Heteromeric MAPPIT: a novel strategy to study modification-dependent protein-protein interactions in mammalian cells

We recently reported a two-hybrid trap for detecting protein-protein interactions in intact mammalian cells (MAPPIT). The bait protein was fused to a STAT recruitment-deficient, homodimeric cytokine receptor and the prey protein to functional STAT recruitment sites. In such a configuration, STAT-dependent responses can be used to monitor a given bait-prey interaction. Using this system, we were able to demonstrate both modification-independent and tyrosine phosphorylation- dependent interactions. Protein modification in this approach is, however, strictly dependent on the receptor-associated JAK tyrosine kinases. We have now extended this concept by using extracellular domains of the heteromeric granulocyte/macrophage colony-stimulating factor receptor (GM-CSFR). Herein, the bait was fused to the (beta)c chain and its modifying enzyme to the GM-CSFRalpha chain (or vice versa). We demonstrate several serine phosphorylation-dependent interactions in the TGFbeta/Smad pathway using the catalytic domains of the ALK4 or ALK6 serine/threonine kinase receptors. In all cases tested, STAT-dependent signaling was completely abolished when mutant baits were used wherein critical serine residues were replaced by alanines. This approach operates both in transient and stable expression systems and may not be limited to serine phosphorylation but has the potential for studying various different types of protein modification-dependent interactions in intact cells.     

A novel approach for the identification of protein–protein interaction with integral membrane proteins

Protein–protein interaction plays a major role in all biological processes. The currently available genetic methods such as the two-hybrid system and the protein recruitment system are relatively limited in their ability to identify interactions with integral membrane proteins. Here we describe the development of a reverse Ras recruitment system (reverse RRS), in which the bait used encodes a membrane protein. The bait is expressed in its natural environment, the membrane, whereas the protein partner (the prey) is fused to a cytoplasmic Ras mutant. Protein–protein interaction between the proteins encoded by the prey and the bait results in Ras membrane translocation and activation of a viability pathway in yeast. We devised the expression of the bait and prey proteins under the control of dual distinct inducible promoters, thus enabling a rapid selection of transformants in which growth is attributed solely to specific protein–protein interaction. The reverse RRS approach greatly extends the usefulness of the protein recruitment systems and the use of integral membrane proteins as baits. The system serves as an attractive approach to explore novel protein–protein interactions with high specificity and selectivity, where other methods fail.     

Regulation of TGF-β receptor hetero-oligomerization and signaling by endoglin

Complex formation among transforming growth factor-β (TGF-β) receptors and its modulation by coreceptors represent an important level of regulation for TGF-β signaling. Oligomerization of ALK5 and the type II TGF-β receptor (TβRII) has been thoroughly investigated, both in vitro and in intact cells. However, such studies, especially in live cells, are missing for the endothelial cell coreceptor endoglin and for the ALK1 type I receptor, which enables endothelial cells to respond to TGF-β by activation of both Smad2/3 and Smad1/5/8. Here we combined immunoglobulin G–mediated immobilization of one cell-surface receptor with lateral mobility studies of a coexpressed receptor by fluorescence recovery after photobleaching (FRAP) to demonstrate that endoglin forms stable homodimers that function as a scaffold for binding TβRII, ALK5, and ALK1. ALK1 and ALK5 bind to endoglin with differential dependence on TβRII, which plays a major role in recruiting ALK5 to the complex. Signaling data indicate a role for the quaternary receptor complex in regulating the balance between TGF-β signaling to Smad1/5/8 and to Smad2/3.     

ZAP-70 Association with T Cell Receptor ζ (TCRζ): Fluorescence Imaging of Dynamic Changes upon Cellular Stimulation

