RESUMEN
Protein kinase C-θ (PKCθ) is a member of the novel PKC subfamily known for its selective and predominant expression in T lymphocytes where it regulates essential functions required for T cell activation and proliferation. Our previous studies provided a mechanistic explanation for the recruitment of PKCθ to the center of the immunological synapse (IS) by demonstrating that a proline-rich (PR) motif within the V3 region in the regulatory domain of PKCθ is necessary and sufficient for PKCθ IS localization and function. Herein, we highlight the importance of Thr335-Pro residue in the PR motif, the phosphorylation of which is key in the activation of PKCθ and its subsequent IS localization. We demonstrate that the phospho-Thr335-Pro motif serves as a putative binding site for the peptidyl-prolyl cis-trans isomerase (PPIase), Pin1, an enzyme that specifically recognizes peptide bonds at phospho-Ser/Thr-Pro motifs. Binding assays revealed that mutagenesis of PKCθ-Thr335-to-Ala abolished the ability of PKCθ to interact with Pin1, while Thr335 replacement by a Glu phosphomimetic, restored PKCθ binding to Pin1, suggesting that Pin1-PKCθ association is contingent upon the phosphorylation of the PKCθ-Thr335-Pro motif. Similarly, the Pin1 mutant, R17A, failed to associate with PKCθ, suggesting that the integrity of the Pin1 N-terminal WW domain is a requisite for Pin1-PKCθ interaction. In silico docking studies underpinned the role of critical residues in the Pin1-WW domain and the PKCθ phospho-Thr335-Pro motif, to form a stable interaction between Pin1 and PKCθ. Furthermore, TCR crosslinking in human Jurkat T cells and C57BL/6J mouse-derived splenic T cells promoted a rapid and transient formation of Pin1-PKCθ complexes, which followed a T cell activation-dependent temporal kinetic, suggesting a role for Pin1 in PKCθ-dependent early activation events in TCR-triggered T cells. PPIases that belong to other subfamilies, i.e., cyclophilin A or FK506-binding protein, failed to associate with PKCθ, indicating the specificity of the Pin1-PKCθ association. Fluorescent cell staining and imaging analyses demonstrated that TCR/CD3 triggering promotes the colocalization of PKCθ and Pin1 at the cell membrane. Furthermore, interaction of influenza hemagglutinin peptide (HA307-319)-specific T cells with antigen-fed antigen presenting cells (APCs) led to colocalization of PKCθ and Pin1 at the center of the IS. Together, we point to an uncovered function for the Thr335-Pro motif within the PKCθ-V3 regulatory domain to serve as a priming site for its activation upon phosphorylation and highlight its tenability to serve as a regulatory site for the Pin1 cis-trans isomerase.
Asunto(s)
Péptidos , Isomerasa de Peptidilprolil , Animales , Ratones , Humanos , Isomerasa de Peptidilprolil/genética , Isomerasa de Peptidilprolil/química , Isomerasa de Peptidilprolil/metabolismo , Proteína Quinasa C-theta/genética , Ratones Endogámicos C57BL , Peptidilprolil Isomerasa de Interacción con NIMA/genética , Receptores de Antígenos de Linfocitos T , Prolina/química , Prolina/metabolismoRESUMEN
BACKGROUND: Our previous studies revealed a critical role of a novel CTLA4-protein kinase C-eta (PKCη) signaling axis in mediating the suppressive activity of regulatory T cells (Tregs) in antitumor immunity. These studies have employed adoptive transfer of germline PKCη-deficient (Prkch-/-) Tregs into Prkch+/+ mice prior to tumor implantation. Here, we extended these findings into a biologically and clinically more relevant context. METHODS: We have analyzed the role of PKCη in antitumor immunity and the tumor microenvironment (TME) in intact tumor-bearing mice with Treg-specific or CD8+ T