Scaffold Proteins IRSp53 and Spinophilin Regulate Localized Rac Activation by T-lymphocyte Invasion and Metastasis Protein 1 (TIAM1)

The Rac exchange factor Tiam1 is involved in diverse cell functions and signaling pathways through multiple protein interactions, raising the question of how signaling and functional specificity are achieved. We have shown that Tiam1 interactions with different scaffold proteins activate different Rac-dependent pathways by recruiting specific Rac effector proteins, and reasoned that there must be regulatory mechanisms governing each interaction. Fibroblasts express at least two Tiam1-interacting proteins, insulin receptor substrate protein 53 kDa (IRSp53) and spinophilin. We used fluorescent resonance energy transfer (FRET) to measure localized Rac activation associated with IRSp53 and spinophilin complexes in individual fibroblasts to test this hypothesis. Pervanadate or platelet-derived growth factor induced localized Rac activation dependent on Tiam1 and IRSp53. Forskolin or epinephrine induced localized Rac activation dependent on Tiam1 and spinophilin. In spinophilin-deficient cells, Tiam1 co-localized with IRSp53 in response to pervanadate or platelet-derived growth factor. In IRSp53-deficient cells, Tiam1 co-localized with spinophilin in response to forskolin or epinephrine. Total cellular levels of activated Rac were affected only in cells with exogenous Tiam1, and were primarily increased in the membrane fraction. Downstream effects of Rac activation were also stimulus and scaffold-specific. Cell ruffling, spreading, and cell adhesion were dependent on IRSp53, but not spinophilin. Epinephrine decreased IRSp53-dependent adhesion and increased cell migration in a Rac and spinophilin-dependent fashion. These results support the idea that Tiam1 interactions with different scaffold proteins couple distinct upstream signals to localized Rac activation and specific downstream pathways, and suggest that manipulating Tiam1-scaffold interactions can modulate Rac-dependent cellular behaviors.     

Molecular targets in immune-mediated diseases: the case of tumour necrosis factor and rheumatoid arthritis

Rheumatoid arthritis is a common autoimmune condition in which, for unknown reasons, synovial joints become the target of a sustained immune response. For many years, rheumatoid arthritis was in the 'too hard basket' in terms of understanding disease mechanisms and providing rational therapy. This has changed dramatically over the last 10 years and rheumatoid arthritis is now at the forefront of biotechnology. In this review, we outline one of the most exciting recent developments, namely antagonists of the cytokine TNF. The preclinical evaluation of TNF in animal models of rheumatoid arthritis, and subsequent clinical trials of TNF inhibitors in patients, provides insight into the 'bench to bedside' paradigm. We therefore briefly review rheumatoid arthritis, animal models of rheumatoid arthritis, the biology of TNF, the pivotal clinical trials of TNF antagonists and the emerging data on side-effects. Tumour necrosis factor inhibitors in rheumatoid arthritis represent the first attempt to achieve sustained blockade of a single cytokine in a human disease. Whilst this approach has been even more successful than might have been predicted, we suggest it is only the beginning of what has become a new therapeutic era.     

Ca2+/calmodulin-dependent kinase II phosphorylates the epidermal growth factor receptor on multiple sites in the cytoplasmic tail and serine 744 within the kinase domain to regulate signal generation.

Down-regulation of receptor tyrosine kinase activity plays an essential role in coordinating and controlling cellular growth/differentiation. Ca2+/calmodulin-dependent kinase II (CaM kinase II)-mediated phosphorylation of threonine 1172 in the cytoplasmic tail of HER2/c-erbB2 can modulate tyrosine kinase activity and consensus phosphorylation sites are also found at serines 1046/1047 in the structurally related epidermal growth factor receptor (EGFR). We show that serines 1046/1047 are sites for CaM kinase II phosphorylation, although there is a preference for serine 1047, which resides within the consensus -R-X-X-S-. In addition, we have identified major phosphorylation sites at serine 1142 and serine 1057, which lie within a novel -S-X-D- consensus. Mutation of serines 1046/1047 in full-length EGFR enhanced both fibroblast transformation and tyrosine autokinase activity that was significantly potentiated by additional mutation of serines 1057 and 1142. A single CaM kinase II site was also identified at serine 744 within sub-kinase domain III, and autokinase activity was significantly affected by mutation of this serine to an aspartic acid making this site appear constitutively phosphorylated. We have addressed the mechanism by which CaM kinase II phosphorylation of the EGFR might regulate receptor autokinase activity and show that this modification can hinder association of the cytoplasmic tail with the kinase domain to prevent an enzyme-substrate interaction. We postulate that the location and greater number of CaM kinase II phosphorylation sites in the EGFR compared with HER-2/c-erbB2, leading to differential regulation of autokinase activity, contributes to differences in the strength of downstream signaling events and may explain the higher relative transforming potential of HER-2/cerbB2.     

