RESUMEN
In this perspective review, the role Hematopoietic Progenitor Kinase 1 (HPK1) in tumor immunity will be reviewed, with special emphasis on how T cells are negatively-regulated at different junctures of cancer-immunity cycle by this regulatory kinase. The review will highlight the strengths and weaknesses of HPK1 as a candidate target for novel immuno-oncology (IO) drug development that is centered on the use of small molecule kinase inhibitor to modulate the immune response against cancer. Such a therapeutic approach, if proven successful, could supplement the cancer cell-centric standard of care therapies in order to fully meet the therapeutic needs of cancer patients.
Asunto(s)
Antineoplásicos/farmacología , Inmunidad , Neoplasias/tratamiento farmacológico , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Humanos , Neoplasias/inmunologíaRESUMEN
Hematopoietic progenitor kinase 1 (HPK1) is a negative regulator of TCR-initiated signal transduction. Both the HPK1-/- mice and the genetically engineered mice with a point mutation that disrupts the catalytic activity of HPK1 possess enhanced antitumor immunity, especially when these mice are treated with anti-PD-L1 immune checkpoint Ab. Because CD4+FOXP3+ regulatory T cells (Tregs) play an important role in suppressing tumor immunity, we investigated whether the loss of HPK1 expression could result in the reduction of Treg functions. We found that the number of HPK1-/- Tregs is elevated relative to the number found in wild-type C57/BL6 mice. However, HPK1-/- Tregs lack the ability to carry out effective inhibition of TCR-induced proliferative responses by effector T cells. Furthermore, HPK1-/- Tregs respond to TCR engagement with an elevated and sustained Erk MAPK and p65/RelA NF-κB phosphorylation in comparison with wild-type Tregs. Also, a multiplex cytokine analysis of HPK1-/- Tregs revealed that they demonstrate an aberrant cytokine expression profile when stimulated by anti-CD3ε and anti-CD28 crosslinking, including the uncharacteristic expression of IL-2 and antitumor proinflammatory cytokines and chemokines such as IFN-γ, CCL3, and CCL4. The aberrant HPK1-/- phenotype observed in these studies suggests that HPK1 may play an important role in maintaining Treg functions with wider implications for HPK1 as a novel immunotherapeutic target.
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FN-kappa B/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Linfocitos T Reguladores/metabolismo , Animales , Línea Celular , Quimiocinas/metabolismo , Citocinas/metabolismo , Ratones , FN-kappa B/genética , Fosforilación , Proteínas Serina-Treonina Quinasas/genética , Transducción de Señal , Linfocitos T Reguladores/citologíaRESUMEN
Hematopoietic progenitor kinase 1 (HPK1 or MAP4K1) is a Ser/Thr kinase that operates via the c-Jun N-terminal kinase (JNK) and extracellular signal-regulated kinase (ERK) signaling pathways to dampen the T-cell response and antitumor immunity. Accordingly, selective HPK1 inhibition is considered a means to enhance antitumor immunity. Sunitinib, a multi-receptor tyrosine kinase (RTK) inhibitor approved for the management of gastrointestinal stromal tumors (GISTs), renal cell carcinoma (RCC), and pancreatic cancer, has been reported to inhibit HPK1 in vitro In this report, we describe the crystal structures of the native HPK1 kinase domain in both nonphosphorylated and doubly phosphorylated states, in addition to a double phosphomimetic mutant (T165E,S171E), each complexed with sunitinib at 2.17-3.00-Å resolutions. The native nonphosphorylated cocrystal structure revealed an inactive dimer in which the activation loop of each monomer partially occupies the ATP- and substrate-binding sites of the partner monomer. In contrast, the structure of the protein with a doubly phosphorylated activation loop exhibited an active kinase conformation with a greatly reduced monomer-monomer interface. Conversely, the phosphomimetic mutant cocrystal structure disclosed an alternative arrangement in which the activation loops are in an extended domain-swapped configuration. These structural results indicate that HPK1 is a highly dynamic kinase that undergoes trans-regulation via dimer formation and extensive intramolecular and intermolecular remodeling of the activation segment.
