Interleukin 4 Regulates Phosphorylation of Serine 756 in the Transactivation Domain of Stat6: ROLES FOR MULTIPLE PHOSPHORYLATION SITES AND Stat6 FUNCTION

Lymphokines interleukin-4 (IL4) and IL13 exert overlapping biological activities via the shared use of the IL4 receptor α-chain and signal transducer and activator of transcription 6 (Stat6). Stat6 is critical for T-helper 2 cell differentiation, B-cell Ig class switch, and allergic diseases; thus, understanding its regulation is of central importance. Phosphorylation is crucial for Stat activity. Whereas Stat6 is phosphorylated on Tyr641, less is known about serine or threonine. We demonstrate in primary human T-cells (>95% CD3+) that IL4 and for the first time IL13 induce Stat6 serine but not threonine phosphorylation that closely paralleled early IL4 receptor α-chain activation (10 min). Stat6 uniquely fails to share a positionally conserved Stat serine phosphorylation sequence; however, known phosphoacceptor sites are proline-flanked. Alanine substitutions of these conserved residues revealed that the transactivation domain, which localized Ser756 but not Ser827 or Ser176, is the IL4-regulated site based on phosphoamino acid analysis. Tyr641 was dispensable for IL4-mediated serine phosphorylation, suggesting that dimerization is not preconditional. Only Stat6 Y641F variant showed a significant effect on IL4-inducible Cϵ DNA-binding and reporter gene expression. Lastly, recent work has shown that protein phosphatase 2A negatively regulates Stat6 (Woetmann, A., Brockdorff, J., Lovato, P., Nielsen, M., Leick, V., Rieneck, K., Svejgaard, A., Geisler, C., and Odum, N. (2003) J. Biol. Chem. 278, 2787–2791). We propose this target residue(s) is distinct from Ser756 and may be proximal to Tyr641 at Thr645, a residue conserved only among Stat6 members. The phosphomimic variants T645E or T645D ablated Stat6 activation, whereas polar uncharged substitutions (Gln or Asn) and additional mutants (Ala, Val, or Phe) showed no effect. These findings suggest that Stat6 has mechanisms of regulation distinct from other Stats

Molecular Basis for the Dual Function of Eps8 on Actin Dynamics: Bundling and Capping

The unusual dual functions of the actin-binding protein EPS8 as an actin capping and actin bundling factor are mapped to distinct structural features of the protein and to distinct physiological activities in vivo

Molecularly Distinct Clathrin-Coated Pits Differentially Impact EGFR Fate and Signaling

International audienc

Requirements for F-BAR Proteins TOCA-1 and TOCA-2 in Actin Dynamics and Membrane Trafficking during Caenorhabditis elegans Oocyte Growth and Embryonic Epidermal Morphogenesis

The TOCA family of F-BAR–containing proteins bind to and remodel lipid bilayers via their conserved F-BAR domains, and regulate actin dynamics via their N-Wasp binding SH3 domains. Thus, these proteins are predicted to play a pivotal role in coordinating membrane traffic with actin dynamics during cell migration and tissue morphogenesis. By combining genetic analysis in Caenorhabditis elegans with cellular biochemical experiments in mammalian cells, we showed that: i) loss of CeTOCA proteins reduced the efficiency of Clathrin-mediated endocytosis (CME) in oocytes. Genetic interference with CeTOCAs interacting proteins WSP-1 and WVE-1, and other components of the WVE-1 complex, produced a similar effect. Oocyte endocytosis defects correlated well with reduced egg production in these mutants. ii) CeTOCA proteins localize to cell–cell junctions and are required for proper embryonic morphogenesis, to position hypodermal cells and to organize junctional actin and the junction-associated protein AJM-1. iii) Double mutant analysis indicated that the toca genes act in the same pathway as the nematode homologue of N-WASP/WASP, wsp-1. Furthermore, mammalian TOCA-1 and C. elegans CeTOCAs physically associated with N-WASP and WSP-1 directly, or WAVE2 indirectly via ABI-1. Thus, we propose that TOCA proteins control tissues morphogenesis by coordinating Clathrin-dependent membrane trafficking with WAVE and N-WASP–dependent actin-dynamics

A Snapshot of the Physical and Functional Wiring of the Eps15 Homology Domain Network in the Nematode

<div><p>Protein interaction modules coordinate the connections within and the activity of intracellular signaling networks. The Eps15 Homology (EH) module, a protein-protein interaction domain that is a key feature of the EH-network, was originally identified in a few proteins involved in endocytosis and vesicle trafficking, and has subsequently also been implicated in actin reorganization, nuclear shuttling, and DNA repair. Here we report an extensive characterization of the physical connections and of the functional wirings of the EH-network in the nematode. Our data show that one of the major physiological roles of the EH-network is in neurotransmission. In addition, we found that the proteins of the network intersect, and possibly coordinate, a number of “territories” of cellular activity including endocytosis/recycling/vesicle transport, actin dynamics, general metabolism and signal transduction, ubiquitination/degradation of proteins, DNA replication/repair, and miRNA biogenesis and processing.</p> </div

Yeast Two Hybrid analysis of EH-proteins in <i>C. elegans</i>.

