ABSTRACT
Proteolytic maturation of proBMP-4 is required to generate an active signaling molecule. We show that proBMP-4 is cleaved by furin in a sequential manner. Cleavage at a consensus furin site adjacent to the mature ligand domain allows for subsequent cleavage at an upstream nonconsensus furin site within the prodomain. BMP-4 synthesized from precursor in which the upstream site is noncleavable is less active, signals at a shorter range, and accumulates at lower levels than does BMP-4 cleaved from native precursor. Conversely, BMP-4 cleaved from precursor in which both sites are rapidly cleaved is more active and signals over a greater range. Differential use of the upstream cleavage site could provide for tissue-specific regulation of BMP-4 activity and signaling range.
Subject(s)
Bone Morphogenetic Proteins/metabolism , Signal Transduction , Amino Acid Sequence , Animals , Binding Sites , Blotting, Western , Bone Morphogenetic Protein 4 , Bone Morphogenetic Proteins/chemistry , DNA, Complementary/metabolism , Embryo, Nonmammalian/metabolism , Furin , Ligands , Molecular Sequence Data , Mutation , Precipitin Tests , Protein Binding , Protein Structure, Tertiary , Subtilisins/chemistry , Time Factors , Xenopus/metabolism , Xenopus Proteins , beta-Galactosidase/metabolismABSTRACT
The c-fes protooncogene encodes a nonreceptor tyrosine kinase (Fes) implicated in cytokine receptor signal transduction, neutrophil survival, and myeloid differentiation. To determine the role of Fes in embryonic development and hematopoiesis, we engineered a null mutation of the murine c-fes locus. c-fes-/- mice are viable but not born in the expected Mendelian ratios. Live born c-fes-/- mice exhibit lymphoid/myeloid homeostasis defects, compromised innate immunity, and increased Stat activation in response to GM-CSF and IL-6 signaling. Therefore, increased cytokine responsiveness in the absence of Fes leads to abnormal myeloid proliferation and functional defects in the macrophage lineage.
Subject(s)
Hematopoietic Stem Cells/enzymology , Hematopoietic Stem Cells/immunology , Homeostasis/genetics , Homeostasis/immunology , Milk Proteins , Protein-Tyrosine Kinases , Proto-Oncogene Proteins/deficiency , Proto-Oncogene Proteins/genetics , Signal Transduction/genetics , Signal Transduction/immunology , Animals , Bone Marrow Cells/enzymology , Bone Marrow Cells/immunology , Bone Marrow Cells/pathology , Cell Adhesion/genetics , Cell Adhesion/immunology , Cell Differentiation/genetics , Cell Differentiation/immunology , Cell Division/genetics , Cell Division/immunology , Cells, Cultured , DNA-Binding Proteins/biosynthesis , DNA-Binding Proteins/metabolism , Female , Gene Targeting , Granulocyte-Macrophage Colony-Stimulating Factor/pharmacology , Hematopoietic Stem Cells/pathology , Immunity, Innate/genetics , Immunologic Deficiency Syndromes/enzymology , Immunologic Deficiency Syndromes/genetics , Immunologic Deficiency Syndromes/metabolism , Immunologic Deficiency Syndromes/pathology , Lymphoid Tissue/enzymology , Lymphoid Tissue/immunology , Lymphoid Tissue/pathology , Macrophages/enzymology , Macrophages/immunology , Macrophages/pathology , Male , Mice , Mice, Inbred C57BL , Mice, Knockout , Mutagenesis, Site-Directed , Proto-Oncogene Proteins c-fes , STAT3 Transcription Factor , STAT5 Transcription Factor , Trans-Activators/biosynthesis , Trans-Activators/metabolismABSTRACT
The STAT1 transcription factor is activated in response to many cytokines and growth factors. To study the requirement for STAT1 in vivo, we disrupted the Stat1 gene in embryonic stem (ES) cells and in mice. Stat1(-1-)ES cells were unresponsive to interferon (IFN), but retained responsiveness to leukemia inhibitory factor (LIF) and remained LIF dependent for undifferentiated growth. Stat1(-1-1) animals were born at normal frequencies and displayed no gross developmental defects. However, these animals failed to thrive and were extremely susceptible to viral disease. Cells and tissues from Stat1(-1-) mice were unresponsive to IFN, but remained responsive to all other cytokines tested. Thus, STAT1 appears to be specific for IFN pathways that are essential for viability in the face of otherwise innocuous pathogens.