Asunto(s)
Proteínas Serina-Treonina Quinasas/metabolismo , Sunitinib/metabolismo , Adenosina Trifosfato/química , Adenosina Trifosfato/metabolismo , Sitios de Unión , Cristalografía por Rayos X , Dimerización , Humanos , Interleucina-2/metabolismo , Simulación de Dinámica Molecular , Mutagénesis Sitio-Dirigida , Fosforilación , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Proteínas Serina-Treonina Quinasas/genética , Estructura Terciaria de Proteína , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Sunitinib/química , Sunitinib/farmacología , Linfocitos T/citología , Linfocitos T/efectos de los fármacos , Linfocitos T/metabolismoRESUMEN
In this issue of Structure, Wu et al. (2018) report several apo and small-molecule inhibitor-bound structures of the kinase domain of hematopoietic progenitor kinase 1, a ser/thr kinase that functions as an inhibitor of T cell activation. The studies reveal that the HPK1 kinase domain exists as a domain-swapped dimer.
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Trasplante de Células Madre Hematopoyéticas , Transducción de Señal , Activación de Linfocitos , Proteínas Serina-Treonina QuinasasRESUMEN
Patients with resected stage II-III cutaneous melanomas remain at high risk for metastasis and death. Biomarker development has been limited by the challenge of isolating high-quality RNA for transcriptome-wide profiling from formalin-fixed and paraffin-embedded (FFPE) primary tumor specimens. Using NanoString technology, RNA from 40 stage II-III FFPE primary melanomas was analyzed and a 53-immune-gene panel predictive of non-progression (area under the curve (AUC)=0.920) was defined. The signature predicted disease-specific survival (DSS P<0.001) and recurrence-free survival (RFS P<0.001). CD2, the most differentially expressed gene in the training set, also predicted non-progression (P<0.001). Using publicly available microarray data from 46 primary human melanomas (GSE15605), a coexpression module enriched for the 53-gene panel was then identified using unbiased methods. A Bayesian network of signaling pathways based on this data identified driver genes. Finally, the proposed 53-gene panel was confirmed in an independent test population of 48 patients (AUC=0.787). The gene signature was an independent predictor of non-progression (P<0.001), RFS (P<0.001), and DSS (P=0.024) in the test population. The identified driver genes are potential therapeutic targets, and the 53-gene panel should be tested for clinical application using a larger data set annotated on the basis of prospectively gathered data.
Asunto(s)
Redes Reguladoras de Genes , Melanoma/inmunología , Teorema de Bayes , Antígenos CD2/análisis , Genes p53 , Humanos , Melanoma/genética , Melanoma/mortalidad , Melanoma/patología , Estadificación de NeoplasiasRESUMEN
HIV-1 Nef is an accessory protein necessary for HIV-1 virulence and rapid AIDS development. Nef promotes viral replication and infection by connecting CD4 and several other cell surface receptors to the clathrin adaptor protein AP2, resulting in the internalization and degradation of the receptors interacting with Nef. We investigated how Nef can mediate constitutive receptor endocytosis through the interaction of the dileucine motif in its C-terminal flexible loop (C-loop) with AP2, whereas AP2 binding of the transmembrane receptors usually results in an equilibrated (recycled) endocytosis. Our results indicated that in addition to the dileucine motif, there is a second motif in the Nef C-loop involved in the Nef-AP2 interaction. Nef-mediated CD4 downregulation was impaired when the residue in the hydrophobic region in the Nef C-loop (LL165HPMSLHGM173) was mutated to a basic residue K/R or an acidic residue E/D or to the rigid residue P, or when M168L170, L170H171, or G172M173 was mutated to AA. A pull-down assay indicated that AP2 was not coprecipitated with Nef mutants that did not downregulate CD4. Molecular modeling of the Nef C-terminal flexible loop in complex with AP2 suggests that M168L170 occupies a pocket in the AP2 σ2 subunit. Our data suggest a new model in the Nef-AP2 interaction in which the hydrophobic region in the Nef C-loop with the dileucine (L164L165) motif and M168L170 motif binds to AP2(σ2), while the acidic motif E174 and D175 binds to AP2(α), which explains how Nef through the flexible loop connects CD4 to AP2 for constitutive CD4 downregulation.
