The Human Formin FHOD1 Contains a Bipartite Structure of FH3 and GTPase-Binding Domains Required for Activation

SummaryFormins induce the nucleation and polymerization of unbranched actin filaments. They share three homology domains required for profilin binding, actin polymerization, and regulation. Diaphanous-related formins (DRFs) are activated by GTPases of the Rho/Rac family, whose interaction with the N-terminal formin domain is thought to displace a C-terminal Diaphanous-autoregulatory domain (DAD). We have determined the structure of the N-terminal domains of FHOD1 consisting of a GTPase-binding domain (GBD) and the DAD-recognition domain FH3. In contrast to the formin mDia1, the FHOD1-GBD reveals a ubiquitin superfold as found similarly in c-Raf1 or PI3 kinase. This GBD is recruited by Rac and Ras GTPases in cells and plays an essential role for FHOD1-mediated actin remodeling. The FHOD1-FH3 domain is composed of five armadillo repeats, similarly to other formins. Mutation of one residue in the predicted DAD-interaction surface efficiently activates FHOD1 in cells. These results demonstrate that DRFs have evolved different molecular solutions to govern their autoregulation and GTPase specificity

The formin FHOD1 and the small GTPase Rac1 promote vaccinia virus actin-based motility

Vaccinia virus dissemination relies on the N-WASP– ARP2/3 pathway, which mediates actin tail formation underneath cell-associated extracellular viruses (CEVs). Here, we uncover a previously unappreciated role for the formin FHOD1 and the small GTPase Rac1 in vaccinia actin tail formation. FHOD1 depletion decreased the number of CEVs forming actin tails and impaired the elongation rate of the formed actin tails. Recruitment of FHOD1 to actin tails relied on its GTPase binding domain in addition to its FH2 domain. In agreement with previous studies showing that FHOD1 is activated by the small GTPase Rac1, Rac1 was enriched and activated at the membrane surrounding actin tails. Rac1 depletion or expression of dominant-negative Rac1 phenocopied the effects of FHOD1 depletion and impaired the recruitment of FHOD1 to actin tails. FHOD1 overexpression rescued the actin tail formation defects observed in cells overexpressing dominant-negative Rac1. Altogether, our results indicate that, to display robust actin-based motility, vaccinia virus integrates the activity of the N-WASP– ARP2/3 and Rac1–FHOD1 pathways.Fil: Alvarez, Diego Ezequiel. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. University of Yale. School of Medicine; Estados UnidosFil: Agaisse, Herve. University of Yale. School of Medicine; Estados Unido

β-Catenin is a pH sensor with decreased stability at higher intracellular pH.

β-Catenin functions as an adherens junction protein for cell-cell adhesion and as a signaling protein. β-catenin function is dependent on its stability, which is regulated by protein-protein interactions that stabilize β-catenin or target it for proteasome-mediated degradation. In this study, we show that β-catenin stability is regulated by intracellular pH (pHi) dynamics, with decreased stability at higher pHi in both mammalian cells and Drosophila melanogaster β-Catenin degradation requires phosphorylation of N-terminal residues for recognition by the E3 ligase β-TrCP. While β-catenin phosphorylation was pH independent, higher pHi induced increased β-TrCP binding and decreased β-catenin stability. An evolutionarily conserved histidine in β-catenin (found in the β-TrCP DSGIHS destruction motif) is required for pH-dependent binding to β-TrCP. Expressing a cancer-associated H36R-β-catenin mutant in the Drosophila eye was sufficient to induce Wnt signaling and produced pronounced tumors not seen with other oncogenic β-catenin alleles. We identify pHi dynamics as a previously unrecognized regulator of β-catenin stability, functioning in coincidence with phosphorylation

Considering Protonation as a Posttranslational Modification Regulating Protein Structure and Function

Post-translational modification of proteins is an evolutionarily conserved mechanism for regulating activity, binding affinities and stability. Compared with established post-translational modifications such as phosphorylation or uniquitination, post-translational modification by protons within physiological pH ranges is a less recognized mechanism for regulating protein function. By changing the charge of amino acid side chains, post-translational modification by protons can drive dynamical changes in protein conformation and function. Addition and removal of a proton is rapid and reversible and in contrast to most other post-translational modifications does not require an enzyme. Signaling specificity is achieved by only a minority of sites in proteins titrating within the physiological pH range. Here, we examine the structural mechanisms and functional consequences of proton post-translational modification of pH-sensing proteins regulating different cellular processes

Septins suppress the release of vaccinia virus from infected cells.

