CIB1 prevents nuclear GAPDH accumulation and non-apoptotic tumor cell death via AKT and ERK signaling.

CIB1 is a 22-kDa regulatory protein previously implicated in cell survival and proliferation. However, the mechanism by which CIB1 regulates these processes is poorly defined. Here, we report that CIB1 depletion in SK-N-SH neuroblastoma and MDA-MB-468 breast cancer cells promotes non-apoptotic, caspase-independent cell death that is not initiated by increased outer mitochondrial membrane permeability or translocation of apoptosis-inducing factor to the nucleus. Instead, cell death requires nuclear GAPDH accumulation. Furthermore, CIB1 depletion disrupts two commonly dysregulated, oncogenic pathways-PI3K/AKT and Ras/MEK/ERK, resulting in a synergistic mechanism of cell death, which was mimicked by simultaneous pharmacological inhibition of both pathways, but not either pathway alone. In defining each pathway's contributions, we found that AKT inhibition alone maximally induced GAPDH nuclear accumulation, whereas MEK/ERK inhibition alone had no effect on GAPDH localization. Concurrent GAPDH nuclear accumulation and ERK inhibition were required, however, to induce a significant DNA damage response, which was critical to subsequent cell death. Collectively, our results indicate that CIB1 is uniquely positioned to regulate PI3K/AKT and MEK/ERK signaling and that simultaneous disruption of these pathways synergistically induces a nuclear GAPDH-dependent cell death. The mechanistic insights into cell death induced by CIB1 interference suggest novel molecular targets for cancer therapy.

Bidirectional transmembrane modulation of integrin alphaIIbbeta3 conformations.

Activation of blood platelets by physiological stimuli (e.g. thrombin, ADP) at sites of vascular injury induces inside-out signaling, resulting in a conformational change of the prototype integrin alphaIIbbeta3 from an inactive to an active state competent to bind soluble fibrinogen. Furthermore, ligand occupancy of alphaIIbbeta3 initiates outside-in signaling and additional conformational changes of the receptor, leading to the exposure of extracellular neoepitopes termed ligand-induced binding sites (LIBS), which are recognized by anti-LIBS monoclonal antibodies. To date, the mechanism of bidirectional transmembrane signaling of alphaIIbbeta3 has not been established. In this study, using our newly developed anti-LIBScyt1 monoclonal antibody, we showed that extracellular ligand binding to alphaIIbbeta3 on blood platelets induces a transmembrane conformational change in alphaIIbbeta3, thereby exposing the LIBScyt1 epitope in the alphaIIb cytoplasmic sequence between Lys994 and Asp1003. In addition, a point mutation at this site (P998A/P999A) renders alphaIIbbeta3 constitutively active to bind extracellular ligands, resulting in fibrinogen-dependent cell-cell aggregation. Taken collectively, these results demonstrated that the extracellular ligand-binding site and a cytoplasmic LIBS epitope in integrin alphaIIbbeta3 are conformationally and functionally coupled. Such bidirectional modulation of alphaIIbbeta3 conformation across the cell membrane may play a key role in inside-out and outside-in signaling via this integrin.

Direct binding of the platelet integrin alphaIIbbeta3 (GPIIb-IIIa) to talin. Evidence that interaction is mediated through the cytoplasmic domains of both alphaIIb and beta3.

As a consequence of platelet activation and fibrinogen binding, glycoprotein (GP)IIb-IIIa (integrin alphaIIbbeta3) becomes associated with the cytoskeleton. Although talin has been suggested to act as a linkage protein mediating the attachment of GPIIb-IIIa to actin filaments, direct binding of GPIIb-IIIa to this cytoskeletal protein has not been demonstrated. In the present study, we examined the interaction of GPIIb-IIIa with purified talin using a solid-phase binding assay. Soluble GPIIb-IIIa bound in a time- and dose-dependent manner to microtiter wells coated with talin but not with BSA. Time course studies demonstrated that steady-state binding was achieved after 4-5 h incubation at 37 degrees C. Binding isotherms with varying concentrations of GPIIb-IIIa showed that half-saturation binding was achieved at approximately 15 nM GPIIb-IIIa. At saturation, there was 211 +/- 8 fmol of GPIIb-IIIa bound per well containing 117 +/- 10 fmol of immobilized talin. Besides binding to immobilized talin, GPIIb-IIIa also bound to talin captured by the anti-talin monoclonal antibody 8d4. Moreover, the interaction of GPIIb-IIIa to 8d4-captured talin was blocked by mAb10B2, a monoclonal antibody raised against a synthetic peptide encompassing the entire cytoplasmic sequence of GPIIb. The interaction of talin with the cytoplasmic domain of GPIIb-IIIa was further investigated using peptide-coated wells. Purified talin was found to bind to both synthetic peptides corresponding to the cytoplasmic sequences of GPIIb (P2b) and GPIIIa (P3a). As expected, the binding of talin to P2b-coated wells was specifically blocked by mAb10B2. Thus, these results demonstrate direct binding of GPIIb-IIIa to talin and suggest a role of the cytoplasmic sequences of both GPIIb and GPIIIa in mediating this interaction.