The nonreceptor protein tyrosine kinase ZAP-70 is a critical enzyme required for successful T lymphocyte activation. After antigenic stimulation, ZAP-70 rapidly associates with T cell receptor (TCR) subunits. The kinetics of its translocation to the cell surface, the properties of its specific interaction with the TCRζ chain expressed as a chimeric protein (TTζ and Tζζ), and its mobility in different intracellular compartments were studied in individual live HeLa cells, using ZAP-70 and Tζζ fused to green fluorescent protein (ZAP-70 GFP and Tζζ–GFP, respectively). Time-lapse imaging using confocal microscopy indicated that the activation-induced redistribution of ZAP-70 to the plasma membrane, after a delayed onset, is of long duration. The presence of the TCRζ chain is critical for the redistribution, which is enhanced when an active form of the protein tyrosine kinase Lck is coexpressed. Binding specificity to TTζ was indicated using mutant ZAP-70 GFPs and a truncated ζ chimera. Photobleaching techniques revealed that ZAP-70 GFP has decreased mobility at the plasma membrane, in contrast to its rapid mobility in the cytosol and nucleus. Tζζ– GFP is relatively immobile, while peripherally located ZAP-70 in stimulated cells is less mobile than cytosolic ZAP-70 in unstimulated cells, a phenotype confirmed by determining the respective diffusion constants. Examination of the specific molecular association of signaling proteins using these approaches has provided new insights into the TCRζ–ZAP-70 interaction and will be a powerful tool for continuing studies of lymphocyte activation.     

Functional Cooperation of the Interleukin-2 Receptor β Chain and Jak1 in Phosphatidylinositol 3-Kinase Recruitment and Phosphorylation

Phosphatidylinositol 3-kinase (PI 3-K) plays an important role in signaling via a wide range of receptors such as those for antigen, growth factors, and a number of cytokines, including interleukin-2 (IL-2). PI 3-K has been implicated in both IL-2-induced proliferation and prevention of apoptosis. A number of potential mechanisms for the recruitment of PI 3-K to the IL-2 receptor have been proposed. We now have found that tyrosine residues in the IL-2 receptor β chain (IL-2Rβ) are unexpectedly not required for the recruitment of the p85 component of PI 3-K. Instead, we find that Jak1, which associates with membrane-proximal regions of the IL-2Rβ cytoplasmic domain, is essential for efficient IL-2Rβ–p85 interaction, although some IL-2Rβ–p85 association can be seen in the absence of Jak1. We also found that Jak1 interacts with p85 in the absence of IL-2Rβ and that IL-2Rβ and Jak1 cooperate for the efficient recruitment and tyrosine phosphorylation of p85. This is the first report of a PI 3-K–Jak1 interaction, and it implicates Jak1 in an essential IL-2 signaling pathway distinct from the activation of STAT proteins.     

The Transforming Growth Factor-β Type III Receptor Mediates Distinct Subcellular Trafficking and Downstream Signaling of Activin-like Kinase (ALK)3 and ALK6 Receptors

Bone morphogenetic proteins (BMPs) signal through the BMP type I and type II receptors to regulate cellular processes, including embryonic development. The type I BMP receptors activin-like kinase (ALK)3 and ALK6 share a high degree of homology, yet possess distinct signaling roles. Here, we report that although the transforming growth factor (TGF)-β type III receptor (TβRIII) enhanced both ALK3 and ALK6 signaling, TβRIII more potently enhanced ALK6-mediated stimulation of the BMP-responsive promoters XVent2 and 3GC2, and up-regulation of the early response gene Smad6. In contrast, TβRIII specifically enhanced ALK3-mediated up-regulation of the early response gene ID-1. TβRIII associated with ALK3 primarily through their extracellular domains, whereas its interaction with ALK6 required both the extracellular and cytoplasmic domains. TβRIII, along with its interacting scaffolding protein β-arrestin2, induced the internalization of ALK6. In contrast, TβRIII colocalized with and resulted in the cell surface retention of ALK3, independently of β-arrestin2. Although complex formation between TβRIII, ALK6, and β-arrestin2 and TβRIII/ALK6 internalization resulted in maximal BMP signaling, the TβRIII mutant unable to interact with β-arrestin2, TβRIII-T841A, was unable to do so. These studies support a novel role for TβRIII in mediating differential ALK3 and ALK6 subcellular trafficking resulting in distinct signaling downstream of ALK3 and ALK6.     