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Antígenos CD4/metabolismo , Proteínas de Unión a Ácidos Grasos/metabolismo , VIH-1/fisiología , VIH-1/patogenicidad , Productos del Gen nef del Virus de la Inmunodeficiencia Humana/química , Productos del Gen nef del Virus de la Inmunodeficiencia Humana/metabolismo , Sitios de Unión , Regulación hacia Abajo , Proteínas de Unión a Ácidos Grasos/química , Genes nef , Infecciones por VIH/etiología , VIH-1/genética , Interacciones Huésped-Patógeno , Humanos , Interacciones Hidrofóbicas e Hidrofílicas , Modelos Moleculares , Complejos Multiproteicos , Mutación , Electricidad Estática , Virulencia , Productos del Gen nef del Virus de la Inmunodeficiencia Humana/genéticaRESUMEN
Identifying the appropriate drug targets for the development of a novel anti-tumor immunotherapy is one of the most risky steps in the drug development cycle. We have identified a hematopoietic cell-restricted serine/threonine kinase, hematopoietic progenitor kinase 1 (HPK1), as a possible target for therapeutic intervention. Targeted disruption of HPK1 alleles confers T cells with an elevated Th1 cytokine production in response to TCR engagement. HPK1 (-/-) T cells proliferate more rapidly than the haplotype-matched wild-type counterpart and are resistant to prostaglandin E2 (PGE(2))-mediated suppression. Most strikingly, mice that received adoptive transfer of HPK1 (-/-) T cells became resistant to lung tumor growth. Also, the loss of HPK1 from dendritic cells (DCs) endows them with superior antigen presentation ability, enabling HPK1 (-/-) DCs to elicit a more potent anti-tumor immune response when used as cancer vaccine. It is probable that blocking the HPK1 kinase activity with a small molecule inhibitor may activate the superior anti-tumor activity of both cell types, resulting in a synergistic amplification of anti-tumor potential. Given that HPK1 is not expressed in any major organs, it is less likely that an inhibitor of HPK1 kinase activity would cause any serious side effects.
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Neoplasias/terapia , Proteínas Serina-Treonina Quinasas/inmunología , Animales , Células Dendríticas/inmunología , Dinoprostona/inmunología , Humanos , Inmunoterapia , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Receptores de Antígenos de Linfocitos T/inmunología , Linfocitos T/inmunologíaRESUMEN
T cell (TC) activation requires the coordinated signaling of the T cell receptor (TCR) and coreceptor molecules, allowing TCs to respond to lower degrees of TCR occupancy. Coreceptor molecules set the threshold for TC activation by controlling different regulatory signaling loops. The Cbl family members prevent undesired activation of T cells by regulating TCR signals. In this report, we show that TC prestimulation by the CD43 coreceptor molecule before TCR engagement inhibits TCR-dependent c-Cbl tyrosine phosphorylation, c-Cbl interaction with the adapter molecule Crk-L and promotes Cbl-b degradation in a PKCθ-dependent manner. Consequently, the prolonged tyrosine phosphorylation and delayed degradation of ZAP-70 and of the ζ chain lead to enhanced mitogen-activated protein kinase activation and robust TC response. These data indicates that CD43-mediated signals lower the threshold for TC activation by restricting the c-Cbl and Cbl-b inhibitory effects on TCR signaling. In addition to the strength and duration of intracellular signals, our data underscore temporality with which certain molecules are engaged as yet another mechanism to fine tune TC signal quality, and ultimately immune function.
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Leucosialina/metabolismo , Activación de Linfocitos/fisiología , Proteínas Proto-Oncogénicas c-cbl/metabolismo , Transducción de Señal/fisiología , Linfocitos T/fisiología , Animales , Proliferación Celular , Humanos , Immunoblotting , Inmunoprecipitación , Células Jurkat , Activación de Linfocitos/inmunología , Ratones , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Fosforilación , Receptores de Antígenos de Linfocitos T/metabolismo , Transducción de Señal/inmunología , Linfocitos T/inmunologíaRESUMEN
BACKGROUND: Maintenance of complex transgenic colonies and labour-intensive techniques pose significant challenges in work involving mouse models for hepatocellular carcinoma (HCC). Other animal models of unusual species are generally impractical for research purposes. AIMS: To develop a highly reproducible orthotopic mouse model for HCC based on the murine α-foetoprotein (AFP), producing cell line Hepa1-6 and to monitor liver tumour progression via in vivo imaging, and measurement of plasma AFP. METHODS: Intrahepatic tumour was induced following subcapsular implantation of 10(+6) Hepa1-6 cells into C57L/J mice. AFP production was examined in vitro and in vivo using immunoblotting. Three confirmatory non-invasive imaging modalities were applied to follow tumour progression over time including ultrasound biomicroscopy (UBM), micromagnetic resonance imaging (microMRI), and bioluminescence. RESULTS: α-foetoprotein expression was confirmed both in vitro and in vivo, with increasing levels in the plasma as tumours progressed. UBM, microMRI and bioluminescence detected intrahepatic tumours to a 2 mm resolution by day 14. Sequential imaging studies demonstrated an intrahepatic pattern of disease progression with an observed median survival of 29 days. Immunosuppression of tumour-bearing mice led to a greater tumour size and decreased survival. CONCLUSIONS: Intrahepatic implantation of Hepa1-6 as a mouse model for HCC is a highly reproducible in vivo system with tumour biology analogous to human disease and is regulated by the presence of an intact host immune system. Tumour progression may be monitored in vivo by UBM, microMRI and bioluminescence. Plasma AFP increases over time, allowing redundancy in non-invasive means of following tumour progression.
