Integrins play a role in fibroblast growth factor (FGF) signaling through cross-talk with FGF receptors (FGFRs), but the mechanism underlying the cross-talk is unknown. We discovered that FGF1 directly bound to soluble and cell-surface integrin ␣v␤3 (K D about 1 M). Antagonists to ␣v␤3 (monoclonal antibody 7E3 and cyclic RGDfV) blocked this interaction. ␣v␤3 was the predominant, if not the only, integrin that bound to FGF1, because FGF1 bound only weakly to several ␤1 integrins tested. We presented evidence that the CYDMKTTC sequence (the specificity loop) within the ligand-binding site of ␤3 plays a role in FGF1 binding. We found that the integrin-binding site of FGF1 overlaps with the heparin-binding site but is distinct from the FGFR-binding site using docking simulation and mutagenesis. We identified an FGF1 mutant (R50E) that was defective in integrin binding but still bound to heparin and FGFR. R50E was defective in inducing DNA synthesis, cell proliferation, cell migration, and chemotaxis, suggesting that the direct integrin binding to FGF1 is critical for FGF signaling. Nevertheless, R50E induced phosphorylation of FGFR1 and FRS2␣ and activation of AKT and ERK1/2. These results suggest that the defect in R50E in FGF signaling is not in the initial activation of FGF signaling pathway components, but in the later steps in FGF signaling. We propose that R50E is a useful tool to identify the role of integrins in FGF signaling. Fibroblast growth factors (FGFs)2 constitute a family of heparin-binding polypeptides involved in the regulation of biological responses such as growth, differentiation, and angiogenesis (1-4). The FGF family currently consists of 22 members with FGF1 (acidic FGF) and FGF2 (basic FGF) the most extensively studied. The biological effects of FGFs are mediated by four structurally related receptor tyrosine kinases designated FGFR1-4. The binding of FGF to its receptor results in receptor dimerization and subsequent autophosphorylation of specific tyrosine residues within the cytoplasmic domain (1-4). Activation of the receptor allows proteins containing Src homology 2 or phosphotyrosine binding domains to bind to sequence recognition motifs in the FGFR, resulting in phosphorylation and activation of these proteins (5). This leads to the activation of intracellular signaling cascades. The main signaling cascade activated through the stimulation of FGFR is the Ras/MAPK pathway.FGF signaling enhances multiple biological processes that promote tumor progression (6). Therapies targeting FGF receptors and/or FGF signaling not only affect the growth of the tumor cells but also modulate tumor angiogenesis (7). FGF1 and FGF2 are responsible for resistance to chemotherapeutic agents in cancer (8 -11) and are also pro-inflammatory growth factors that play a role in pathological angiogenesis in chronic inflammatory diseases (12). Thus FGF signaling is a potential therapeutic target for cancer and pathological angiogenesis in chronic inflammatory diseases.It has been proposed that cross-talk between i...

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Several small molecule ligands for amyloid‐β (Aβ) fibrils deposited in brain have been developed to facilitate radiological diagnosis of Alzheimer’s disease (AD). Recently, the build‐up of Aβ oligomers (AβO) in brain has been recognized as an additional hallmark of AD and may play a more significant role in early stages. Evidence suggests that quantitative assessment of AβO would provide a more accurate index of therapeutic effect of drug trials. Therefore, there is an urgent need to develop methods for efficient identification as well as structural analysis of AβO. We found that some well established amyloid ligands, analogs of Congo red and thioflavin‐T (ThT), bind AβO with high affinity and detect AβO in vitro and in vivo. Binding studies revealed the presence of binding sites for Congo red‐ and thioflavin‐T‐analogs on AβO. Furthermore, these ligands can be used for imaging intracellular AβO in living cells and animals and as positive contrast agent for ultrastructural imaging of AβO, two applications useful for structural analysis of AβO in cells. We propose that by improving the binding affinity of current ligands, in vivo imaging of AβO is feasible by a ‘signal subtraction’ procedure. This approach may facilitate the identification of individuals with early AD.

