The guanine nucleotide exchange factor (GEF) Vav1 is essential for transducing T cell antigen receptor (TCR) signals and therefore plays a critical role in the development and activation of T cells. It has been presumed that the GEF activity of Vav1 is important for its function; however, there has been no direct demonstration of this. Here, we generated mice expressing enzymatically inactive, but normally folded, Vav1 protein. Analysis of these mice showed that the GEF activity of Vav1 was necessary for the selection of thymocytes and for the optimal activation of T cells, including signal transduction to Rac1, Akt, and integrins. In contrast, the GEF activity of Vav1 was not required for TCR-induced calcium flux, activation of extracellular signal–regulated kinase (ERK) and protein kinase D1 (PKD1), and cell polarization. Thus, in T cells, the GEF activity of Vav1 is essential for some, but not all, of its functions.
LUBAC synthesizes linear ubiquitin chains via a thioester intermediate
The N-terminus of the LUBAC catalytic subunit is shown to be autoinhibitory and counteracted by the other subunits of the complex. Linear ubiquitination proceeds through a thioesther intermediate, indicative of a RING/HECT hybrid mechanism.
The linear ubiquitin chain assembly complex (LUBAC) is a RING E3 ligase that regulates immune and inflammatory signalling pathways. Unlike classical RING E3 ligases, LUBAC determines the type of ubiquitin chain being formed, an activity normally associated with the E2 enzyme. We show that the RING-in-between-RING (RBR)-containing region of HOIP—the catalytic subunit of LUBAC—is sufficient to generate linear ubiquitin chains. However, this activity is inhibited by the N-terminal portion of the molecule, an inhibition that is released upon complex formation with HOIL-1L or SHARPIN. Furthermore, we demonstrate that HOIP transfers ubiquitin to the substrate through a thioester intermediate formed by a conserved cysteine in the RING2 domain, supporting the notion that RBR ligases act as RING/HECT hybrids.
E3 ligase; mechanism; thioester; ubiquitination
The expression, purification and crystallization of an N-terminal fragment of SHARPIN are reported. Diffraction-quality crystals were obtained using a two-dimensional grid-screen seeding technique.
An N-terminal fragment of human SHARPIN was recombinantly expressed in Escherichia coli, purified and crystallized. Crystals suitable for X-ray diffraction were obtained by a one-step optimization of seed dilution and protein concentration using a two-dimensional grid screen. The crystals belonged to the primitive tetragonal space group P43212, with unit-cell parameters a = b = 61.55, c = 222.81 Å. Complete data sets were collected from native and selenomethionine-substituted protein crystals at 100 K to 2.6 and 2.0 Å resolution, respectively.
Background: SHARPIN is a subunit of the E3 ligase complex LUBAC and the gene that is mutated in chronic proliferative dermatitis mice.
Results: The N-terminal portion of SHARPIN adopts the PH superfold and mediates homodimerization.
Conclusion: The PH superfold can be used as a protein dimerization module.
Significance: This study highlights the versatility of the PH superfold and its function as a protein interaction module.
SHARPIN (SHANK-associated RH domain interacting protein) is part of a large multi-protein E3 ubiquitin ligase complex called LUBAC (linear ubiquitin chain assembly complex), which catalyzes the formation of linear ubiquitin chains and regulates immune and apoptopic signaling pathways. The C-terminal half of SHARPIN contains ubiquitin-like domain and Npl4-zinc finger domains that mediate the interaction with the LUBAC subunit HOIP and ubiquitin, respectively. In contrast, the N-terminal region does not show any homology with known protein interaction domains but has been suggested to be responsible for self-association of SHARPIN, presumably via a coiled-coil region. We have determined the crystal structure of the N-terminal portion of SHARPIN, which adopts the highly conserved pleckstrin homology superfold that is often used as a scaffold to create protein interaction modules. We show that in SHARPIN, this domain does not appear to be used as a ligand recognition domain because it lacks many of the surface properties that are present in other pleckstrin homology fold-based interaction modules. Instead, it acts as a dimerization module extending the functional applications of this superfold.
Protein Structure; Protein-Protein Interactions; Signal Transduction; Ubiquitin Ligase; Ubiquitination; X-ray Crystallography
SHARPIN is a ubiquitin-binding and ubiquitin-like domain-containing protein which, when mutated in mice, results in immune system disorders and multiorgan inflammation1,2. Here we report that SHARPIN functions as a novel component of the Linear Ubiquitin Chain Assembly Complex (LUBAC) and that the absence of SHARPIN causes disregulation of NF-κB and apoptotic signalling pathways, explaining the severe phenotypes displayed by chronic proliferative dermatitis in SHARPIN deficient mice. Upon binding to the LUBAC subunit HOIP, SHARPIN stimulates the formation of linear ubiquitin chains in vitro and in vivo. Co-expression of SHARPIN and HOIP promotes linear ubiquitylation of NEMO, an adaptor of the IκB kinases (IKKs) and subsequent activation of NF-κB signalling, while SHARPIN deficiency in mice causes an impaired activation of the IKK complex and NF-κB in B cells, macrophages, and mouse embryonic fibroblasts (MEFs). This effect is further enhanced upon concurrent downregulation of HOIL-1L, another HOIP-binding component of LUBAC. In addition, SHARPIN deficiency leads to rapid cell death upon TNFα stimulation via FADD- and Caspase-8-dependent pathways. SHARPIN thus activates NF-κB and inhibits apoptosis via distinct pathways in vivo.