Asunto(s)
Carcinoma Hepatocelular/diagnóstico , Diagnóstico por Imagen , Inmunocompetencia , Neoplasias Hepáticas Experimentales/diagnóstico , Animales , Western Blotting , Carcinoma Hepatocelular/inmunología , Carcinoma Hepatocelular/metabolismo , Carcinoma Hepatocelular/patología , Línea Celular Tumoral , Proliferación Celular , Diagnóstico por Imagen/métodos , Neoplasias Hepáticas Experimentales/inmunología , Neoplasias Hepáticas Experimentales/metabolismo , Neoplasias Hepáticas Experimentales/patología , Luciferasas de Luciérnaga/biosíntesis , Luciferasas de Luciérnaga/genética , Mediciones Luminiscentes , Imagen por Resonancia Magnética , Ratones , Ratones Endogámicos , Microscopía Acústica , Trasplante de Neoplasias , Factores de Tiempo , Transfección , Carga Tumoral , alfa-Fetoproteínas/metabolismoRESUMEN
HIV-1 and SIV Nef proteins downregulate cell surface CD4 and MHC class I (MHC-I) molecules of infected cells, which are necessary for efficient viral replication and pathogenicity. We previously reported that K144 in HIV-1 Nef is di-ubiquitinated, and K144R substitution impairs Nef-mediated CD4 downregulation. In this report, we extend the role of ubiquitination at this lysine residue from Nef-mediated CD4 downregulation to Nef-mediated MHC-I downregulation and from HIV Nef to SIV Nef. All HIV-1 Nef mutants that contain K144R substitution are inactive in MHC-I downregulation. Tested MHC-I alleles include HLA-ABC endogenously expressed and HLA-A2 exogenously expressed in Jurkat T cells. CD4 downregulation by SIV Nef involves K176 that aligns with K144 in HIV-1 Nef, as well as an N-terminal tyrosine motif Y28Y39 not present in HIV-1 Nef. Dual mutation at K176 and Y28Y39 completely impaired SIV Nef-mediated CD4 and MHC-I downregulation, whereas a single mutation at K176 or Y28Y39 did not. The involvement of tyrosine motif in SIV Nef-mediated CD4 and MHC-I downregulation prompted us to investigate a putative tyrosine motif (Y202Y/F203) in HIV-1 Nef that is conserved among HIV-1 species. Single mutation at the tyrosine motif Y202F203 in HIV-1 Nef (NA7) greatly impaired Nef-mediated CD4 downregulation, which is similar to what we observed previously with the single mutation at lysine K144. Thus, our study demonstrated that Nef-mediated receptor endocytosis involves the ubiquitination motif and tyrosine motif.