The Second Catalytic Domain of Protein Tyrosine Phosphatase δ (PTPδ) Binds to and Inhibits the First Catalytic Domain of PTPς

The LAR family protein tyrosine phosphatases (PTPs), including LAR, PTPδ, and PTPς, are transmembrane proteins composed of a cell adhesion molecule-like ectodomain and two cytoplasmic catalytic domains: active D1 and inactive D2. We performed a yeast two-hybrid screen with the first catalytic domain of PTPς (PTPς-D1) as bait to identify interacting regulatory proteins. Using this screen, we identified the second catalytic domain of PTPδ (PTPδ-D2) as an interactor of PTPς-D1. Both yeast two-hybrid binding assays and coprecipitation from mammalian cells revealed strong binding between PTPς-D1 and PTPδ-D2, an association which required the presence of the wedge sequence in PTPς-D1, a sequence recently shown to mediate D1-D1 homodimerization in the phosphatase RPTPα. This interaction was not reciprocal, as PTPδ-D1 did not bind PTPς-D2. Addition of a glutathione S-transferase (GST)–PTPδ-D2 fusion protein (but not GST alone) to GST–PTPς-D1 led to ~50% inhibition of the catalytic activity of PTPς-D1, as determined by an in vitro phosphatase assay against p-nitrophenylphosphate. A similar inhibition of PTPς-D1 activity was obtained with coimmunoprecipitated PTPδ-D2. Interestingly, the second catalytic domains of LAR (LAR-D2) and PTPς (PTPς-D2), very similar in sequence to PTPδ-D2, bound poorly to PTPς-D1. PTPδ-D1 and LAR-D1 were also able to bind PTPδ-D2, but more weakly than PTPς-D1, with a binding hierarchy of PTPς-D1>>PTPδ-D1>LAR-D1. These results suggest that association between PTPς-D1 and PTPδ-D2, possibly via receptor heterodimerization, provides a negative regulatory function and that the second catalytic domains of this and likely other receptor PTPs, which are often inactive, may function instead to regulate the activity of the first catalytic domains.     

Marburg Virus Evades Interferon Responses by a Mechanism Distinct from Ebola Virus

Previous studies have demonstrated that Marburg viruses (MARV) and Ebola viruses (EBOV) inhibit interferon (IFN)-α/β signaling but utilize different mechanisms. EBOV inhibits IFN signaling via its VP24 protein which blocks the nuclear accumulation of tyrosine phosphorylated STAT1. In contrast, MARV infection inhibits IFNα/β induced tyrosine phosphorylation of STAT1 and STAT2. MARV infection is now demonstrated to inhibit not only IFNα/β but also IFNγ-induced STAT phosphorylation and to inhibit the IFNα/β and IFNγ-induced tyrosine phosphorylation of upstream Janus (Jak) family kinases. Surprisingly, the MARV matrix protein VP40, not the MARV VP24 protein, has been identified to antagonize Jak and STAT tyrosine phosphorylation, to inhibit IFNα/β or IFNγ-induced gene expression and to inhibit the induction of an antiviral state by IFNα/β. Global loss of STAT and Jak tyrosine phosphorylation in response to both IFNα/β and IFNγ is reminiscent of the phenotype seen in Jak1-null cells. Consistent with this model, MARV infection and MARV VP40 expression also inhibit the Jak1-dependent, IL-6-induced tyrosine phosphorylation of STAT1 and STAT3. Finally, expression of MARV VP40 is able to prevent the tyrosine phosphorylation of Jak1, STAT1, STAT2 or STAT3 which occurs following over-expression of the Jak1 kinase. In contrast, MARV VP40 does not detectably inhibit the tyrosine phosphorylation of STAT2 or Tyk2 when Tyk2 is over-expressed. Mutation of the VP40 late domain, essential for efficient VP40 budding, has no detectable impact on inhibition of IFN signaling. This study shows that MARV inhibits IFN signaling by a mechanism different from that employed by the related EBOV. It identifies a novel function for the MARV VP40 protein and suggests that MARV may globally inhibit Jak1-dependent cytokine signaling.     

