth factor, or antigen receptor stimulation, p97 becomes tyrosyl phosphorylated and associates with several SH2 domain-containing proteins, including SHP2. Expression of p97 mutants unable to bind SHP2 blocks cytokine-induced c-fos promoter activation, inhibiting Elk1-mediated and STAT5-mediated transactivation. Surprisingly, such mutants do not inhibit MAPK activation. Our results identify p97 as an important regulator of receptor signaling that controls a novel pathway to immediate-early gene activation and suggest multiple functions for SHP2 in cytokine receptor signaling.

The Small GTP-Binding Protein Rho Potentiates AP-1 Transcription in T Cells

ABSTRACT The Rho family of small GTP-binding proteins is involved in the regulation of cytoskeletal structure, gene transcription, specific cell fate development, and transformation. We demonstrate in this report that overexpression of an activated form of Rho enhances AP-1 activity in Jurkat T cells in the presence of phorbol myristate acetate (PMA), but activated Rho (V14Rho) has little or no effect on NFAT, Oct-1, and NF-κB enhancer element activities under similar conditions. Overexpression of a V14Rho construct incapable of membrane localization (CAAX deleted) abolishes PMA-induced AP-1 transcriptional activation. The effect of Rho on AP-1 is independent of the mitogen-activated protein kinase pathway, as a dominant-negative MEK and a MEK inhibitor (PD98059) did not affect Rho-induced AP-1 activity. V14Rho binds strongly to protein kinase Cα (PKCα) in vivo; however, deletion of the CAAX site on V14Rho severely diminished this association. Evidence for a role for PKCα as an effector of Rho was obtained by the observation that coexpression of the N-terminal domain of PKCα blocked the effects of activated Rho plus PMA on AP-1 transcriptional activity. These data suggest that Rho potentiates AP-1 transcription during T-cell activation.

Evidence for a Physical Association between the Shc-PTB Domain and the βc Chain of the Granulocyte-Macrophage Colony-stimulating Factor Receptor

Granulocyte-macrophage colony-stimulating factor (GM-CSF) regulates the growth and function of several myeloid cell types at different stages of maturation. The effects of GM-CSF are mediated through a high affinity receptor that is composed of two chains: a unique, ligand-specific α chain and a β common chain (βc) that is also a component of the receptors for interleukin 3 (IL-3) and IL-5. βc plays an essential role in the transduction of extracellular signals to the nucleus through its recruitment of secondary messengers. Several downstream signaling events induced by GM-CSF stimulation have been described, including activation of tyrosine kinases and tyrosine phosphorylation of cellular proteins (including βc) and activation of the Ras/mitogen-activated protein kinase and the JAK/STAT pathways. A region within the βc cytoplasmic tail (amino acids 517-763) has been reported to be necessary for tyrosine phosphorylation of the adapter protein, Shc, and for the subsequent GM-CSF-induced activation of Ras. In this paper, we describe a physical association between the tyrosine phosphorylated GM-CSF receptor (GMR)-βc chain and Shc in vivo. Using a series of cytoplasmic truncation mutants of βc and various mutant Shc proteins, we demonstrate that the N-terminal phosphotyrosine-binding (PTB) domain of Shc binds to a short region of βc (amino acids 549-656) that contains Tyr577. Addition of a specific phosphopeptide encoding amino acids surrounding this tyrosine inhibited the interaction between βc and Shc. Moreover, mutation of a key residue within the phosphotyrosine binding pocket of the Shc-PTB domain abrogated its association with βc. These observations provide an explanation for the previously described requirement for Tyr577 of βc for GM-CSF-induced tyrosine phosphorylation of Shc and have implications for Ras activation through the GM-CSF, IL-3, and IL-5 receptors.

Cutting Edge: Gab2 Mediates an Inhibitory Phosphatidylinositol 3′-Kinase Pathway in T Cell Antigen Receptor Signaling

Phosphatidylinositol 3′-kinase (PI3K) is a key component of multiple signaling pathways, where it typically promotes survival, proliferation, and/or adhesion. Here, we show that in TCR signaling, the scaffolding adapter Gab2 delivers an inhibitory signal via PI3K. Overexpression of Gab2 in T cell lines inhibits TCR-evoked activation of the IL-2 promoter, blocking NF-AT- and NF-κB-directed transcription. Inhibition is abrogated by mutating the Gab2 p85-binding sites, by treatment with PI3K inhibitors or by cotransfection of phosphatase homolog of tensin. Our findings provide the first evidence of a negative function for a scaffolding adapter in T cells and identify Gab2/PI3K-containing complexes as novel regulators of TCR signaling.

