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1.
Nat Struct Mol Biol ; 28(9): 762-770, 2021 09.
Artigo em Inglês | MEDLINE | ID: mdl-34518698

RESUMO

Kinases play central roles in signaling cascades, relaying information from the outside to the inside of mammalian cells. De novo designed protein switches capable of interfacing with tyrosine kinase signaling pathways would open new avenues for controlling cellular behavior, but, so far, no such systems have been described. Here we describe the de novo design of two classes of protein switch that link phosphorylation by tyrosine and serine kinases to protein-protein association. In the first class, protein-protein association is required for phosphorylation by the kinase, while in the second class, kinase activity drives protein-protein association. We design systems that couple protein binding to kinase activity on the immunoreceptor tyrosine-based activation motif central to T-cell signaling, and kinase activity to reconstitution of green fluorescent protein fluorescence from fragments and the inhibition of the protease calpain. The designed switches are reversible and function in vitro and in cells with up to 40-fold activation of switching by phosphorylation.


Assuntos
Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Tirosina Quinases/metabolismo , Motivos de Aminoácidos , Ligação Competitiva , Proteínas de Ligação ao Cálcio/farmacologia , Calpaína/antagonistas & inibidores , Calpaína/metabolismo , Catálise , Domínio Catalítico , Linhagem Celular , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Desenho de Fármacos , Genes Sintéticos , Proteínas de Fluorescência Verde/química , Proteínas de Fluorescência Verde/metabolismo , Humanos , Ligação de Hidrogênio , Modelos Moleculares , Fosforilação , Fosfotirosina/metabolismo , Ligação Proteica , Conformação Proteica , Domínios Proteicos , Mapeamento de Interação de Proteínas , Processamento de Proteína Pós-Traducional , Proteínas Serina-Treonina Quinases/química , Proteínas Tirosina Quinases/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Transdução de Sinais , Relação Estrutura-Atividade , Quinases da Família src/metabolismo
2.
Biochim Biophys Acta Gen Subj ; 1865(11): 129977, 2021 11.
Artigo em Inglês | MEDLINE | ID: mdl-34391832

RESUMO

BACKGROUND: Phosphotyrosine Binding (PTB) Domains, usually found on scaffold proteins, are pervasive in many cellular signaling pathways. These domains are the second-largest family of phosphotyrosine recognition domains and since their initial discovery, dozens of PTB domains have been structurally determined. SCOPE OF REVIEW: Due to its signature sequence flexibility, PTB domains can bind to a large variety of ligands including phospholipids. PTB peptide binding is divided into classical binding (canonical NPXY motifs) and non-classical binding (all other motifs). The first atypical PTB domain was discovered in cerebral cavernous malformation 2 (CCM2) protein, while only one third in size of the typical PTB domain, it remains functionally equivalent. MAJOR CONCLUSIONS: PTB domains are involved in numerous signaling processes including embryogenesis, neurogenesis, and angiogenesis, while dysfunction is linked to major disorders including diabetes, hypercholesterolemia, Alzheimer's disease, and strokes. PTB domains may also be essential in infectious processes, currently responsible for the global pandemic in which viral cellular entry is suspected to be mediated through PTB and NPXY interactions. GENERAL SIGNIFICANCE: We summarize the structural and functional updates in the PTB domain over the last 20 years in hopes of resurging interest and further analyzing the importance of this versatile domain.


Assuntos
Doença de Alzheimer/metabolismo , Diabetes Mellitus/metabolismo , Hipercolesterolemia/metabolismo , Fosfotirosina/metabolismo , Acidente Vascular Cerebral/metabolismo , Sítios de Ligação , Humanos , Fosfotirosina/química
3.
Sci Rep ; 11(1): 16951, 2021 08 20.
Artigo em Inglês | MEDLINE | ID: mdl-34417497

RESUMO

T-cell activation and cellular expansion by common gamma chain cytokines such as Interleukin-2 is necessary for adaptive immunity. However, when unregulated these same pathways promote pathologies ranging from autoimmune disorders to cancer. While the functional role of Interleukin-2 and downstream effector molecules is relatively clear, the repertoire of phosphoregulatory proteins downstream of this pathway is incomplete. To identify phosphoproteins downstream of common gamma chain receptor, YT cells were radiolabeled with [32P]-orthophosphate and stimulated with Interleukin-2. Subsequently, tyrosine phosphorylated proteins were immunopurified and subjected to tandem mass spectrometry-leading to the identification of CrkL. Phosphoamino acid analysis revealed concurrent serine phosphorylation of CrkL and was later identified as S114 by mass spectrometry analysis. S114 was inducible through stimulation with Interleukin-2 or T-cell receptor stimulation. Polyclonal antibodies were generated against CrkL phospho-S114, and used to show its inducibility by multiple stimuli. These findings confirm CrkL as an Interleukin-2 responsive protein that becomes phosphorylated at S114 by a kinase/s downstream of PI3K and MEK/ERK signaling.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Citocinas/metabolismo , Subunidade gama Comum de Receptores de Interleucina/metabolismo , Interleucina-2/metabolismo , Fosfosserina/metabolismo , Receptores de Antígenos de Linfócitos T/metabolismo , Transdução de Sinais , Proteínas Adaptadoras de Transdução de Sinal/química , Sequência de Aminoácidos , Anticorpos/metabolismo , Linhagem Celular , Humanos , Sistema de Sinalização das MAP Quinases , Peptídeos/química , Peptídeos/metabolismo , Fosforilação , Fosfotirosina/metabolismo , Processamento de Proteína Pós-Traducional , Serina-Treonina Quinases TOR/metabolismo
4.
PLoS Biol ; 19(6): e3001281, 2021 06.
Artigo em Inglês | MEDLINE | ID: mdl-34077419