Characterization of interactions within the Igα/Igβ transmembrane domains of the human B-cell receptor provides insights into receptor assembly

The B-cell receptor (BCR), a complex comprised of a membrane-associated immunoglobulin and the Igα/β heterodimer, is one of the most important immune receptors in humans and controls B-cell development, activity, selection, and death. BCR signaling plays key roles in autoimmune diseases and lymphoproliferative disorders, yet, despite the clinical significance of this protein complex, key regions (i.e., the transmembrane domains) have yet to be structurally characterized. The mechanism for BCR signaling also remains unclear and has been variously described by the mutually exclusive cross-linking and dissociation activation models. Common to these models is the significance of local plasma membrane composition, which implies that interactions between BCR transmembrane domains (TMDs) play a role in receptor functionality. Here we used an in vivo assay of TMD oligomerization called GALLEX alongside spectroscopic and computational methods to characterize the structures and interactions of human Igα and Igβ TMDs in detergent micelles and natural membranes. We observed weak self-association of the Igβ TMD and strong self-association of the Igα TMD, which scanning mutagenesis revealed was entirely stabilized by an E–X₁₀–P motif. We also demonstrated strong heterotypic interactions between the Igα and Igβ TMDs both in vitro and in vivo, which scanning mutagenesis and computational models suggest is multiconfigurational but can accommodate distinct interaction sites for self-interactions and heterotypic interactions of the Igα TMD. Taken together, these results demonstrate that the TMDs of the human BCR are sites of strong protein–protein interactions that may direct BCR assembly, endoplasmic reticulum retention, and immune signaling.     

Structural Basis for the Binding Specificity of Human Recepteur d'Origine Nantais (RON) Receptor Tyrosine Kinase to Macrophage-stimulating Protein

Recepteur d''origine nantais (RON) receptor tyrosine kinase and its ligand, serum macrophage-stimulating protein (MSP), play important roles in inflammation, cell growth, migration, and epithelial to mesenchymal transition during tumor development. The binding of mature MSPαβ (disulfide-linked α- and β-chains) to RON ectodomain modulates receptor dimerization, followed by autophosphorylation of tyrosines in the cytoplasmic receptor kinase domains. Receptor recognition is mediated by binding of MSP β-chain (MSPβ) to the RON Sema. Here we report the structure of RON Sema-PSI-IPT₁ (SPI₁) domains in complex with MSPβ at 3.0 Å resolution. The MSPβ serine protease-like β-barrel uses the degenerate serine protease active site to recognize blades 2, 3, and 4 of the β-propeller fold of RON Sema. Despite the sequence homology between RON and MET receptor tyrosine kinase and between MSP and hepatocyte growth factor, it is well established that there is no cross-reactivity between the two receptor-ligand systems. Comparison of the structure of RON SPI₁ in complex with MSPβ and that of MET receptor tyrosine kinase Sema-PSI in complex with hepatocyte growth factor β-chain reveals the receptor-ligand selectivity determinants. Analytical ultracentrifugation studies of the SPI₁-MSPβ interaction confirm the formation of a 1:1 complex. SPI₁ and MSPαβ also associate primarily as a 1:1 complex with a binding affinity similar to that of SPI₁-MSPβ. In addition, the SPI₁-MSPαβ ultracentrifuge studies reveal a low abundance 2:2 complex with ∼10-fold lower binding affinity compared with the 1:1 species. These results support the hypothesis that the α-chain of MSPαβ mediates RON dimerization.     

SH2 Modified STAT1 Induces HLA-I Expression and Improves IFN-γ Signaling in IFN-α Resistant HCV Replicon Cells

Background

We have developed multiple stable cell lines containing subgenomic HCV RNA that are resistant to treatment with interferon alpha (IFN-α. Characterization of these IFN-α resistant replicon cells showed defects in the phosphorylation and nuclear translocation of STAT1 and STAT2 proteins due to a defective Jak-STAT pathway.