cell-specific Prkch deletion, including in a therapeutic model of combinatorial treatment. In addition to measuring tumor growth, we analyzed the phenotype and functional attributes of tumor-infiltrating immune cells, particularly Tregs and dendritic cells (DCs). RESULTS: Using two models of mouse transplantable cancer and a genetically engineered autochthonous hepatocellular carcinoma (HCC) model, we found, first, that mice with Treg-specific Prkch deletion displayed a significantly reduced growth of B16-F10 melanoma and TRAMP-C1 adenocarcinoma tumors. Tumor growth reduction was associated with a less immunosuppressive TME, indicated by increased numbers and function of tumor-infiltrating CD8+ effector T cells and elevated expression of the costimulatory ligand CD86 on intratumoral DCs. In contrast, CD8+ T cell-specific Prkch deletion had no effect on tumor growth or the abundance and functionality of CD8+ effector T cells, consistent with findings that Prkch-/- CD8+ T cells proliferated normally in response to in vitro polyclonal or specific antigen stimulation. Similar beneficial antitumor effects were found in mice with germline or Treg-specific Prkch deletion that were induced to develop an autochthonous HCC. Lastly, using a therapeutic model, we found that monotherapies consisting of Treg-specific Prkch deletion or vaccination with irradiated Fms-like tyrosine kinase 3 ligand (Flt3L)-expressing B16-F10 tumor cells post-tumor implantation significantly delayed tumor growth. This effect was more pronounced in mice receiving a combination of the two immunotherapies. CONCLUSION: These findings demonstrate the potential utility of PKCη inhibition as a viable clinical approach to treat patients with cancer, especially when combined with adjuvant therapies.
Asunto(s)
Antígeno CTLA-4/metabolismo , Inmunoterapia/métodos , Neoplasias/tratamiento farmacológico , Animales , Humanos , Ratones , Neoplasias/genética , Linfocitos T ReguladoresRESUMEN
We reported that protein kinase C-η (PKCη) forms a novel (to our knowledge) signaling complex with the checkpoint inhibitory protein CTLA-4 in regulatory T cells (Tregs). This complex is required for the contact-dependent suppressive activity of Tregs, including suppression of antitumor immunity. However, the importance of PKCη in protective immunity mediated by T effector cells remains unclear. We used mice with germline or conditional Treg-specific deletion of Prkch, the PKCη-encoding gene, to explore CD8+ T cell-dependent antiviral immunity using the lymphocytic choriomeningitis virus Armstrong strain acute infection model as well as the in vitro activation of murine or human CD8+ T cells. Five days following infection, germline Prkch -/- mice displayed enhanced viral clearance compared with control mice. Similarly, Prkch Treg-specific conditional knockout mice also showed improved viral clearance and displayed enhanced expression of granzyme B and IFN-γ by both virus-specific and total CD8+ T cells, demonstrating that enhanced viral clearance in germline Prkch -/- mice is caused by PKCη deficiency in Tregs and the resulting functional defect of Prkch -/- Tregs. In addition, purified Prkch -/- mouse CD8+ T cells as well as PRKCH knockdown human CD8+ T cells displayed intact, or even enhanced, T cell activation in vitro as measured by proliferation and expression of granzyme B and IFN-γ. Thus, global PKCη deletion does not impair overall CD8+ T cell-mediated immunity, including antiviral immunity, implying that selective pharmacological PKCη inhibition could be safely used in vivo to inhibit undesired contact-dependent suppression by Tregs and, thus, enhance tumor-specific and, likely, virus-specific immunity.
