Efficient T-cell receptor signaling requires a high-affinity interaction between the Gads C-SH3 domain and the SLP-76 RxxK motif

The relationship between the binding affinity and specificity of modular interaction domains is potentially important in determining biological signaling responses. In signaling from the T-cell receptor (TCR), the Gads C-terminal SH3 domain binds a core RxxK sequence motif in the SLP-76 scaffold. We show that residues surrounding this motif are largely optimized for binding the Gads C-SH3 domain resulting in a high-affinity interaction (K(D)=8-20 nM) that is essential for efficient TCR signaling in Jurkat T cells, since Gads-mediated signaling declines with decreasing affinity. Furthermore, the SLP-76 RxxK motif has evolved a very high specificity for the Gads C-SH3 domain. However, TCR signaling in Jurkat cells is tolerant of potential SLP-76 crossreactivity, provided that very high-affinity binding to the Gads C-SH3 domain is maintained. These data provide a quantitative argument that the affinity of the Gads C-SH3 domain for SLP-76 is physiologically important and suggest that the integrity of TCR signaling in vivo is sustained both by strong selection of SLP-76 for the Gads C-SH3 domain and by a capacity to buffer intrinsic crossreactivity.     

Reciprocal cross talk between gonadotropin-releasing hormone (GnRH) and prostaglandin receptors regulates GnRH receptor expression and differential gonadotropin secretion

The asynchronous secretion of gonadotrope LH and FSH under the control of GnRH is crucial for ovarian cyclicity but the underlying mechanism is not fully resolved. Because prostaglandins (PG) are autocrine regulators in many tissues, we determined whether they have this role in gonadotropes. We first demonstrated that GnRH stimulates PG synthesis by induction of cyclooxygenase-2, via the protein kinase C/c-Src/phosphatidylinositol 3'-kinase/MAPK pathway in the LbetaT2 gonadotrope cell line. We then demonstrated that PGF(2alpha) and PGI2, but not PGE2 inhibited GnRH receptor expression by inhibition of phosphoinositide turnover. PGF(2alpha), but not PGI2 or PGE2, reduced GnRH-induction of LHbeta gene expression, but not the alpha-gonadotropin subunit or the FSHbeta subunit genes. The prostanoid receptors EP1, EP2, FP, and IP were expressed in rat gonadotropes. Incubations of rat pituitaries with PGF(2alpha), but not PGI2 or PGE2, inhibited GnRH-induced LH secretion, whereas the cyclooxygenase inhibitor, indomethacin, stimulated GnRH-induced LH secretion. None of these treatments had any effect on GnRH-induced FSH secretion. The findings have thus elaborated a novel GnRH signaling pathway mediated by PGF(2alpha)-FP and PGI2-IP, which acts through an autocrine/paracrine modality to limit autoregulation of the GnRH receptor and differentially inhibit LH and FSH release. These findings provide a mechanism for asynchronous LH and FSH secretions and suggest the use of combination therapies of GnRH and prostanoid analogs to treat infertility, diseases with unbalanced LH and FSH secretion and in hormone-dependent diseases such as prostatic cancer.     

The K15 protein of Kaposi's sarcoma-associated herpesvirus recruits the endocytic regulator intersectin 2 through a selective SH3 domain interaction

Kaposi's sarcoma-associated herpesvirus, also known as human herpesvirus 8, is closely associated with several cancers including Kaposi's sarcoma, primary effusion lymphoma, and multicentric Castleman's disease. The rightmost end of the KSHV genome encodes a protein, K15, with multiple membrane-spanning segments and an intracellular carboxy-terminal tail that contains several conserved motifs with the potential to recruit interaction domains (i.e., SH2, SH3, TRAF) of host cell proteins. K15 has been implicated in downregulating B cell receptor (BCR) signaling through its conserved motifs and may thereby play a role in maintaining viral latency and/or preventing apoptosis of the infected B cells. However, K15's mode of action is largely unknown. We have used mass spectrometry, domain and peptide arrays, and surface plasmon resonance to identify binding partners for a conserved proline-rich sequence (PPLP) in the K15 cytoplasmic tail. We show that the PPLP motif selectively binds the SH3-C domain of an endocytic adaptor protein, Intersectin 2 (ITSN2). This interaction can be observed both in vitro and in cells, where K15 and ITSN2 colocalize to discrete compartments within the B cell. The ability of K15 to associate with ITSN2 suggests a new role for the K15 viral protein in intracellular protein trafficking.     