Sumoylation has emerged as a major post-translational modification of cellular proteins, affecting a variety of cellular processes. Viruses have exploited the sumoylation pathway to advance their own replication by evolving several ways to perturb the host sumoylation apparatus. However, there has been no report of virally encoded enzymes directly involved in catalyzing the sumoylation reaction. Here, we report that the K-bZIP protein encoded by Kaposi's sarcoma-associated herpesvirus (KSHV) is a SUMO E3 ligase with specificity toward SUMO2/3. K-bZIP is a nuclear factor that functions to modulate viral gene expression and to prolong the G1 phase, allowing viral transcription and translation to proceed at the early stage of infection. In addition to functioning as a transcriptional factor, we show that K-bZIP carries a SIM (SUMO-interacting motif), which specifically binds to SUMO-2/3 but not SUMO-1. K-bZIP catalyzes its own SUMO modification as well as that of its interacting partners such as the cellular tumor suppressor proteins p53 and Rb, both in vitro and in vivo. This reaction depends on an intact SIM. Sumoylation of p53 leads to its activation and K-bZIP is recruited to several p53 target chromatin sites in a SIM-dependent manner. In addition to the identification of a viral SUMO-2/3 E3 ligase, our results provide additional insights into the mechanisms whereby K-bZIP induces cell cycle arrest.Increasing evidence indicates that sumoylation, i.e. posttranslational modification of proteins by the small ubiquitinlike modifier (SUMO) 2 plays a central role in cellular signal transduction. Like phosphorylation, sumoylation is rapid and reversible. In a manner similar to the binding of phosphorylated tyrosine by signal molecules carrying Src homology 2 (SH2) and phosphotyrosine binding (PTB) domains, sumoylated proteins are specifically engaged by proteins with a SUMO-interacting motif (SIM). Modulation of sumoylation has a profound effect on protein-protein interactions and the propagation of cellular signals. Viruses have evolved different mechanisms to exploit the host sumoylation pathway to create a cellular environment that is favorable for viral replication by modulating the functions of viral and cellular proteins (reviewed in Ref. 1). Many viral proteins are themselves sumoylated, and this post-translational modification affects specific functions of these targets. For DNA tumor viruses, the immediate-early and early gene products, which include transcriptional factors, are often sumoylated. Examples include immediate-early 1 (IE 1) and immediate-early 2 (IE 2) proteins of cytomegalovirus (CMV) (2, 3), E1 and E2 of human papillomavirus (HPV) (4, 5), BZLF1 of Epstein-Barr virus (EBV) (6), and K-bZIP of Kaposi's sarcomaassociated herpesvirus (KSHV) (7). Some viral proteins indirectly modulate the sumoylation status of specific cellular proteins. For example, the HPV E7 protein and adenovirus E1A protein block sumoylation of the cellular tumor suppressor Rb (8). Additionally, the KSHV viral protein kinase ...

Secretory phospholipase A2 group IIA (sPLA2-IIA) plays an important role in the pathogenesis of inflammatory diseases. Catalytic activity of this enzyme that generates arachidonic acid is a major target for development of anti-inflammatory agents. Independent of its catalytic activity, sPLA2-IIA induces pro-inflammatory signals in a receptor-mediated mechanism (e.g. through the M-type receptor). However, the M-type receptor is species-specific: sPLA2-IIA binds to the M-type receptor in rodents and rabbits, but not in human. Thus sPLA2-IIA receptors in human have not been established. Here we demonstrated that sPLA2-IIA bound to integrin ␣v␤3 at a high affinity (K D ‫؍‬ 2 ؋ 10 ؊7 M). We identified amino acid residues in sPLA2-IIA (Arg-74 and Arg-100) that are critical for integrin binding using docking simulation and mutagenesis. The integrin-binding site did not include the catalytic center or the M-type receptor-binding site. sPLA2-IIA also bound to ␣4␤1. We showed that sPLA2-IIA competed with VCAM-1 for binding to ␣4␤1, and bound to a site close to those for VCAM-1 and CS-1 in the ␣4 subunit. Wild type and the catalytically inactive H47Q mutant of sPLA2-IIA induced cell proliferation and ERK1/2 activation in monocytic cells, but the integrin binding-defective R74E/R100E mutant did not. This indicates that integrin binding is required, but catalytic activity is not required, for sPLA2-IIA-induced proliferative signaling. These results suggest that integrins ␣v␤3 and ␣4␤1 may serve as receptors for sPLA2-IIA and mediate pro-inflammatory action of sPLA2-IIA, and that integrinsPLA2-IIA interaction is a novel therapeutic target. The phospholipase A2 (PLA2)2 family is a group of intracellular and secreted enzymes that hydrolyzes the sn-2 ester bond in the glyceroacyl phospholipids present in lipoproteins and cell membranes to form nonesterified fatty acids and lysophospholipids. These products act as intracellular second messengers or are further metabolized into potent mediators of a broad range of cellular processes, including inflammation, apoptosis, and atherogenesis (1). The mammalian secretory PLA2 isoforms are comprised of the groups named IB, IIA, IIC, IID, IIE, IIF, V, X, and XII (2, 3). All secretory PLA2 isoforms have in common a Ca 2ϩ -dependent catalytic mechanism, a low molecular mass (13-16 kDa), several disulfide bridges, and a wellconserved overall three-dimensional structure (2, 4, 5). Secretory PLA2 type IIA (sPLA2-IIA) was first isolated and purified from rheumatoid synovial fluid. sPLA2-IIA is an acute phase reactant and is found in markedly increased plasma concentrations in diseases that involve systemic inflammation such as sepsis, rheumatoid arthritis, and cardiovascular disease (up to 1000-fold and Ͼ1 g/ml). Inflammatory cytokines such as IL-6, TNF-␣, and IL-1␤ induce synthesis and release of sPLA2-IIA in arterial smooth muscle cells and hepatocytes, which are the major sources of the plasma sPLA2-IIA in these systemic inflammatory conditions (6, 7). In addition to being a pro-inflammatory ...