Methodology/Principal Findings

In this study, we have developed an alternative strategy to overcome interferon resistance in a cell culture model by improving intracellular STAT1 signaling. An engineered STAT1-CC molecule with double cysteine substitutions in the Src-homology 2 (SH2) domains of STAT1 (at Ala-656 and Asn-658) efficiently phosphorylates and translocates to the nucleus of IFN-resistant cells in an IFN-γ dependent manner. Transfection of a plasmid clone containing STAT1-CC significantly activated the GAS promoter compared to wild type STAT1 and STAT3. The activity of the engineered STAT1-CC is dependent upon the phosphorylation of tyrosine residue 701, since the construct with a substituted phenylalanine residue at position 701 (STAT1-CC-Y701F) failed to activate GAS promoter in the replicon cells. Intracellular expression of STAT1-CC protein showed phosphorylation and nuclear translocation in the resistant cell line after IFN-γ treatment. Transient transfection of STAT1-CC plasmid clone into an interferon resistant cell line resulted in inhibition of viral replication and viral clearance in an IFN-γ dependent manner. Furthermore, the resistant replicon cells transfected with STAT1-CC constructs significantly up regulated surface HLA-1 expression when compared to the wild type and Y to F mutant controls.

Conclusions

These results suggest that modification of the SH2 domain of the STAT1 molecule allows for improved IFN-γ signaling through increased STAT1 phosphorylation, nuclear translocation, HLA-1 surface expression, and prolonged interferon antiviral gene activation.     

Nitration of protein kinase G-Iα modulates cyclic nucleotide crosstalk via phosphodiesterase 3A: Implications for acute lung injury

Phosphodiesterase 3A (PDE3A) selectively cleaves the phosphodiester bond of cAMP and is inhibited by cGMP, making it an important regulator of cAMP–cGMP signaling crosstalk in the pulmonary vasculature. In addition, the nitric oxide–cGMP axis is known to play an important role in maintaining endothelial barrier function. However, the potential role of protein kinase G-Iα (PKG-Iα) in this protective process is unresolved and was the focus of our study. We describe here a novel mechanism regulating PDE3A activity, which involves a PKG-Iα–dependent inhibitory phosphorylation of PDE3A at serine 654. We also show that this phosphorylation is critical for maintaining intracellular cAMP levels in the pulmonary endothelium and endothelial barrier integrity. In an animal model of acute lung injury (ALI) induced by challenging mice with lipopolysaccharide (LPS), an increase in PDE3 activity and a decrease in cAMP levels in lung tissue was associated with reduced PKG activity upon PKG-Iα nitration at tyrosine 247. The peroxynitrite scavenger manganese (III) tetrakis(1-methyl-4-pyridyl)porphyrin prevented this increase in PDE3 activity in LPS-exposed lungs. In addition, site-directed mutagenesis of PDE3A to replace serine 654 with alanine yielded a mutant protein that was insensitive to PKG-dependent regulation. Taken together, our data demonstrate a novel functional link between nitrosative stress induced by LPS during ALI and the downregulation of barrier-protective intracellular cAMP levels. Our data also provide new evidence that PKG-Iα is critical for endothelial barrier maintenance and that preservation of its catalytic activity may be efficacious in ALI therapy.     

ALK1 regulates the internalization of endoglin and the type III TGF-β receptor

Complex formation and endocytosis of transforming growth factor-β (TGF-β) receptors play important roles in signaling. However, their interdependence remained unexplored. Here, we demonstrate that ALK1, a TGF-β type I receptor prevalent in endothelial cells, forms stable complexes at the cell surface with endoglin and with type III TGF-β receptors (TβRIII). We show that ALK1 undergoes clathrin-mediated endocytosis (CME) faster than ALK5, type II TGF-β receptor (TβRII), endoglin, or TβRIII. These complexes regulate the endocytosis of the TGF-β receptors, with a major effect mediated by ALK1. Thus, ALK1 enhances the endocytosis of TβRIII and endoglin, while ALK5 and TβRII mildly enhance endoglin, but not TβRIII, internalization. Conversely, the slowly endocytosed endoglin has no effect on the endocytosis of either ALK1, ALK5, or TβRII, while TβRIII has a differential effect, slowing the internalization of ALK5 and TβRII, but not ALK1. Such effects may be relevant to signaling, as BMP9-mediated Smad1/5/8 phosphorylation is inhibited by CME blockade in endothelial cells. We propose a model that links TGF-β receptor oligomerization and endocytosis, based on which endocytosis signals are exposed/functional in specific receptor complexes. This has broad implications for signaling, implying that complex formation among various receptors regulates their surface levels and signaling intensities.