ABSTRACT
Protein-protein interactions (PPIs) are central in cell metabolism but research tools for the structural and functional characterization of these PPIs are often missing. Here we introduce broadly applicable immunization (Cross-link PPIs and immunize llamas, ChILL) and selection strategies (Display and co-selection, DisCO) for the discovery of diverse nanobodies that either stabilize or disrupt PPIs in a single experiment. We apply ChILL and DisCO to identify competitive, connective, or fully allosteric nanobodies that inhibit or facilitate the formation of the SOS1â¢RAS complex and modulate the nucleotide exchange rate on this pivotal GTPase in vitro as well as RAS signalling in cellulo. One of these connective nanobodies fills a cavity that was previously identified as the binding pocket for a series of therapeutic lead compounds. The long complementarity-determining region (CDR3) that penetrates this binding pocket serves as pharmacophore for extending the repertoire of potential leads.
Subject(s)
Protein Binding , SOS1 Protein , Single-Domain Antibodies , Single-Domain Antibodies/chemistry , Single-Domain Antibodies/immunology , Single-Domain Antibodies/metabolism , SOS1 Protein/metabolism , SOS1 Protein/chemistry , SOS1 Protein/genetics , SOS1 Protein/immunology , Humans , Animals , Allosteric Regulation , ras Proteins/metabolism , ras Proteins/chemistry , Complementarity Determining Regions/chemistry , Complementarity Determining Regions/immunology , Binding Sites , Camelids, New World/immunology , Immunization , Signal Transduction , Models, MolecularABSTRACT
JAK-dependent activation of the rho module of integrin affinity triggering mediates chemokine-induced leukocyte adhesion. However, the signaling events linking JAKs to rho small GTPase activation by chemokines is still incompletely described. In this study, we show that son of sevenless 1 (SOS1), rho guanine nucleotide exchange factor (GEF)1 (ARHGEF1), and dedicator of cytokinesis (DOCK)2 GEFs mediate CXCL12-induced LFA-1 activation in human primary T lymphocytes. Downregulated expression of SOS1, ARHGEF1, and DOCK2 impairs LFA-1-mediated rapid T lymphocyte adhesion as well as underflow arrest on ICAM-1 induced by CXCL12. Moreover, LFA-1 affinity triggering by CXCL12 is impaired by SOS1, ARHGEF1, and DOCK2 downregulation. Notably, the three GEFs are all critically involved in chemokine-induced RhoA and Rac1 activation, thus suggesting the occurrence of a SOS1 specificity shift in the context of chemokine signaling. Accordingly, SOS1, ARHGEF1, and DOCK2 are tyrosine phosphorylated upon chemokine signaling with timing coherent with rapid LFA-1 affinity activation. Importantly, chemokine-induced tyrosine phosphorylation of these GEFs is fully mediated by JAK protein tyrosine kinases. Unexpectedly, and differently from VAV1, tyrosine phosphorylation of SOS1, ARHGEF1, and DOCK2 is completely inhibited by pertussis toxin pretreatment, thus suggesting different routes of rho-GEF triggering upon CXCR4 engagement. Taken together, these findings reveal a deeper level of complexity in the rho-signaling module, with at least four different rho-GEFs cooperating in the regulation of chemokine-induced integrin activation, possibly suggesting the emergence of stochastic concurrency in signaling mechanisms controlling leukocyte trafficking.