Time Space Translation: A Hox Mechanism for Vertebrate A-P Patterning

The vertebrate A-P axis is a time axis. The head is made first and more and more posterior levels are made at later and later stages. This is different to the situation in most other animals, for example, in Drosophila. Central to this timing is Hox temporal collinearity (see below). This occurs rarely in the animal kingdom but is characteristic of vertebrates and is used to generate the primary axial Hox pattern using time space translation and to integrate successive derived patterns (see below). This is thus a different situation than in Drosophila, where the primary pattern guiding Hox spatial collinearity is generated externally, by the gap and segmentation genes.     

Histone recognition and large-scale structural analysis of the human bromodomain family

Bromodomains (BRDs) are protein interaction modules that specifically recognize ε-N-lysine acetylation motifs, a key event in the reading process of epigenetic marks. The 61 BRDs in the human genome cluster into eight families based on structure/sequence similarity. Here, we present 29 high-resolution crystal structures, covering all BRD families. Comprehensive crossfamily structural analysis identifies conserved and family-specific structural features that are necessary for specific acetylation-dependent substrate recognition. Screening of?more than 30 representative BRDs against systematic histone-peptide arrays identifies new BRD substrates and reveals a strong influence of flanking posttranslational modifications, such as acetylation and phosphorylation, suggesting that BRDs recognize combinations of marks rather than singly acetylated sequences. We further uncovered a structural mechanism for the simultaneous binding and recognition of diverse diacetyl-containing peptides by BRD4. These data provide a foundation for structure-based drug design of specific inhibitors for this emerging target family.     

Network medicine

To more effectively target complex diseases like cancer, diabetes and schizophrenia, we may need to rethink our strategies for drug development and the selection of molecular targets for pharmacological treatments. Here, we discuss the potential use of protein signaling networks as the targets for new therapeutic intervention. We argue that by targeting the architecture of aberrant signaling networks associated with cancer and other diseases new therapeutic strategies can be implemented. Transforming medicine into a network driven endeavour will require quantitative measurements of cell signaling processes; we will describe how this may be performed and combined with new algorithms to predict the trajectories taken by a cellular system either in time or through disease states. We term this approach, network medicine.     

The synthetic triterpenoid 2-cyano-3,12-dioxooleana-1,9-dien-28-oic acid-imidazolide alters transforming growth factor beta-dependent signaling and cell migration by affecting the cytoskeleton and the polarity complex

The anti-tumor synthetic triterpenoid 2-cyano-3,12-dioxooleana-1,9-dien-28-oic acid (CDDO)-imidazolide (CDDO-Im) ectopically activates the transforming growth factor beta (TGFbeta)-Smad pathway and extends the duration of signaling by an undefined mechanism. Here we show that CDDO-Imdependent persistence of Smad2 phosphorylation is independent of Smad2 phosphatase activity and correlates with delayed TGFbeta receptor degradation and trafficking. Altered TGFbeta trafficking parallels the dispersal of EEA1-positive endosomes from the perinuclear region of CDDO-Im-treated cells. The effect of CDDO-Im on the EEA1 compartment led to an analysis of the cytoskeleton, and we observed that CDDO-Im alters microtubule dynamics by disrupting the microtubule-capping protein, Clip-170. Interestingly, biotinylated triterpenoid was found to localize to the polarity complex at the leading edge of migrating cells. Furthermore, CDDO-Im disrupted the localization of IQGAP1, PKCzeta, Par6, and TGFbeta receptors from the leading edge of migrating cells and inhibited TGFbeta-dependent cell migration. Thus, the synthetic triterpenoid CDDO-Im interferes with TGFbeta receptor trafficking and turnover and disrupts cell migration by severing the link between members of the polarity complex and the microtubule network.     

Saccharomyces cerevisiae Ste50 binds the MAPKKK Ste11 through a head-to-tail SAM domain interaction

In Saccharomyces cerevisiae, signal transduction through pathways governing mating, osmoregulation, and nitrogen starvation depends upon a direct interaction between the sterile alpha motif (SAM) domains of the Ste11 mitogen-activated protein kinase kinase kinase (MAPKKK) and its regulator Ste50. Previously, we solved the NMR structure of the SAM domain from Ste11 and identified two mutants that diminished binding to the Ste50 SAM domain. Building upon the Ste11 study, we present the NMR structure of the monomeric Ste50 SAM domain and a series of mutants bearing substitutions at surface-exposed hydrophobic amino acid residues. The mid-loop (ML) region of Ste11-SAM, defined by helices H3 and H4 and the end-helix (EH) region of Ste50-SAM, defined by helix H5, were sensitive to substitution, indicating that these two surfaces contribute to the high-affinity interaction. The combination of two mutants, Ste11-SAM-L72R and Ste50-SAM-L69R, formed a high-affinity heterodimer unencumbered by competing homotypic interactions that had prevented earlier NMR studies of the wild-type complex. Yeast bearing mutations that prevented the heterotypic Ste11-Ste50 association in vitro presented signaling defects in the mating and high-osmolarity growth pathways.