Asunto(s)
Linfocitos T CD8-positivos , Activación de Linfocitos , Proteína Quinasa C , Linfocitos T Reguladores , Virosis , Animales , Humanos , Ratones , Linfocitos T CD8-positivos/efectos de los fármacos , Linfocitos T CD8-positivos/inmunología , Línea Celular , Proliferación Celular/efectos de los fármacos , Antígeno CTLA-4/inmunología , Granzimas/inmunología , Células HEK293 , Inmunidad Celular/efectos de los fármacos , Inmunidad Celular/inmunología , Interferón gamma/inmunología , Activación de Linfocitos/efectos de los fármacos , Activación de Linfocitos/inmunología , Virus de la Coriomeningitis Linfocítica/inmunología , Ratones Noqueados , Proteína Quinasa C/deficiencia , Proteína Quinasa C/inmunología , Inhibidores de Proteínas Quinasas/inmunología , Inhibidores de Proteínas Quinasas/farmacología , Linfocitos T Reguladores/efectos de los fármacos , Linfocitos T Reguladores/inmunología , Virosis/inmunologíaAsunto(s)
Inmunoterapia , Neoplasias , Antígeno CTLA-4 , Humanos , Concentración de Iones de Hidrógeno , LisosomasRESUMEN
Right ventricle to pulmonary artery (RV-PA) conduits have been used for the surgical repair of congenital heart defects. These conduits frequently become stenosed or develop insufficiency with time, necessitating reoperations. Percutanous pulmonary valve implantation (PPVI) can delay the need for repeated surgeries in patients with congenital heart defects and degenerated RV-PA conduits. We presented our first experience with PPVI and described in detail the procedural methods and the considerations that are needed for this intervention to be successful. Immediate and short-term clinical outcomes of our patients were reported. Good haemodynamic results were obtained, both angiographically and on echocardiography. PPVI provides an excellent alternative to repeat open-heart surgery for patients with congenital heart defects and degenerated RV-PA conduits. This represents a paradigm shift in the management of congenital heart disease, which is traditionally managed by open-heart surgery.
Asunto(s)
Cateterismo Cardíaco/métodos , Implantación de Prótesis de Válvulas Cardíacas/métodos , Estenosis de la Válvula Pulmonar/cirugía , Válvula Pulmonar/cirugía , Adulto , Cateterismo Cardíaco/instrumentación , Vena Femoral , Implantación de Prótesis de Válvulas Cardíacas/instrumentación , Humanos , Masculino , Diseño de Prótesis , Singapur , Adulto JovenRESUMEN
Follicular helper T (TFH) cells represent a highly specialized CD4+ T cell subpopulation that supports the generation of germinal centers (GC) and provides B cells with critical signals promoting antibody class switching, generation of high affinity antibodies, and memory formation. TFH cells are characterized by the expression of the chemokine receptor CXCR5, the transcription factor Bcl-6, costimulatory molecules ICOS, and PD-1, and the production of cytokine IL-21. The acquisition of a TFH phenotype is a complex and multistep process that involves signals received through engagement of the TCR along with a multitude of costimulatory molecules and cytokines receptors. Members of the Tumor necrosis factor Receptor Associated Factors (TRAF) represent one of the major classes of signaling mediators involved in the differentiation and functions of TFH cells. TRAF molecules are the canonical adaptor molecules that physically interact with members of the Tumor Necrosis Factor Receptor Superfamily (TNFRSF) and actively modulate their downstream signaling cascades through their adaptor function and/or E3 ubiquitin ligase activity. OX-40, GITR, and 4-1BB are the TRAF-dependent TNFRSF members that have been implicated in the differentiation and functions of TFH cells. On the other hand, emerging data demonstrate that TRAF proteins also participate in signaling from the TCR and CD28, which deliver critical signals leading to the differentiation of TFH cells. More intriguingly, we recently showed that the cytoplasmic tail of ICOS contains a conserved TANK-binding kinase 1 (TBK1)-binding motif that is shared with TBK1-binding TRAF proteins. The presence of this TRAF-mimicking signaling module downstream of ICOS is required to mediate the maturation step during TFH differentiation. In addition, JAK-STAT pathways emanating from IL-2, IL-6, IL-21, and IL-27 cytokine receptors affect TFH development, and crosstalk between TRAF-mediated pathways and the JAK-STAT pathways can contribute to generate integrated signals required to drive and sustain TFH differentiation. In this review, we will introduce the molecular interactions and the major signaling pathways controlling the differentiation of TFH cells. In each case, we will highlight the contributions of TRAF proteins to these signaling pathways. Finally, we will discuss the role of individual TRAF proteins in the regulation of T cell-dependent humoral responses.