Asunto(s)
Antígenos CD4/metabolismo , Endocitosis , Productos del Gen nef/metabolismo , VIH-1/metabolismo , Virus de la Inmunodeficiencia de los Simios/metabolismo , Productos del Gen nef del Virus de la Inmunodeficiencia Humana/metabolismo , Secuencias de Aminoácidos , Antígenos CD4/genética , Regulación hacia Abajo , Productos del Gen nef/química , Productos del Gen nef/genética , Genes MHC Clase I , VIH-1/química , VIH-1/genética , VIH-1/inmunología , Antígeno HLA-A2 , Células HeLa , Humanos , Immunoblotting , Células Jurkat , Virus de la Inmunodeficiencia de los Simios/química , Virus de la Inmunodeficiencia de los Simios/genética , Virus de la Inmunodeficiencia de los Simios/inmunología , Ubiquitinación , Productos del Gen nef del Virus de la Inmunodeficiencia Humana/química , Productos del Gen nef del Virus de la Inmunodeficiencia Humana/genéticaRESUMEN
BACKGROUND: Nef is a 27 KDa HIV-1 accessory protein. It downregulates CD4 from infected cell surface, a mechanism critical for efficient viral replication and pathogenicity. Agents that antagonize the Nef-mediated CD4 downregulation may offer a new class of drug to combat HIV infection and disease. TPCK (N-alpha-p-tosyl-L-phenylalanine chloromethyl ketone) and TLCK (N-alpha-p-tosyl-L-lysine chloromethyl ketone) are alkylation reagents that chemically modify the side chain of His or Cys residues in a protein. In search of chemicals that inhibit Nef function, we discovered that TPCK and TLCK alkylated HIV Nef. METHODS: Nef modification by TPCK was demonstrated on reducing SDS-PAGE. The specific cysteine residues modified were determined by site-directed mutagenesis and mass spectrometry (MS). The effect of TPCK modification on Nef-CD4 interaction was studied using fluorescence titration of a synthetic CD4 tail peptide with recombinant Nef-His protein. The conformational change of Nef-His protein upon TPCK-modification was monitored using CD spectrometry RESULTS: Incubation of Nef-transfected T cells, or recombinant Nef-His protein, with TPCK resulted in mobility shift of Nef on SDS-PAGE. Mutagenesis analysis indicated that the modification occurred at Cys55 and Cys206 in Nef. Mass spectrometry demonstrated that the modification was a covalent attachment (alkylation) of TPCK at Cys55 and Cys206. Cys55 is next to the CD4 binding motif (A56W57L58) in Nef required for Nef-mediated CD4 downregulation and for AIDS development. This implies that the addition of a bulky TPCK molecule to Nef at Cys55 would impair Nef function and reduce HIV pathogenicity. As expected, Cys55 modification reduced the strength of the interaction between Nef-His and CD4 tail peptide by 50%. CONCLUSIONS: Our data suggest that this Cys55-specific alkylation mechanism may be exploited to develop a new class of anti HIV drugs.
RESUMEN
Lung cancer is the leading cause of cancer-related mortality in the world, resulting in over a million deaths each year. Non-small cell lung cancers (NSCLCs) are characterized by a poor immunogenic response, which may be the result of immunosuppressive factors such as prostaglandin E2 (PGE(2)) present in the tumor environment. The effect of PGE(2) in the suppression of anti-tumor immunity and its promotion of tumor survival has been established for over three decades, but with limited mechanistic understanding. We have previously reported that PGE(2) activates hematopoietic progenitor kinase 1 (HPK1), a hematopoietic-specific kinase known to negatively regulate T-cell receptor signaling. Here, we report that mice genetically lacking HPK1 resist the growth of PGE(2)-producing Lewis lung carcinoma (LLC). The presence of tumor-infiltrating lymphocytes (TILs) and T-cell transfer into T cell-deficient mice revealed that tumor rejection is T cell mediated. Further analysis demonstrated that this may be significantly due to the ability of HPK1 (-/-) T cells to withstand PGE(2)-mediated suppression of T-cell proliferation, IL-2 production, and apoptosis. We conclude that PGE(2) utilizes HPK1 to suppress T cell-mediated anti-tumor responses.
