The Guanine-Nucleotide Exchange Factor Caldag Gefi Fine-Tunes Functional Properties of Regulatory T Cells

Using quantitative phosphopeptide sequencing of unstimulated versus stimulated primary murine Foxp3+ regulatory and Foxp3– conventional T cells (Tregs and Tconv, respectively), we detected a novel and differentially regulated tyrosine phosphorylation site within the C1 domain of the guanine-nucleotide exchange factor CalDAG GEFI. We hypothesized that the Treg-specific and activation-dependent reduced phosphorylation at Y523 allows binding of CalDAG GEFI to diacylglycerol, thereby impacting the formation of a Treg-specific immunological synapse. However, diacylglycerol binding assays of phosphomutant C1 domains of CalDAG GEFI could not confirm this hypothesis. Moreover, CalDAG GEFI–/– mice displayed normal Treg numbers in thymus and secondary lymphoid organs, and CalDAG GEFI–/– Tregs showed unaltered in vitro suppressive capacity when compared to CalDAG GEFI+/+ Tregs. Interestingly, when tested in vivo, CalDAG GEFI–/– Tregs displayed a slightly reduced suppressive ability in the transfer colitis model when compared to CalDAG GEFI+/+ Tregs. Additionally, CRISPR-Cas9-generated CalDAG GEFI–/– Jurkat T cell clones showed reduced adhesion to ICAM-1 and fibronectin when compared to CalDAG GEFI-competent Jurkat T cells. Therefore, we speculate that deficiency in CalDAG GEFI impairs adherence of Tregs to antigen-presenting cells, thereby impeding formation of a fully functional immunological synapse, which finally results in a reduced suppressive potential.

[1]  J. Sondek,et al.  A negative-feedback loop regulating ERK1/2 activation and mediated by RasGPR2 phosphorylation. , 2016, Biochemical and biophysical research communications.

[2]  Megan L. Peach,et al.  Structural Basis for the Failure of the C1 Domain of Ras Guanine Nucleotide Releasing Protein 2 (RasGRP2) to Bind Phorbol Ester with High Affinity* , 2016, The Journal of Biological Chemistry.

[3]  M. Beckstette,et al.  Foxp3+ T cells expressing RORγt represent a stable regulatory T-cell effector lineage with enhanced suppressive capacity during intestinal inflammation , 2015, Mucosal Immunology.

[4]  Feng Zhang,et al.  Genome engineering using CRISPR-Cas9 system. , 2015, Methods in molecular biology.

[5]  G. Stein,et al.  Epigenetic landscape during osteoblastogenesis defines a differentiation-dependent Runx2 promoter region. , 2014, Gene.

[6]  W. Baumgärtner,et al.  Induced and thymus‐derived Foxp3+ regulatory T cells share a common niche , 2014, European journal of immunology.

[7]  R. Zamoyska,et al.  T cell receptor signalling networks: branched, diversified and bounded , 2013, Nature Reviews Immunology.

[8]  Suet-Mien Tan,et al.  The leucocyte β2 (CD18) integrins: the structure, functional regulation and signalling properties. , 2012, Bioscience reports.

[9]  N. Hogg,et al.  The insider's guide to leukocyte integrin signalling and function , 2011, Nature Reviews Immunology.

[10]  R. Alon,et al.  Occupancy of Lymphocyte LFA-1 by Surface-Immobilized ICAM-1 Is Critical for TCR- but Not for Chemokine-Triggered LFA-1 Conversion to an Open Headpiece High-Affinity State , 2010, The Journal of Immunology.

[11]  Sudha Kumari,et al.  Protein Kinase C-θ Mediates Negative Feedback on Regulatory T Cell Function , 2010, Science.

[12]  Jason S. Mitchell,et al.  The WAVE2 complex regulates T cell receptor signaling to integrins via Abl- and CrkL–C3G-mediated activation of Rap1 , 2008, The Journal of cell biology.

[13]  Shimon Sakaguchi,et al.  Foxp3+ natural regulatory T cells preferentially form aggregates on dendritic cells in vitro and actively inhibit their maturation , 2008, Proceedings of the National Academy of Sciences.

[14]  David A. Williams,et al.  Rapid lentiviral transduction preserves the engraftment potential of Fanca(-/-) hematopoietic stem cells. , 2008, Molecular therapy : the journal of the American Society of Gene Therapy.

[15]  T. Nomura,et al.  Regulatory T Cells and Immune Tolerance , 2008, Cell.

[16]  C. Hsieh,et al.  A two-step process for thymic regulatory T cell development. , 2008, Immunity.

[17]  D. Wagner,et al.  CalDAG-GEFI and protein kinase C represent alternative pathways leading to activation of integrin alphaIIbbeta3 in platelets. , 2007, Blood.

[18]  T. Mayadas,et al.  Essential role for Rap1 GTPase and its guanine exchange factor CalDAG-GEFI in LFA-1 but not VLA-4 integrin mediated human T-cell adhesion. , 2007, Blood.

[19]  R. Cornell,et al.  Differential membrane binding and diacylglycerol recognition by C1 domains of RasGRPs. , 2007, The Biochemical journal.

[20]  Jim Miller,et al.  LFA-1-Mediated T Cell Costimulation through Increased Localization of TCR/Class II Complexes to the Central Supramolecular Activation Cluster and Exclusion of CD45 from the Immunological Synapse1 , 2007, The Journal of Immunology.