Asunto(s)
Formación de Anticuerpos/inmunología , Linfocitos B/inmunología , Linfocitos B/metabolismo , Diferenciación Celular/inmunología , Transducción de Señal , Linfocitos T Colaboradores-Inductores/inmunología , Linfocitos T Colaboradores-Inductores/metabolismo , Péptidos y Proteínas Asociados a Receptores de Factores de Necrosis Tumoral/metabolismo , Animales , Comunicación Celular/inmunología , Citocinas/metabolismo , Humanos , Activación de Linfocitos , FN-kappa B/metabolismo , Linfocitos T Colaboradores-Inductores/citologíaRESUMEN
The ability of Tregs to control the development of immune responses is essential for maintaining immune system homeostasis. However, Tregs also inhibit the development of efficient antitumor responses. Here, we explored the characteristics and mechanistic basis of the Treg-intrinsic CTLA4/PKCη signaling pathway that we recently found to be required for contact-dependent Treg-mediated suppression. We show that PKCη is required for the Treg-mediated suppression of tumor immunity in vivo. The presence of PKCη-deficient (Prkch-/-) Tregs in the tumor microenvironment was associated with a significantly increased expression of the costimulatory molecule CD86 on intratumoral CD103+ DCs, enhanced priming of antigen-specific CD8+ T cells, and greater levels of effector cytokines produced by these cells. Similar to mouse Tregs, the GIT/PAK/PIX complex also operated downstream of CTLA4 and PKCη in human Tregs, and GIT2 knockdown in Tregs promoted antitumor immunity. Collectively, our data suggest that targeting the CTLA4/PKCη/GIT/PAK/PIX signaling pathway in Tregs could represent a novel immunotherapeutic strategy to alleviate the negative impact of Tregs on antitumor immune responses.
Asunto(s)
Antígeno CTLA-4/inmunología , Proteína Quinasa C/inmunología , Linfocitos T Reguladores/inmunología , Escape del Tumor/inmunología , Microambiente Tumoral/inmunología , Animales , Antígeno B7-2/metabolismo , Femenino , Xenoinjertos , Humanos , Tolerancia Inmunológica , Linfocitos Infiltrantes de Tumor/inmunología , Masculino , Melanoma Experimental/inmunología , Ratones Endogámicos C57BL , Ratones Transgénicos , Trasplante de Neoplasias , Neoplasias de la Próstata/inmunología , Transducción de Señal/inmunologíaRESUMEN
The protein kinase C (PKC) family, discovered in the late 1970s, is composed of at least 10 serine/threonine kinases, divided into three groups based on their molecular architecture and cofactor requirements. PKC enzymes have been conserved throughout evolution and are expressed in virtually all cell types; they represent critical signal transducers regulating cell activation, differentiation, proliferation, death, and effector functions. PKC family members play important roles in a diverse array of hematopoietic and immune responses. This review covers the discovery and history of this enzyme family, discusses the roles of PKC enzymes in the development and effector functions of major hematopoietic and immune cell types, and points out gaps in our knowledge, which should ignite interest and further exploration, ultimately leading to better understanding of this enzyme family and, above all, its role in the many facets of the immune system.
Asunto(s)
Hematopoyesis , Sistema Inmunológico , Proteína Quinasa C/metabolismo , Animales , Coenzimas/metabolismo , Activación Enzimática/inmunología , Humanos , Proteína Quinasa C/inmunología , Transducción de SeñalRESUMEN
Signaling via the inducible costimulator ICOS fuels the stepwise development of follicular helper T cells (TFH cells). However, a signaling pathway unique to ICOS has not been identified. We found here that the kinase TBK1 associated with ICOS via a conserved motif, IProx, that shares homology with the tumor-necrosis-factor receptor (TNFR)-associated factors TRAF2 and TRAF3. Disruption of this motif abolished the association of TBK1 with ICOS, TRAF2 and TRAF3, which identified a TBK1-binding consensus. Alteration of this motif in ICOS or depletion of TBK1 in T cells severely impaired the differentiation of germinal center (GC) TFH cells and the development of GCs, interfered with B cell differentiation and disrupted the development of antibody responses, but the IProx motif and TBK1 were dispensable for the early differentiation of TFH cells. These results reveal a previously unknown ICOS-TBK1 signaling pathway that specifies the commitment of GC TFH cells.