Subject(s)
Lymphocyte Activation/immunology , Signal Transduction/immunology , T-Lymphocytes/immunology , Blotting, Western , Chemokines/immunology , GTPase-Activating Proteins , Guanine Nucleotide Exchange Factors/immunology , Humans , Immunoprecipitation , Janus Kinases/immunology , Lymphocyte Function-Associated Antigen-1/immunology , Rho Guanine Nucleotide Exchange Factors/immunology , SOS1 Protein/immunologyABSTRACT
Podosomes are protrusive structures implicated in macrophage extracellular matrix degradation and three-dimensional migration through cell barriers and the interstitium. Podosome formation and assembly are regulated by cytoskeleton remodeling requiring cytoplasmic tyrosine kinases of the Src and the Abl families. Considering that Abl has been reported to phosphorylate the guanine nucleotide exchange factor Sos1, eliciting its Rac-guanine nucleotide exchange factor activity, and Rac regulates podosome formation in myeloid cells and invadopodia formation in cancer cells, we addressed whether Sos1 is implicated in podosome formation and function in macrophages. We found that ectopically expressed Abl or the Src kinase Fgr phosphorylate Sos1, and the Src kinases Hck and Fgr are required for Abl and Sos1 phosphorylation and Abl/Sos1 interaction in macrophages. Sos1 localizes to podosomes in both murine and human macrophages, and its silencing by small interfering RNA results in disassembly of murine macrophage podosomes and a marked reduction of GTP loading on Rac. Matrix degradative capacity, three-dimensional migration through Matrigel, and transmigration through an endothelial cell monolayer of Sos1-silenced macrophages were inhibited. In addition, Sos1- or Abl-silenced macrophages, or macrophages treated with the selective Abl inhibitor imatinib mesylate had a reduced capability to migrate into breast tumor spheroids, the majority of cells remaining at the margin and the outer layers of the spheroid itself. Because of the established role of Src and Abl kinases to regulate also invadopodia formation in cancer cells, our findings suggest that targeting the Src/Abl/Sos1/Rac pathway may represent a double-edged sword to control both cancer-invasive capacities and cancer-related inflammation.
Subject(s)
Cell Movement/immunology , Macrophages/immunology , Neoplasms/immunology , Proto-Oncogene Proteins/immunology , SOS1 Protein/immunology , src-Family Kinases/immunology , Animals , COS Cells , Cell Movement/drug effects , Cell Movement/genetics , Chlorocebus aethiops , Humans , Imatinib Mesylate/pharmacology , Macrophages/pathology , Mice , Neoplasm Invasiveness/genetics , Neoplasm Invasiveness/immunology , Neoplasms/genetics , Neoplasms/pathology , Phosphorylation/drug effects , Phosphorylation/genetics , Phosphorylation/immunology , Podosomes , Proto-Oncogene Proteins/genetics , Proto-Oncogene Proteins c-abl/antagonists & inhibitors , Proto-Oncogene Proteins c-abl/genetics , Proto-Oncogene Proteins c-abl/immunology , SOS1 Protein/genetics , rac GTP-Binding Proteins/genetics , rac GTP-Binding Proteins/immunology , src-Family Kinases/geneticsABSTRACT
Sos-1 and Sos-2 are ubiquitously expressed Ras-guanine exchange factors involved in Erk-MAP kinase pathway activation. Using mice lacking genes encoding Sos-1 and Sos-2, we evaluated the role of these proteins in peripheral T-cell signaling and function. Our results confirmed that TCR-mediated Erk activation in peripheral CD4(+) T cells does not depend on Sos-1 and Sos-2, although IL-2-mediated Erk activation does. Unexpectedly, however, we show an increase in AKT phosphorylation in Sos-1/2dKO CD4(+) T cells upon TCR and IL-2 stimulation. Activation of AKT was likely a consequence of increased recruitment of PI3K to Grb2 upon TCR and/or IL-2 stimulation in Sos-1/2dKO CD4(+) T cells. The increased activity of the PI3K/AKT pathway led to downregulation of the surface receptor CD62L in Sos-1/2dKO T cells and a subsequent impairment in T-cell migration.