<p>(A) Schematic diagram of the five EH-containing proteins in <i>C. elegans</i>. Note that several isoforms are reported in wormbase. Here, we show the isoforms cloned, sequenced and used for the described experiments. Baits used for the Y2H are indicated by black lines. For EHS-1, two distinct baits were used in the screens, since a bait spanning the three EH domains showed self-activation. CC, coiled-coil region; SH3, region containing multiple SH3s in ITSN-1; PxxP, region containing multiple SH3-binding sites in EHS-1; DPFs, region containing multiple AP-2-binding sites in EHS-1; P-loop, nucleotide-binding domain in RME-1. (B) Results of the Y2H screen. The 26 identified EH-interactors are listed. Potential EH-binding motifs are indicated. Black, interactions detected in the initial screen; gray, interactions detected in the re-transformation assay (see text). The number of clones identified in the initial screen is also shown. No interactions were detected for R10E11.6. (C) The indicated genes were tested by quantitative PCR in the yeast library used for the Y2H screening. The number of EH-interacting motifs (NPF) and the frequency of identification in the Y2H (H, high; In, intermediate; L, low; No, no interaction) are shown at the bottom. The estimated number of copies present in the cDNA library is shown, by grey bars, in arbitrary units relative to the level of representation of <i>epn</i>-1 that was set to 100. As a comparison we show, using black bars, the frequency of isolation of the various clones in Y2H, again relative to the frequency of isolation of <i>epn</i>-1 that was set to 100 ( = 45 clones).</p

Effect of RNAi of EH interactors in various genetic backgrounds.

<p>Down-regulation of the EH-interactors was achieved by feeding RNA interference (RNAi), in the indicated strains, and animals were tested for aldicarb sensitivity. (A) In the column N2, the effect of RNAi on aldicarb sensitivity in wild type (N2) animals is reported (H, hypersensitive to aldicarb, R, resistant to aldicarb). In the other columns, the type of genetic interaction, detected in the various strains, is reported (S, suppressing; W, worsening; Rv, reverting; A, asynthetic; L, lethal; Ep, possibly RNAi epistatic; see also <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0056383#pone-0056383-t001" target="_blank">Table 1</a>). (B) Examples of the detected genetic interactions. Results are expressed as the change in the λ parameter in the best-fitting Weibull distribution with respect to WT. “KO strain”, null mutant for the EH-containing gene; “RNAi in WT”, N2 worms in which the EH-interactor was silenced by RNAi; “RNAi in KO strain”, null mutants for the EH-containing gene in which the EH-interactor was silenced by RNAi; Null hypothesis, mathematical sum of the observed phenotypes in the “KO strain” and “RNAi in WT conditions”. Details of the analysis are in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0056383#s2" target="_blank">Materials and Methods</a>.</p

In vitro binding assays.

<p>Sixteen interactors, identified by Y2H (listed at the bottom), were expressed as GST-fusion proteins and used for <i>in vitro</i> binding assays with FLAG-EH proteins expressed in Phoenix cells. Results are the average of three independent experiments (examples are shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0056383#pone.0056383.s003" target="_blank">Figure S3</a>), and are expressed in arbitrary units on a scale 0–100, in which 100 represents the efficiency of the pull-down for the strongest interacting protein in each panel.</p

The EH network in <i>C. elegans</i>. An interaction diagram is shown representing <i>C. elegans</i> EH proteins (red circles) together with their interactors (blue circles); the interactors are further grouped into functional categories that were derived from the Wormbase and the Gene Ontology databases, from the literature, or inferred from functions of the mammalian homologues.

<p>Interactions uncovered in this study by Y2H are shown by light blue lines. Interactions confirmed by <i>in vitro</i> binding assays are shown by dark blue lines. Interactions not fully depending on the EH domain are shown with dashed lines. Additional interactions, derived from the BioGRID database (<a href="http://thebiogrid.org/" target="_blank">http://thebiogrid.org/</a>) and from the literature, are shown by red lines. The picture was initially generated using the Osprey software <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0056383#pone.0056383-Breitkreutz1" target="_blank">[96]</a> and then edited with Adobe Illustrator.</p