Asunto(s)
Linfocitos B/fisiología , Linfocitos T CD4-Positivos/fisiología , Centro Germinal/inmunología , Proteína Coestimuladora de Linfocitos T Inducibles/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Animales , Formación de Anticuerpos/genética , Diferenciación Celular/genética , Células Cultivadas , Proteína Coestimuladora de Linfocitos T Inducibles/genética , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Transgénicos , Unión Proteica , Proteínas Serina-Treonina Quinasas/genética , Transducción de Señal , Factor 2 Asociado a Receptor de TNF/genética , Factor 3 Asociado a Receptor de TNF/genéticaRESUMEN
Pseudomonas aeruginosa is a dreaded pathogen in many clinical settings. Its inherent and acquired antibiotic resistance thwarts therapy. In particular, derepression of the AmpC ß-lactamase is a common mechanism of ß-lactam resistance among clinical isolates. The inducible expression of ampC is controlled by the global LysR-type transcriptional regulator (LTTR) AmpR. In the present study, we investigated the genetic and structural elements that are important for ampC induction. Specifically, the ampC (PampC) and ampR (PampR) promoters and the AmpR protein were characterized. The transcription start sites (TSSs) of the divergent transcripts were mapped using 5' rapid amplification of cDNA ends-PCR (RACE-PCR), and strong σ(54) and σ(70) consensus sequences were identified at PampR and PampC, respectively. Sigma factor RpoN was found to negatively regulate ampR expression, possibly through promoter blocking. Deletion mapping revealed that the minimal PampC extends 98 bp upstream of the TSS. Gel shifts using membrane fractions showed that AmpR binds to PampC in vitro whereas in vivo binding was demonstrated using chromatin immunoprecipitation-quantitative PCR (ChIP-qPCR). Additionally, site-directed mutagenesis of the AmpR helix-turn-helix (HTH) motif identified residues critical for binding and function (Ser38 and Lys42) and critical for function but not binding (His39). Amino acids Gly102 and Asp135, previously implicated in the repression state of AmpR in the enterobacteria, were also shown to play a structural role in P. aeruginosa AmpR. Alkaline phosphatase fusion and shaving experiments suggest that AmpR is likely to be membrane associated. Lastly, an in vivo cross-linking study shows that AmpR dimerizes. In conclusion, a potential membrane-associated AmpR dimer regulates ampC expression by direct binding.
Asunto(s)
Proteínas Bacterianas/metabolismo , Regulación Bacteriana de la Expresión Génica/fisiología , Pseudomonas aeruginosa/metabolismo , Secuencias de Aminoácidos , Antibacterianos/farmacología , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Mapeo Cromosómico , Cromosomas Bacterianos , Secuencia de Consenso , Farmacorresistencia Bacteriana , Regiones Promotoras Genéticas , Unión Proteica , Conformación Proteica , Pseudomonas aeruginosa/efectos de los fármacos , Pseudomonas aeruginosa/genética , Resistencia betalactámica , beta-Lactamasas/genética , beta-Lactamasas/metabolismo , beta-Lactamas/farmacologíaRESUMEN
Protein kinase C (PKC) proteins are a group of well-conserved, intracellular signaling enzymes expressed in all cells and tissues, including immune cells. Much of the molecular insight into PKC immunobiology has been gleaned from studies using PKC gene (Prkc) knockout mice and the analysis of different disease models in these animals. More-recent studies have revealed that PKCs also have crucial roles in the pathogenesis of human immune disorders. Therefore, strategies to modulate the functions of PKC enzymes could have a major impact on the treatment and therapies of autoimmune diseases and other immune disorders.