Interaction of a nuclear factor 1-like protein with a cAMP response element-binding protein in rat liver.

1. The existence of both cAMP-responsive element binding factor and a nuclear factor 1-like (NF-1-like) protein in nuclear extracts from liver of cAMP-treated rat has been revealed. 2. Binding of these proteins to a DNA fragment containing both elements was cooperative, and 50% binding was achieved with considerably less protein than with a fragment bearing either element alone. 3. Cleavage of the fragment between the two elements abolished the apparent cooperative interaction. 4. Southwestern blot analysis showed that the NF-1-like protein has a molecular weight in the 28-30-kDa range. 5. The NF-1-like binding activity was very stable.     

Genomewide linkage study in 1,176 affected sister pair families identifies a significant susceptibility locus for endometriosis on chromosome 10q26

Endometriosis is a common gynecological disease that affects up to 10% of women in their reproductive years. It causes pelvic pain, severe dysmenorrhea, and subfertility. The disease is defined as the presence of tissue resembling endometrium in sites outside the uterus. Its cause remains uncertain despite >50 years of hypothesis-driven research, and thus the therapeutic options are limited. Disease predisposition is inherited as a complex genetic trait, which provides an alternative route to understanding the disease. We seek to identify susceptibility loci, using a positional-cloning approach that starts with linkage analysis to identify genomic regions likely to harbor these genes. We conducted a linkage study of 1,176 families (931 from an Australian group and 245 from a U.K. group), each with at least two members--mainly affected sister pairs--with surgically diagnosed disease. We have identified a region of significant linkage on chromosome 10q26 (maximum LOD score [MLS] of 3.09; genomewide P = .047) and another region of suggestive linkage on chromosome 20p13 (MLS = 2.09). Minor peaks (with MLS > 1.0) were found on chromosomes 2, 6, 7, 8, 12, 14, 15, and 17. This is the first report of linkage to a major locus for endometriosis. The findings will facilitate discovery of novel positional genetic variants that influence the risk of developing this debilitating disease. Greater understanding of the aberrant cellular and molecular mechanisms involved in the etiology and pathophysiology of endometriosis should lead to better diagnostic methods and targeted treatments.     

CHE-3, a cytosolic dynein heavy chain, is required for sensory cilia structure and function in Caenorhabditis elegans

Forward genetic screens using novel assays of nematode chemotaxis to soluble compounds identified three independent transposon-insertion mutations in the gene encoding the Caenorhabditis elegans dynein heavy chain (DHC) 1b isoform. These disruptions were mapped and cloned using a newly developed PCR-based transposon display. The mutations were demonstrated to be allelic to the che-3 genetic locus. This isoform of dynein shows temporally and spatially restricted expression in ciliated sensory neurons, and mutants show progressive developmental defects of the chemosensory cilia. These results are consistent with a role for this motor protein in the process of intraflagellar transport; DHC 1b acts in concert with a number of other proteins to establish and maintain the structural integrity of the ciliated sensory endings in C. elegans.     

The influence of temperature and habitat on the distribution of chiselmouth, Acrocheilus alutaceus, in British Columbia

We intensively sampled fish in rivers and streams within a single major drainage basin (the Blackwater River) and across major drainages in British Columbia to assess the factors influencing distribution of chiselmouth, Acrocheilus alutaceus, and to develop models for predicting chislemouth presence. Chiselmouth were typically absent from sites with maximum temperatures below 20°C or 2100 annual degree days, both within a single drainage and between larger drainages. Indices of stream size (bankfull channel width and basin area) were the most significant predictors of chiselmouth presence within the Blackwater drainage (p=0.016 and p=0.032, respectively), and inclusion of thermal variables only marginally increased classification success. In contrast, bankfull channel width and basin area were poor predictors of chiselmouth presence in mainstem habitat within larger drainage basins throughout British Columbia. Inclusion of thermal variables (particularly degree days > 12°C) doubled correct classification rates of chiselmouth presence across larger drainage basins. These habitat associations suggest that water temperature is the primary constraint on presence of chiselmouth populations in larger drainages across a landscape, while selection of different habitat types (mainstem habitat over smaller tributaries) determines distribution within any given basin.     

Characterization of a human synovial cell antigen: VCAM-1 and inflammatory arthritis

The contribution of synovial cells to the pathogenesis of rheumatoid arthritis (RA) is only partly understood. Monoclonal antibody (mAb) 1D5 is one of very few mAb ever raised against RA synovial cells in order to study the biology of these cells. Studies on the expression pattern and structural features of the 1D5 Ag suggest that 1D5 recognizes human vascular cell adhesion molecule-1 (VCAM-1), which is an intercellular adhesion molecule. Vascular cell adhesion molecule-1 may be involved in a number of crucial intercellular interactions in RA.