Asunto(s)
Carcinoma Pulmonar de Lewis/inmunología , Dinoprostona/fisiología , Proteínas Serina-Treonina Quinasas/fisiología , Linfocitos T/inmunología , Escape del Tumor , Animales , Tolerancia Inmunológica , Ratones , Ratones Endogámicos BALB C , Ratones Noqueados , Proteínas Serina-Treonina Quinasas/genéticaRESUMEN
Hematopoietic progenitor kinase 1 (HPK1) is a hematopoietic cell-restricted member of the Ste20 kinases that acts as a negative regulator of T cell functions through the AP-1, NFAT, and NFkappaB pathways. Using HPK1-deficient (HPK1(-/-)) mice, we report in this study a novel role for HPK1 in dendritic cells (DCs). Specifically, we observed that matured HPK1(-/-) bone marrow-derived DCs (BMDCs) are superior to their wild-type (WT) counterpart in stimulating T cell proliferation in vivo and in vitro. Several characteristics of HPK1(-/-) BMDCs may account for this enhanced activity: Matured HPK1(-/-) BMDCs express higher levels of costimulatory molecules CD80, CD86, and I-A(b) as well as produce more proinflammatory cytokines IL-12, IL-1beta, TNF-alpha, and IL-6 than their WT littermates. The role of HPK1 as a proapoptotic molecule was assessed post activation with LPS, and results indicated that HPK1(-/-) BMDCs are significantly resistant to LPS-induced apoptosis. Our results led us to investigate the role of HPK1(-/-) BMDCs in tumor immunotherapy. Using a s.c. murine model of Lewis Lung Carcinoma, we found that HPK1(-/-) BMDCs eliminate established s.c. Lewis Lung Carcinoma more efficiently than their WT counterpart. Our data reveal a novel role for HPK1 as a negative regulator of DC functions, identifying its potential as a molecular target for DC-based immunotherapy against cancers.
Asunto(s)
Presentación de Antígeno/inmunología , Células Dendríticas/inmunología , Proteínas Serina-Treonina Quinasas/inmunología , Animales , Apoptosis/inmunología , Células de la Médula Ósea/citología , Células de la Médula Ósea/inmunología , Carcinoma Pulmonar de Lewis/inmunología , Diferenciación Celular/inmunología , Citocinas/biosíntesis , Células Dendríticas/citología , Ensayo de Inmunoadsorción Enzimática , Citometría de Flujo , Activación de Linfocitos/inmunología , Prueba de Cultivo Mixto de Linfocitos , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Proteínas Serina-Treonina Quinasas/genética , Linfocitos T/inmunologíaRESUMEN
Interleukin-3 (IL-3) regulates cell growth by affecting various processes such as cell death, survival, and proliferation. Cues from the external environment are sensed by surface receptors, and complex signaling mechanisms arise within the cells, leading to specific functional outcomes. In this study, we demonstrate that the cytokine IL-3 induces the activation of the Ca(2+)-dependent phosphatase, calcineurin (Cn). Furthermore Cn dephosphorylates Gab2, resulting in c-fos activation and cell proliferation. We also report that there is a direct interaction between Cn and Gab2 upon IL-3 stimulation, and Akt can regulate this interaction.
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Calcineurina/metabolismo , Interleucina-3/metabolismo , Fosfoproteínas/metabolismo , Proteínas Proto-Oncogénicas c-fos/metabolismo , Transducción de Señal/fisiología , Proteínas Adaptadoras Transductoras de Señales , Animales , Calcio/metabolismo , Muerte Celular/fisiología , Línea Celular , Proliferación Celular , Supervivencia Celular/fisiología , Activación Enzimática/fisiología , Ratones , Fosforilación , Proteínas Proto-Oncogénicas c-akt/metabolismoRESUMEN
Nef is a HIV-1 accessory protein critical for the replication of the virus and the development of AIDS. The major pathological activity of Nef is the down-regulation of CD4, the primary receptor of HIV-1 infection. The mechanism underlying Nef-mediated CD4 endocytosis and degradation remains incompletely understood. Since protein ubiquitination is the predominant sorting signal in receptor endocytosis, we investigated whether Nef is ubiquitinated. The in vivo ubiquitination assay showed that both HIV-1 and SIV Nef proteins expressed in Jurkat T cells and 293T cells were multiple ubiquitinated by ubiquitin-His. The lysine-free HIV-1 Nef mutant (Delta10K) generated by replacing all 10 lysines with arginines was not ubiquitinated and the major ubiquitin-His attachment sites in HIV-1 Nef were determined to be lysine 144 (di-ubiquitinated) and lysine 204 (mono-ubiquitinated). Lysine-free HIV-1 Nef was completely inactive in Nef-mediated CD4 down-regulation, so was the Nef mutant with a single arginine substitution at K144 but not at K204. A mutant HIV-1 provirion NL4-3 with a single arginine substitution in Nef at K144 was also inactive in Nef-mediated CD4 down-regulation. Lysine-free Nef mutant reintroduced with lysine 144 (DeltaK10 + K144) was shown active in CD4 down-regulation. These data suggest that ubiquitination of Nef, particularly diubiquitination of the lysine 144, is necessary for Nef-mediated CD4 down-regulation.