Subject(s)
CD4-Positive T-Lymphocytes/immunology , Cell Movement/immunology , Phosphatidylinositol 3-Kinases/immunology , SOS1 Protein/immunology , Signal Transduction/immunology , Son of Sevenless Proteins/immunology , Animals , Cell Movement/genetics , Enzyme Activation/genetics , Enzyme Activation/immunology , GRB2 Adaptor Protein/genetics , GRB2 Adaptor Protein/immunology , Interleukin-2/genetics , Interleukin-2/immunology , L-Selectin/genetics , L-Selectin/immunology , Mice , Mice, Knockout , Phosphatidylinositol 3-Kinases/genetics , Phosphorylation/genetics , Phosphorylation/immunology , Proto-Oncogene Proteins c-akt/genetics , Proto-Oncogene Proteins c-akt/immunology , Receptors, Antigen, T-Cell/genetics , Receptors, Antigen, T-Cell/immunology , SOS1 Protein/genetics , Signal Transduction/genetics , Son of Sevenless Proteins/geneticsABSTRACT
The duration and/or the magnitude of Ras-Erk activation are known to be crucial for cell-fate decisions. In T cells, sustained Erk activation correlates with differentiation/proliferation, whereas transient Erk activation parallels with unresponsiveness/apoptosis. The mechanism by which Son of sevenless (Sos) proteins and Ras guanyl-releasing protein 1 (RasGRP1) contribute to dynamics of Erk activation in mature T cells is not yet known. Here, we have assessed this issue using stimuli inducing either transient or sustained TCR signaling and RNA interference mediated suppression of Sos1, Sos2, and RasGRP1 expression in primary human T cells. We found that transient Erk activation depends on RasGRP1 but not on Sos. Conversely, sustained Erk signaling and T-cell activation depend on both Sos1 and RasGRP1. In summary, our data show for the first time that the two guanine nucleotide exchange factors expressed in T cells are differentially involved in the regulation of the duration of Erk phosphorylation and T-cell activation.
Subject(s)
Extracellular Signal-Regulated MAP Kinases/immunology , Lymphocyte Activation/physiology , MAP Kinase Signaling System/physiology , Receptors, Antigen, T-Cell/immunology , SOS1 Protein/immunology , T-Lymphocytes/immunology , DNA-Binding Proteins/biosynthesis , DNA-Binding Proteins/genetics , DNA-Binding Proteins/immunology , Enzyme Activation/physiology , Extracellular Signal-Regulated MAP Kinases/biosynthesis , Extracellular Signal-Regulated MAP Kinases/genetics , Female , Gene Expression Regulation/physiology , Guanine Nucleotide Exchange Factors/biosynthesis , Guanine Nucleotide Exchange Factors/genetics , Guanine Nucleotide Exchange Factors/immunology , Humans , Male , Phosphorylation/physiology , Receptors, Antigen, T-Cell/genetics , Receptors, Antigen, T-Cell/metabolism , SOS1 Protein/genetics , SOS1 Protein/metabolism , Son of Sevenless Proteins/genetics , Son of Sevenless Proteins/immunology , Son of Sevenless Proteins/metabolism , T-Lymphocytes/cytology , T-Lymphocytes/metabolismABSTRACT
Activation of the small G protein Ras is required for thymocyte differentiation. In thymocytes, Ras is activated by the Ras guanine exchange factors (RasGEFs) Sos1, Sos2, and RasGRP1. We report the development of a floxed allele of sos1 to assess the role of Sos1 during thymocyte development. Sos1 was required for pre-T-cell receptor (pre-TCR)- but not TCR-stimulated developmental signals. Sos1 deletion led to a partial block at the DN-to-DP transition. Sos1-deficient thymocytes showed reduced pre-TCR-stimulated proliferation, differentiation, and ERK phosphorylation. In contrast, TCR-stimulated positive selection, and negative selection under strong stimulatory conditions, remained intact in Sos1-deficient mice. Comparison of RasGEF expression at different developmental stages showed that relative to Sos2 and RasGRP1, Sos1 is most abundant in DN thymocytes, but least abundant in DP thymocytes. These data reveal that Sos1 is uniquely positioned to affect signal transduction early in thymocyte development.