RESUMO
GTPases act as important switches in many signaling events in cells. Although small and heterotrimeric G proteins are subjects of intensive studies, little is known about the large IFN-inducible GTPases. In this article, we show that the IFN-γ-inducible guanylate binding protein 1 (GBP-1) is a regulator of T cell activation. Silencing of GBP-1 leads to enhanced activation of early T cell Ag receptor/CD3 signaling molecules, including Lck, that is translated to higher IL-2 production. Mass spectrometry analyses showed that regulatory cytoskeletal proteins, like plastin-2 that bundles actin fibers and spectrin ß-chain, brain 1 that links the plasma membrane to the actin cytoskeleton, are binding partners of GBP-1. The spectrin cytoskeleton influences cell spreading and surface expression of TCR/CD3 and the leukocyte phosphatase CD45. We found higher cell spreading and enhanced surface expression of TCR/CD3 and CD45 in GBP-1 silenced T cells that explain their enhanced TCR/CD3 signaling. We conclude that GBP-1 is a downstream processor of IFN-γ via which T cells regulate cytoskeleton-dependent cell functions.
Assuntos
Citoesqueleto/metabolismo , Proteínas de Ligação ao GTP/metabolismo , Complexo Receptor-CD3 de Antígeno de Linfócitos T/metabolismo , Receptores de Antígenos de Linfócitos T/metabolismo , Complexo CD3/genética , Complexo CD3/metabolismo , Linhagem Celular , Linhagem Celular Tumoral , Citoesqueleto/genética , Citosol/metabolismo , Proteínas de Ligação ao GTP/genética , Células HEK293 , Humanos , Interferon gama/genética , Interferon gama/metabolismo , Interleucina-2/genética , Interleucina-2/metabolismo , Células Jurkat , Antígenos Comuns de Leucócito , Leucócitos/metabolismo , Ativação Linfocitária/genética , Proteína Tirosina Quinase p56(lck) Linfócito-Específica/genética , Proteína Tirosina Quinase p56(lck) Linfócito-Específica/metabolismo , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/metabolismo , Proteínas dos Microfilamentos/genética , Proteínas dos Microfilamentos/metabolismo , Complexo Receptor-CD3 de Antígeno de Linfócitos T/genética , Receptores de Antígenos de Linfócitos T/genética , Transdução de Sinais/genética , Linfócitos T/metabolismo , Regulação para Cima/genéticaRESUMO
Gamma interferon (IFN-γ) regulates immune defenses against viruses, intracellular pathogens, and tumors by modulating cell proliferation, migration, invasion, and vesicle trafficking processes. The large GTPase guanylate binding protein 1 (GBP-1) is among the cellular proteins that is the most abundantly induced by IFN-γ and mediates its cell biologic effects. As yet, the molecular mechanisms of action of GBP-1 remain unknown. Applying an interaction proteomics approach, we identified actin as a strong and specific binding partner of GBP-1. Furthermore, GBP-1 colocalized with actin at the subcellular level and was both necessary and sufficient for the extensive remodeling of the fibrous actin structure observed in IFN-γ-exposed cells. These effects were dependent on the oligomerization and the GTPase activity of GBP-1. Purified GBP-1 and actin bound to each other, and this interaction was sufficient to impair the formation of actin filaments in vitro, as demonstrated by atomic force microscopy, dynamic light scattering, and fluorescence-monitored polymerization. Cosedimentation and band shift analyses demonstrated that GBP-1 binds robustly to globular actin and slightly to filamentous actin. This indicated that GBP-1 may induce actin remodeling via globular actin sequestering and/or filament capping. These results establish GBP-1 as a novel member within the family of actin-remodeling proteins specifically mediating IFN-γ-dependent defense strategies.
Assuntos
Proteínas de Ligação ao GTP/genética , Interferon gama/fisiologia , Fibras de Estresse/metabolismo , Actinas/química , Actinas/metabolismo , Proteínas de Ligação ao GTP/química , Proteínas de Ligação ao GTP/metabolismo , Células HeLa , Células Endoteliais da Veia Umbilical Humana/enzimologia , Humanos , Polimerização , Ligação Proteica , Mapeamento de Interação de Proteínas , Multimerização Proteica , Transporte Proteico , Ativação TranscricionalRESUMO
The human guanylate-binding protein 1 (hGBP1) is a large GTP-binding protein belonging to the dynamin family, a common feature of which is nucleotide-dependent assembly to homotypic oligomers. Assembly leads to stimulation of GTPase activity, which, in the case of dynamin, is responsible for scission of vesicles from membranes. By yeast two-hybrid and biochemical experiments we addressed intermolecular interactions between all subdomains of hGBP1 and identified the C-terminal subdomain, α12/13, as a new interaction site for self-assembly. α12/13 represents a stable subdomain of hGBP1, as shown by CD spectroscopy. In addition to contacts between GTPase domains leading to dimer formation, the interaction between two α12/13 subdomains, in the course of GTP hydrolysis, results in tetramer formation of the protein. With the help of CD spectroscopy we showed coiled-coil formation of two α12/13 subdomains and concentration-dependent measurements allow estimating a value for the dissociation constant of 7.3 µM. We suggest GTP hydrolysis-driven release of the α12/13 subdomain, making it available for coiled-coil formation. Furthermore, we can demonstrate the biological relevance of hGBP1 tetramer formation in living cells by chemical cross-link experiments.