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Enfermedades del Sistema Inmune/tratamiento farmacológico , Proteína Quinasa C/antagonistas & inhibidores , Inhibidores de Proteínas Quinasas/farmacología , Inhibidores de Proteínas Quinasas/uso terapéutico , Animales , Humanos , Enfermedades del Sistema Inmune/metabolismo , Proteína Quinasa C/metabolismoRESUMEN
Regulatory T (Treg) cells, which maintain immune homeostasis and self-tolerance, form an immunological synapse (IS) with antigen-presenting cells (APCs). However, signaling events at the Treg cell IS remain unknown. Here we show that the kinase PKC-η associated with CTLA-4 and was recruited to the Treg cell IS. PKC-η-deficient Treg cells displayed defective suppressive activity, including suppression of tumor immunity but not of autoimmune colitis. Phosphoproteomic and biochemical analysis revealed an association between CTLA-4-PKC-η and the GIT2-αPIX-PAK complex, an IS-localized focal adhesion complex. Defective activation of this complex in PKC-η-deficient Treg cells was associated with reduced depletion of CD86 from APCs by Treg cells. These results reveal a CTLA-4-PKC-η signaling axis required for contact-dependent suppression and implicate this pathway as a potential cancer immunotherapy target.
Asunto(s)
Antígeno CTLA-4/metabolismo , Sinapsis Inmunológicas/metabolismo , Inmunoterapia/tendencias , Melanoma Experimental/inmunología , Melanoma Experimental/terapia , Complejos Multiproteicos/metabolismo , Proteína Quinasa C/metabolismo , Linfocitos T Reguladores/inmunología , Animales , Humanos , Tolerancia Inmunológica/genética , Células Jurkat , Activación de Linfocitos , Ratones , Ratones Endogámicos C57BL , Unión Proteica , Proteína Quinasa C/genética , Proteómica , Transducción de SeñalRESUMEN
The immunological synapse (IS) formed between immune cells and antigen-presenting cells (APCs) provides a platform for signaling. Protein kinase C (PKC)θ localizes in the T cell IS within the central supramolecular activation cluster (cSMAC), where it associates with CD28 and mediates T cell receptor (TCR)/CD28 signals leading to effector T (Teff) cell activation. In regulatory T (Treg) cells, PKCθ is sequestered away from the IS, and inhibits suppressive function. Other PKCs localizing in the IS mediate additional functions in various immune cells. Further work is needed to identify mechanisms underlying PKC recruitment or exclusion at the IS, potential redundancy among IS-localized PKCs, and the relevance of PKC localization for IS dynamics and lymphocyte activation.
Asunto(s)
Sinapsis Inmunológicas/inmunología , Proteína Quinasa C/metabolismo , Subgrupos de Linfocitos T/inmunología , Linfocitos T Reguladores/inmunología , Animales , Antígenos CD28/metabolismo , Humanos , Isoenzimas/inmunología , Activación de Linfocitos , Complejos Multiproteicos/metabolismo , Proteína Quinasa C/inmunología , Proteína Quinasa C-theta , Receptores de Antígenos de Linfocitos T/metabolismo , Transducción de Señal/inmunologíaRESUMEN
Protein kinase C-theta (PKCθ) is a protein kinase C (PKC) family member expressed predominantly in T lymphocytes, and extensive studies addressing its function have been conducted. PKCθ is the only T cell-expressed PKC that localizes selectively to the center of the immunological synapse (IS) following conventional T cell antigen stimulation, and this unique localization is essential for PKCθ-mediated downstream signaling. While playing a minor role in T cell development, early in vitro studies relying, among others, on the use of PKCθ-deficient (Prkcq(-/-)) T cells revealed that PKCθ is required for the activation and proliferation of mature T cells, reflecting its importance in activating the transcription factors nuclear factor kappa B, activator protein-1, and nuclear factor of activated T cells, as well as for the survival of activated T cells. Upon subsequent analysis of in vivo immune responses in Prkcq(-/-) mice, it became clear that PKCθ has a selective role in the immune system: it is required for experimental Th2- and Th17-mediated allergic and autoimmune diseases, respectively, and for alloimmune responses, but is dispensable for protective responses against pathogens and for graft-versus-leukemia responses. Surprisingly, PKCθ was recently found to be excluded from the IS of regulatory T cells and to negatively regulate their suppressive function. These attributes of PKCθ make it an attractive target for catalytic or allosteric inhibitors that are expected to selectively suppress harmful inflammatory and alloimmune responses without interfering with beneficial immunity to infections. Early progress in developing such drugs is being made, but additional studies on the role of PKCθ in the human immune system are urgently needed.