Septins are conserved components of the cytoskeleton that play important roles in many fundamental cellular processes including division, migration, and membrane trafficking. Septins can also inhibit bacterial infection by forming cage-like structures around pathogens such as Shigella We found that septins are recruited to vaccinia virus immediately after its fusion with the plasma membrane during viral egress. RNA interference-mediated depletion of septins increases virus release and cell-to-cell spread, as well as actin tail formation. Live cell imaging reveals that septins are displaced from the virus when it induces actin polymerization. Septin loss, however, depends on the recruitment of the SH2/SH3 adaptor Nck, but not the activity of the Arp2/3 complex. Moreover, it is the recruitment of dynamin by the third Nck SH3 domain that displaces septins from the virus in a formin-dependent fashion. Our study demonstrates that septins suppress vaccinia release by "entrapping" the virus at the plasma membrane. This antiviral effect is overcome by dynamin together with formin-mediated actin polymerization

pHocal adhesion kinase regulation is on a FERM foundation

Increases in intracellular pH (pHi) occur upon integrin receptor binding to matrix proteins and in tumor cells. In this issue, Choi et al. (2013. J. Cell Biol. http://dx.doi.org/10.1083/jcb.201308034) show that pHi increase activates FAK by causing deprotonation of histidine 58 in its FERM (band 4.1, ezrin, radixin, moesin) homology domain, which exposes a region important for FAK autophosphorylation. This model of FAK activation could contribute to motility of tumor cells by promoting focal adhesion turnover

Temperature-induced changes in the wheat phosphoproteome reveal temperature-regulated interconversion of phosphoforms

Wheat (Triticum ssp.) is one of the most important human food sources. However, this crop is very sensitive to temperature changes. Specifically, processes during wheat leaf, flower, and seed development and photosynthesis, which all contribute to the yield of this crop, are affected by high temperature. While this has to some extent been investigated on physiological, developmental, and molecular levels, very little is known about early signalling events associated with an increase in temperature. Phosphorylation-mediated signalling mechanisms, which are quick and dynamic, are associated with plant growth and development, also under abiotic stress conditions. Therefore, we probed the impact of a short-term and mild increase in temperature on the wheat leaf and spikelet phosphoproteome. In total, 3822 (containing 5178 phosphosites) and 5581 phosphopeptides (containing 7023 phosphosites) were identified in leaf and spikelet samples, respectively. Following statistical analysis, the resulting data set provides the scientific community with a first large-scale plant phosphoproteome under the control of higher ambient temperature. This community resource on the high temperature-mediated wheat phosphoproteome will be valuable for future studies. Our analyses also revealed a core set of common proteins between leaf and spikelet, suggesting some level of conserved regulatory mechanisms. Furthermore, we observed temperature-regulated interconversion of phosphoforms, which probably impacts protein activity

Talin as a mechanosensitive signaling hub

Cell adhesion to the extracellular matrix (ECM), mediated by transmembrane receptors of the integrin family, is exquisitely sensitive to biochemical, structural, and mechanical features of the ECM. Talin is a cytoplasmic protein consisting of a globular head domain and a series of ?-helical bundles that form its long rod domain. Talin binds to the cytoplasmic domain of integrin ?-subunits, activates integrins, couples them to the actin cytoskeleton, and regulates integrin signaling. Recent evidence suggests switch-like behavior of the helix bundles that make up the talin rod domains, where individual domains open at different tension levels, exerting positive or negative effects on different protein interactions. These results lead us to propose that talin functions as a mechanosensitive signaling hub that integrates multiple extracellular and intracellular inputs to define a major axis of adhesion signaling