[21]  Ann M Graybiel,et al.  Mice lacking the signaling molecule CalDAG-GEFI represent a model for leukocyte adhesion deficiency type III. , 2007, The Journal of clinical investigation.

[22]  Michael Loran Dustin,et al.  Force as a facilitator of integrin conformational changes during leukocyte arrest on blood vessels and antigen-presenting cells. , 2007, Immunity.

[23]  L. Turka,et al.  Defective Activation of Protein Kinase C and Ras-ERK Pathways Limits IL-2 Production and Proliferation by CD4+CD25+ Regulatory T Cells1 , 2006, The Journal of Immunology.

[24]  M. Kazanietz,et al.  C1 domains exposed: from diacylglycerol binding to protein-protein interactions. , 2006, Biochimica et biophysica acta.

[25]  N. Câmara,et al.  Altered proximal T cell receptor (TCR) signaling in human CD4+CD25+ regulatory T cells , 2006, Journal of leukocyte biology.

[26]  Michael Loran Dustin,et al.  A dynamic view of the immunological synapse. , 2005, Seminars in immunology.

[27]  P. Stork,et al.  Regulation of the Small GTPase Rap1 and Extracellular Signal-Regulated Kinases by the Costimulatory Molecule CTLA-4 , 2005, Molecular and Cellular Biology.

[28]  H. Ohigashi,et al.  Tumor promoter binding of the protein kinase C C1 homology domain peptides of RasGRPs, chimaerins, and Unc13s. , 2004, Bioorganic & Medicinal Chemistry.

[29]  A. Graybiel,et al.  CalDAG-GEFI integrates signaling for platelet aggregation and thrombus formation , 2004, Nature Medicine.

[30]  F. Sánchez‐Madrid,et al.  F-actin-dependent Translocation of the Rap1 GDP/GTP Exchange Factor RasGRP2*[boxs] , 2004, Journal of Biological Chemistry.

[31]  K. Katagiri,et al.  Rap1-mediated Lymphocyte Function-associated Antigen-1 Activation by the T Cell Antigen Receptor Is Dependent on Phospholipase C-γ1* , 2004, Journal of Biological Chemistry.

[32]  A. Rudensky,et al.  Control of immune homeostasis by naturally arising regulatory CD4+ T cells. , 2003, Current opinion in immunology.

[33]  N. Minato,et al.  Rap1 GTPase: functions, regulation, and malignancy. , 2003, Journal of biochemistry.

[34]  J. Stone,et al.  Integration of DAG signaling systems mediated by PKC-dependent phosphorylation of RasGRP3. , 2003, Blood.

[35]  H. Ochs,et al.  Immune dysregulation, polyendocrinopathy, enteropathy, and X-linked inheritance (IPEX), a syndrome of systemic autoimmunity caused by mutations of FOXP3, a critical regulator of T-cell homeostasis , 2003, Current opinion in rheumatology.

[36]  F. Ramsdell,et al.  An essential role for Scurfin in CD4+CD25+ T regulatory cells , 2003, Nature Immunology.

[37]  A. Rudensky,et al.  Foxp3 programs the development and function of CD4+CD25+ regulatory T cells , 2003, Nature Immunology.

[38]  T. Nomura,et al.  Control of Regulatory T Cell Development by the Transcription Factor Foxp3 , 2002 .

[39]  M. Gold,et al.  The Rap GTPases Regulate B Cell Migration Toward the Chemokine Stromal Cell-Derived Factor-1 (CXCL12): Potential Role for Rap2 in Promoting B Cell Migration1 , 2002, The Journal of Immunology.

[40]  A. Sali,et al.  RasGRP4, a New Mast Cell-restricted Ras Guanine Nucleotide-releasing Protein with Calcium- and Diacylglycerol-binding Motifs , 2002, Journal of Biological Chemistry.

[41]  M. Kazanietz.  Novel "nonkinase" phorbol ester receptors: the C1 domain connection. , 2002, Molecular pharmacology.

[42]  A. Rudensky,et al.  Homeostasis and anergy of CD4+CD25+ suppressor T cells in vivo , 2002, Nature Immunology.

[43]  H. Ochs,et al.  The immune dysregulation, polyendocrinopathy, enteropathy, X-linked syndrome (IPEX) is caused by mutations of FOXP3 , 2001, Nature Genetics.

[44]  D. Galas,et al.  Disruption of a new forkhead/winged-helix protein, scurfin, results in the fatal lymphoproliferative disorder of the scurfy mouse , 2001, Nature Genetics.

[45]  M. Matsuda,et al.  CalDAG-GEFIII Activation of Ras, R-Ras, and Rap1* , 2000, The Journal of Biological Chemistry.

[46]  P. Blumberg,et al.  RasGRP links T-cell receptor signaling to Ras. , 2000, Blood.

[47]  A. Graybiel,et al.  A Rap guanine nucleotide exchange factor enriched highly in the basal ganglia. , 1998, Proceedings of the National Academy of Sciences of the United States of America.

[48]  T. Mak,et al.  Distinct roles for LFA-1 and CD28 during activation of naive T cells: adhesion versus costimulation. , 1997, Immunity.

[49]  Y. Nishizuka,et al.  Taxonomy and function of C1 protein kinase C homology domains , 1997, Protein science : a publication of the Protein Society.