Asunto(s)
Enfermedades del Sistema Inmune/inmunología , Tolerancia Inmunológica , Sinapsis Inmunológicas/metabolismo , Isoenzimas/metabolismo , Proteína Quinasa C/metabolismo , Transducción de Señal , Animales , Enfermedades Autoinmunes/tratamiento farmacológico , Enfermedades Autoinmunes/inmunología , Enfermedades Autoinmunes/metabolismo , Humanos , Enfermedades del Sistema Inmune/tratamiento farmacológico , Enfermedades del Sistema Inmune/metabolismo , Tolerancia Inmunológica/efectos de los fármacos , Sinapsis Inmunológicas/efectos de los fármacos , Sinapsis Inmunológicas/inmunología , Inmunosupresores/farmacología , Inmunosupresores/uso terapéutico , Isoenzimas/antagonistas & inhibidores , Isoenzimas/química , Terapia Molecular Dirigida , Proteína Quinasa C/antagonistas & inhibidores , Proteína Quinasa C/química , Proteína Quinasa C-theta , Transducción de Señal/efectos de los fármacosRESUMEN
Protein kinase Cθ (PKCθ) is a novel PKC that plays a key role in T lymphocyte activation. To understand how PKCθ is regulated in T cells, we investigated the properties of its N-terminal C2 domain that functions as an autoinhibitory domain. Our measurements show that a Tyr(P)-containing peptide derived from CDCP1 binds the C2 domain of PKCθ with high affinity and activates the enzyme activity of the intact protein. The Tyr(P) peptide also binds the C2 domain of PKCδ tightly, but no enzyme activation was observed with PKCδ. Mutations of PKCθ-C2 residues involved in Tyr(P) binding abrogated the enzyme activation and association of PKCθ with Tyr-phosphorylated full-length CDCP1 and severely inhibited the T cell receptor/CD28-mediated activation of a PKCθ-dependent reporter gene in T cells. Collectively, these studies establish the C2 domain of PKCθ as a Tyr(P)-binding domain and suggest that the domain may play a major role in PKCθ activation via its Tyr(P) binding.
Asunto(s)
Isoenzimas/química , Péptidos/química , Fosfotirosina/química , Proteína Quinasa C/química , Activación Enzimática , Humanos , Isoenzimas/genética , Isoenzimas/metabolismo , Péptidos/genética , Péptidos/metabolismo , Fosforilación/fisiología , Fosfotirosina/genética , Fosfotirosina/metabolismo , Unión Proteica/fisiología , Proteína Quinasa C/genética , Proteína Quinasa C/metabolismo , Proteína Quinasa C-delta/química , Proteína Quinasa C-delta/genética , Proteína Quinasa C-delta/metabolismo , Proteína Quinasa C-theta , Estructura Terciaria de ProteínaAsunto(s)
Rechazo de Injerto/tratamiento farmacológico , Enfermedad Injerto contra Huésped/tratamiento farmacológico , Isoenzimas/metabolismo , Proteína Quinasa C/metabolismo , Psoriasis/tratamiento farmacológico , Linfocitos T/metabolismo , Animales , Rechazo de Injerto/inmunología , Enfermedad Injerto contra Huésped/inmunología , Humanos , Terapia de Inmunosupresión , Isoenzimas/genética , Isoenzimas/inmunología , Ratones , Ratones Noqueados , Terapia Molecular Dirigida , FN-kappa B/metabolismo , Proteína Quinasa C/genética , Proteína Quinasa C/inmunología , Proteína Quinasa C-theta , Inhibidores de Proteínas Quinasas/uso terapéutico , Psoriasis/inmunología , Pirroles/uso terapéutico , Quinazolinas/uso terapéutico , Transducción de Señal , Linfocitos T/inmunología , Linfocitos T/patologíaRESUMEN
Protein kinase C-θ (PKC-θ) translocates to the center of the immunological synapse, but the underlying mechanism and its importance in T cell activation are unknown. Here we found that the V3 domain of PKC-θ was necessary and sufficient for localization to the immunological synapse mediated by association with the coreceptor CD28 and dependent on the kinase Lck. We identified a conserved proline-rich motif in V3 required for association with CD28 and immunological synapse localization. We found association with CD28 to be essential for PKC-θ-mediated downstream signaling and the differentiation of T helper type 2 cells (T(H)2 cells) and interleukin 17-producing helper T cells (T(H)17 cells) but not of T helper type 1 cells (T(H)1 cells). Ectopic expression of V3 sequestered PKC-θ from the immunological synapse and interfered with its functions. Our results identify a unique mode of CD28 signaling, establish a molecular basis for the immunological synapse localization of PKC-θ and indicate V3-based 'decoys' may be therapeutic modalities for T cell-mediated inflammatory diseases.