Asunto(s)
Antígenos CD4/metabolismo , Regulación hacia Abajo/inmunología , VIH-1/fisiología , Lisina/fisiología , Ubiquitina/metabolismo , Acoplamiento Viral , Productos del Gen nef del Virus de la Inmunodeficiencia Humana/fisiología , Sustitución de Aminoácidos/genética , Sustitución de Aminoácidos/inmunología , Arginina/genética , Línea Celular , Regulación hacia Abajo/genética , Endocitosis/genética , Endocitosis/inmunología , Productos del Gen nef/genética , Productos del Gen nef/metabolismo , VIH-1/genética , Células HeLa , Humanos , Hibridomas , Células Jurkat , Lisina/genética , Lisina/metabolismo , Ubiquitinación , Productos del Gen nef del Virus de la Inmunodeficiencia Humana/genética , Productos del Gen nef del Virus de la Inmunodeficiencia Humana/metabolismoRESUMEN
Hematopoietic progenitor kinase 1 (HPK1) is a hematopoietic cell-restricted member of the Ste20 serine/threonine kinase super family. We recently reported that the immunosuppressive eicosanoid, prostaglandin E(2) (PGE(2)), is capable of activating HPK1 in T cells. In this report, we demonstrate that unlike the TCR-induced activation of HPK1 kinase activity, the induction of HPK1 catalytic activity by PGE(2) does not require the presence of phosphotyrosine-based signaling molecules such as Lck, ZAP-70, SLP-76, and Lat. Nor does the PGE(2)-induced HPK1 activation require the intermolecular interaction between its proline-rich regions and the SH3 domain-containing adaptor proteins, as required by the signaling from the TCR to HPK1. Instead, our study reveals that PGE(2) signal to HPK1 via a 3' -5 '-cyclic adenosine monophosphate-regulated, PKA-dependent pathway. Consistent with this observation, changing the serine 171 residue that forms the optimal PKA phosphorylation site within the "activation loop" of HPK1 to alanine completely prevents this mutant from responding to PGE(2)-generated stimulation signals. Moreover, the inability of HPK1 to respond to PGE(2) stimulation in PKA-deficient S49 cells further supports the importance of PKA in this signaling pathway. We speculate that this unique signaling pathway enables PGE(2) signals to engage a proven negative regulator of TCR signal transduction pathway and uses it to inhibit T cell activation.
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Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Dinoprostona/fisiología , Regulación Enzimológica de la Expresión Génica , Proteínas Serina-Treonina Quinasas/biosíntesis , Secuencia de Aminoácidos , Catálisis , Dinoprostona/metabolismo , Activación Enzimática , Humanos , Inmunosupresores/farmacología , Células Jurkat , Datos de Secuencia Molecular , Fosforilación , Prolina/química , Homología de Secuencia de Aminoácido , Transducción de SeñalAsunto(s)
Investigación Biomédica/métodos , Biología Computacional/métodos , Bases de Datos Factuales , Oncología Médica/métodos , Proteínas de Neoplasias/metabolismo , Neoplasias/fisiopatología , Proyectos de Investigación , Integración de Sistemas , Investigación Biomédica/tendencias , Biología Computacional/tendencias , Oncología Médica/tendencias , Investigación/tendenciasRESUMEN
Efficient development of thymocytes requires participation of a CD8 or CD4 coreceptor in the TCR:MHC interaction. Both CD8 and CD4 coreceptor cytoplasmic domains associate with Lck. In this study, we attempted to delineate the role of CD8alpha-associated Lck in driving CD8 single positive (SP) thymocyte development. We used a chimeric molecule encoding the extracellular and transmembrane domains of CD8alpha fused to full-length Lck. In mice deficient for CD8alpha and transgenic for 2C, a MHC class I-restricted TCR, robust reconstitution of CD8 SP thymocytes occurred both centrally and peripherally. The reconstituted CD8 SP population was phenotypically and functionally comparable to 2C wild-type counterparts expressing endogenous CD8alpha. A CD8alpha/Lck kinase-dead chimera also resulted in reconstitution of CD8 SP thymocytes. Our results suggest that CD8alpha-associated Lck is sufficient to drive CD8 SP thymocyte development. Furthermore, this CD8 SP development may not necessarily depend on Lck kinase activity.