Caspase recruitment domains (CARDs) are homotypic protein interaction modules that link the stimulus-dependent assembly of large signaling platforms such as inflammasomes to the activation of downstream effectors that often include caspases and kinases and thereby play an important role in the regulation of inflammatory and apoptotic signaling pathways. NOD2 belongs to the NOD-like (NLR) family of intracellular pattern recognition receptors (PRR) and induces activation of the NF-κB pathway in response to the recognition of bacterial components. This process requires the specific recognition of the CARD of the protein kinase RIP2 by the tandem CARDs of NOD2. Here we demonstrate that the tandem CARDs of NOD2 are engaged in an intramolecular interaction that is important for the structural stability of this region. Using a combination of ITC and pull-down experiments we identify distinct surface areas that are involved in the intramolecular tandem CARD interaction and the interaction with the downstream effector RIP2. Our findings indicate that while CARDa of NOD2 might be the primary binding partner of RIP2 the two CARDs of NOD2 do not act independently of one another but may cooperate to from a binding surface that is distinct from that of single CARDs.
Reactive oxygen species (ROS) have been known for a long time to play important roles in host defense against microbial infections. In addition, it has become apparent that they also perform regulatory roles in signal transduction and cell proliferation. The source of these chemicals are members of the NOX family of NADPH oxidases that are found in a variety of tissues. NOX1, an NADPH oxidase homologue that is most abundantly expressed in colon epithelial cells, requires the regulatory subunits NOXO1 (NOX organizing protein 1) and NOXA1 (NOX activating protein 1), as well as the flavocytochrome component p22phox for maximal activity. Unlike NOX2, the phagocytic NADPH oxidase whose activity is tightly repressed in the resting state, NOX1 produces superoxide constitutively at low levels. These levels can be further increased in a stimulus-dependent manner, yet the molecular details regulating this activity are not fully understood. Here we present the first quantitative characterization of the interactions made between the cytosolic regulators NOXO1 and NOXA1 and membrane-bound p22phox. Using isothermal titration calorimetry we show that the isolated tandem SH3 domains of NOXO1 bind to p22phox with high affinity, most likely adopting a superSH3 domain conformation. In contrast, complex formation is severely inhibited in the presence of the C-terminal tail of NOXO1, suggesting that this region competes for binding to p22phox and thereby contributes to the regulation of superoxide production. Furthermore, we provide data indicating that the molecular details of the interaction between NOXO1 and NOXA1 is significantly different from that between the homologous proteins of the phagocytic oxidase, suggesting that there are important functional differences between the two systems. Taken together, this study provides clear evidence that the assembly of the NOX1 oxidase complex can be regulated through reversible protein-protein interactions.
The IKK [IκB (inhibitory κB) kinase] complex is a key regulatory component of NF-κB (nuclear factor κB) activation and is responsible for mediating the degradation of IκB, thereby allowing nuclear translocation of NF-κB and transcription of target genes. NEMO (NF-κB essential modulator), the regulatory subunit of the IKK complex, plays a pivotal role in this process by integrating upstream signals, in particular the recognition of polyubiquitin chains, and relaying these to the activation of IKKα and IKKβ, the catalytic subunits of the IKK complex. The oligomeric state of NEMO is controversial and the mechanism by which it regulates activation of the IKK complex is poorly understood. Using a combination of hydrodynamic techniques we now show that apo-NEMO is a highly elongated, dimeric protein that is in weak equilibrium with a tetrameric assembly. Interaction with peptides derived from IKKβ disrupts formation of the tetrameric NEMO complex, indicating that interaction with IKKα and IKKβ and tetramerization are mutually exclusive. Furthermore, we show that NEMO binds to linear di-ubiquitin with a stoichiometry of one molecule of di-ubiquitin per NEMO dimer. This stoichiometry is preserved in a construct comprising the second coiled-coil region and the leucine zipper and in one that essentially spans the full-length protein. However, our data show that at high di-ubiquitin concentrations a second weaker binding site becomes apparent, implying that two different NEMO–di-ubiquitin complexes are formed during the IKK activation process. We propose that the role of these two complexes is to provide a threshold for activation, thereby ensuring sufficient specificity during NF-κB signalling.
analytical ultracentrifugation (AUC); IKK (inhibitory κB kinase) complex; isothermal titration calorimetry (ITC); multi-angle light scattering (MALS); protein–protein interaction; ubiquitin; AUC, analytical ultracentrifugation; CoZi, C-terminal portion of NEMO encompassing the second coiled-coil region and the leucine zipper domain; IκB, inhibitory κB; IKK, IκB kinase; IKKNBD, IKKβ NEMO-binding domain; ITC, isothermal titration calorimetry; LZ, leucine zipper; MALS, multi-angle laser light scattering; NF-κB, nuclear factor κB; NEMO, NF-κB essential modulator; NEMO355, NEMO residues 1–355, with mutations C54S and K285N; SEC, size-exclusion chromatography; TCEP, tris-(2-carboxyethyl)phosphine; RI, refractive index; RIP1, receptor interaction protein kinase; vFLIP, viral FLICE [FADD (Fas-associated death domain)-like interleukin 1β-converting enzyme]-inhibitory protein; ZF, zinc finger
The Vav family of proteins are guanine nucleotide exchange factors (GEFs) for the Rho family of GTPases, which regulate various cellular functions, including T-cell activation. They contain a catalytic Dbl homology (DH) domain that is invariably followed by a pleckstrin homology (PH) domain, which is often required for catalytic activity. Vav proteins are the first GEFs for which an additional C1 domain is required for full biological activity. Here, we present the structure of a Vav1 fragment comprising the DH–PH–C1 domains bound to Rac1. This structure shows that the PH and C1 domains form a single structural unit that packs against the carboxy-terminal helix of the DH domain to stabilize its conformation and to promote nucleotide exchange. In contrast to previous reports, this structure shows that there are no direct contacts between the GTPase and C1 domain but instead suggests new mechanisms for the regulation of Vav1 activity.
C1 domain; exchange factor; GTPase; Vav1; X-ray crystallography