Asunto(s)
Antígenos CD28/metabolismo , Isoenzimas/metabolismo , Proteína Quinasa C/metabolismo , Subgrupos de Linfocitos T/metabolismo , Células Th17/metabolismo , Células Th2/metabolismo , Secuencias de Aminoácidos/genética , Animales , Antígenos CD28/inmunología , Diferenciación Celular/inmunología , Células Cultivadas , Sinapsis Inmunológicas , Inmunomodulación , Isoenzimas/genética , Isoenzimas/inmunología , Activación de Linfocitos , Ratones , Ratones Noqueados , Dominios Proteicos Ricos en Prolina/genética , Unión Proteica/inmunología , Proteína Quinasa C/genética , Proteína Quinasa C/inmunología , Proteína Quinasa C-theta , Transporte de Proteínas/inmunología , Transducción de Señal/inmunología , Subgrupos de Linfocitos T/inmunología , Subgrupos de Linfocitos T/patología , Células Th17/inmunología , Células Th17/patología , Células Th2/inmunología , Células Th2/patologíaAsunto(s)
Histona Desacetilasas/metabolismo , Fosfoproteínas/metabolismo , Proteómica/métodos , Transducción de Señal , Linfocitos T Citotóxicos/metabolismo , Animales , Núcleo Celular/metabolismo , Células Cultivadas , Citosol/metabolismo , Perfilación de la Expresión Génica , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Histona Desacetilasas/genética , Ratones , Ratones Transgénicos , Microscopía Confocal , Análisis de Secuencia por Matrices de Oligonucleótidos , Fosfoproteínas/genética , Fosforilación , Receptores de Antígenos de Linfocitos T/genética , Receptores de Antígenos de Linfocitos T/metabolismo , Linfocitos T Citotóxicos/citologíaRESUMEN
Biofilms are surface-attached agglomerations of microorganisms embedded in an extracellular matrix. Biofilm-associated infections are difficult to eradicate and represent a significant reservoir for disseminating and recurring serious infections. Infections involving biofilms frequently develop on indwelling medical devices in hospitalized patients, and Staphylococcus epidermidis is the leading cause of infection in this setting. However, the molecular determinants of biofilm dissemination are unknown. Here we have demonstrated that specific secreted, surfactant-like S. epidermidis peptides--the ß subclass of phenol-soluble modulins (PSMs)--promote S. epidermidis biofilm structuring and detachment in vitro and dissemination from colonized catheters in a mouse model of device-related infection. Our study establishes in vivo significance of biofilm detachment mechanisms for the systemic spread of biofilm-associated infection and identifies the effectors of biofilm maturation and detachment in a premier biofilm-forming pathogen. Furthermore, by demonstrating that antibodies against PSMß peptides inhibited bacterial spread from indwelling medical devices, we have provided proof of principle that interfering with biofilm detachment mechanisms may prevent dissemination of biofilm-associated infection.