Role of MEKK2-MEK5 in the regulation of TNF-α gene expression and MEKK2-MKK7 in the activation of c-Jun N-terminal kinase in mast cells

Cross-linking of the high-affinity IgE receptor (FcɛRI) on mast cells with IgE and multivalent antigen triggers mitogen-activated protein (MAP) kinase activation and cytokine gene expression. We report here that MAP kinase kinase 4 (MKK4) gene disruption does not affect either MAP kinase activation or cytokine gene expression in response to cross-linking of FcɛRI in embryonic stem cell-derived mast cells. MKK7 is activated in response to cross-linking of FcɛRI, and this activation is inhibited by MAP/ERK kinase (MEK) kinase 2 (MEKK2) gene disruption. In addition, expression of kinase-inactive MKK7 in the murine mast cell line MC/9 inhibits c-Jun NH2-terminal kinase (JNK) activation in response to cross-linking of FcɛRI, whereas expression of kinase-inactive MKK4 does not affect JNK activation by this stimulus. However, FcɛRI-induced activation of the tumor necrosis factor-α (TNF-α) gene promoter is not affected by expression of kinase-inactive MKK7. We describe an alternative pathway by which MEKK2 activates MEK5 and big MAP kinase1/extracellular signal-regulated kinase 5 in addition to MKK7 and JNK, and interruption of this pathway inhibits TNF-α promoter activation. These findings suggest that JNK activation by antigen cross-linking is dependent on the MEKK2-MKK7 pathway, and cytokine production in mast cells is regulated in part by the signaling complex MEKK2-MEK5-ERK5.

Dynamics of RASSF1A/MOAP-1 Association with Death Receptors†

ABSTRACT RASSF1A is a tumor suppressor protein involved in death receptor-dependent apoptosis utilizing the Bax-interacting protein MOAP-1 (previously referred to as MAP-1). However, the dynamics of death receptor recruitment of RASSF1A and MOAP-1 are still not understood. We have now detailed recruitment to death receptors (tumor necrosis factor receptor 1 [TNF-R1] and TRAIL-R1/DR4) and identified domains of RASSF1A and MOAP-1 that are required for death receptor interaction. Upon TNF-α stimulation, the C-terminal region of MOAP-1 associated with the death domain of TNF-R1; subsequently, RASSF1A was recruited to MOAP-1/TNF-R1 complexes. Prior to recruitment to TNF-R1/MOAP-1 complexes, RASSF1A homodimerization was lost. RASSF1A associated with the TNF-R1/MOAP-1 or TRAIL-R1/MOAP-1 complex via its N-terminal cysteine-rich (C1) domain containing a potential zinc finger binding motif. Importantly, TNF-R1 association domains on both MOAP-1 and RASSF1A were essential for death receptor-dependent apoptosis. The association of RASSF1A and MOAP-1 with death receptors involves an ordered recruitment to receptor complexes to promote cell death and inhibit tumor formation.

Direct evidence for the existence of nominal antigen binding sites on T cell surface Ti α-β heterodimers of MHC-restricted T cell clones

Abstract The binding of nominal antigen to Ti α-β heterodimers on MHC-restricted human T cell clones specific for fluorescein-5-isothiocyanate (FL) was detected by flow cytometry and affinity chromatography. The FL-Ti interaction is of physiologic significance, since T cell activation is induced by cross-linked arrays of FL in the absence of the specific MHC recognition. High antigen valence is required to achieve stable binding to cells and subsequent activation, which is consistent with estimated Ti-FL association constants of 5 l/mol. In addition to providing direct evidence that the Ti α-β heterodimer is the receptor for antigen, these data suggest that nominal antigen binding sites exist on the Ti molecules of at least some MHC-restricted clones.

The Olin curriculum: thinking toward the future

In 1997, the F. W. Olin Foundation of New York established the Franklin W. Olin College of Engineering, Needham, MA, with the mission of creating an engineering school for the 21st century. Over the last five years, the college has transformed from an idea to a functioning entity that admitted its first freshman class in fall 2002. This paper describes the broad outlines of the Olin curriculum with some emphasis on the electrical and computer engineering degree. The curriculum incorporates the best practices from many other institutions as well as new ideas and approaches in an attempt to address the future of engineering education.

Interleukin-3 (IL-3)-induced c-fos Activation Is Modulated by Gab2-Calcineurin Interaction

Interleukin-3 (IL-3) regulates cell growth by affecting various processes such as cell death, survival, and proliferation. Cues from the external environment are sensed by surface receptors, and complex signaling mechanisms arise within the cells, leading to specific functional outcomes. In this study, we demonstrate that the cytokine IL-3 induces the activation of the Ca2+-dependent phosphatase, calcineurin (Cn). Furthermore Cn dephosphorylates Gab2, resulting in c-fos activation and cell proliferation. We also report that there is a direct interaction between Cn and Gab2 upon IL-3 stimulation, and Akt can regulate this interaction.