RESUMO

Nutrient-responsive protein kinases control the balance between anabolic growth and catabolic processes such as autophagy. Aberrant regulation of these kinases is a major cause of human disease. We report here that the vertebrate nonreceptor tyrosine kinase Src-related kinase lacking C-terminal regulatory tyrosine and N-terminal myristylation sites (SRMS) inhibits autophagy and promotes growth in a nutrient-responsive manner. Under nutrient-replete conditions, SRMS phosphorylates the PHLPP scaffold FK506-binding protein 51 (FKBP51), disrupts the FKBP51-PHLPP complex, and promotes FKBP51 degradation through the ubiquitin-proteasome pathway. This prevents PHLPP-mediated dephosphorylation of AKT, causing sustained AKT activation that promotes growth and inhibits autophagy. SRMS is amplified and overexpressed in human cancers where it drives unrestrained AKT signaling in a kinase-dependent manner. SRMS kinase inhibition activates autophagy, inhibits cancer growth, and can be accomplished using the FDA-approved tyrosine kinase inhibitor ibrutinib. This illuminates SRMS as a targetable vulnerability in human cancers and as a new target for pharmacological induction of autophagy in vertebrates.


Assuntos
Autofagia , Neoplasias/metabolismo , Neoplasias/patologia , Proteínas de Ligação a Tacrolimo/metabolismo , Quinases da Família src/metabolismo , Adenina/análogos & derivados , Adenina/farmacologia , Animais , Autofagia/efeitos dos fármacos , Proteína Beclina-1/metabolismo , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Senescência Celular/efeitos dos fármacos , Ativação Enzimática/efeitos dos fármacos , Camundongos , Proteínas Nucleares/metabolismo , Fosfoproteínas Fosfatases/metabolismo , Fosforilação/efeitos dos fármacos , Fosfotirosina/metabolismo , Piperidinas/farmacologia , Ligação Proteica/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-akt/metabolismo , Transdução de Sinais/efeitos dos fármacos , Quinases da Família src/antagonistas & inibidores
5.
Nature ; 595(7867): 404-408, 2021 07.
Artigo em Inglês | MEDLINE | ID: mdl-34163073

RESUMO

Congenital myasthenia (CM) is a devastating neuromuscular disease, and mutations in DOK7, an adaptor protein that is crucial for forming and maintaining neuromuscular synapses, are a major cause of CM1,2. The most common disease-causing mutation (DOK71124_1127 dup) truncates DOK7 and leads to the loss of two tyrosine residues that are phosphorylated and recruit CRK proteins, which are important for anchoring acetylcholine receptors at synapses. Here we describe a mouse model of this common form of CM (Dok7CM mice) and a mouse with point mutations in the two tyrosine residues (Dok72YF). We show that Dok7CM mice had severe deficits in neuromuscular synapse formation that caused neonatal lethality. Unexpectedly, these deficits were due to a severe deficiency in phosphorylation and activation of muscle-specific kinase (MUSK) rather than a deficiency in DOK7 tyrosine phosphorylation. We developed agonist antibodies against MUSK and show that these antibodies restored neuromuscular synapse formation and prevented neonatal lethality and late-onset disease in Dok7CM mice. These findings identify an unexpected cause for disease and a potential therapy for both DOK7 CM and other forms of CM caused by mutations in AGRIN, LRP4 or MUSK, and illustrate the potential of targeted therapy to rescue congenital lethality.


Assuntos
Proteínas Musculares/genética , Mutação , Síndromes Miastênicas Congênitas/tratamento farmacológico , Síndromes Miastênicas Congênitas/genética , Envelhecimento , Agrina/genética , Agrina/metabolismo , Animais , Animais Recém-Nascidos , Anticorpos/imunologia , Modelos Animais de Doenças , Feminino , Proteínas Relacionadas a Receptor de LDL/genética , Proteínas Relacionadas a Receptor de LDL/metabolismo , Masculino , Camundongos , Terapia de Alvo Molecular , Fibras Musculares Esqueléticas/química , Fibras Musculares Esqueléticas/metabolismo , Proteínas Musculares/química , Proteínas Musculares/metabolismo , Síndromes Miastênicas Congênitas/imunologia , Fosforilação , Fosfotirosina/genética , Fosfotirosina/metabolismo , Proteínas Proto-Oncogênicas c-crk/metabolismo , Receptores Proteína Tirosina Quinases/agonistas , Receptores Proteína Tirosina Quinases/genética , Receptores Proteína Tirosina Quinases/imunologia , Receptores Proteína Tirosina Quinases/metabolismo , Recidiva , Sinapses/metabolismo
6.
Biochem Biophys Res Commun ; 562: 139-145, 2021 07 12.
Artigo em Inglês | MEDLINE | ID: mdl-34052659

RESUMO

We recently isolated a novel co-activator of peroxisome proliferator-activated receptor γ, helicase with zinc finger 2 (HELZ2). HELZ2 null mice were resistant to diet-induced obesity and NAFFL/NASH, and HELZ2 was phosphorylated at tyrosine residues. In order to find a factor related to HELZ2, we analyzed products co-immunoprecipitated with phosphorylated HELZ2 by mass spectrometry analyses. We identified proline- and glutamine-rich (SFPQ) as a protein associating with tyrosine-phosphorylated HELZ2. The knockdown of SFPQ in 3T3-L1 cells downregulated mRNA levels of transcription factors including Krox20, Cebpß, and Cebpδ: key factors for early-stage adipocyte differentiation. In addition, knockdown of SFPQ inhibited 3T3-L1 cell differentiation to mature adipocytes. These findings demonstrated that SFPQ associating with HELZ2 is an important novel transcriptional regulator of adipocyte differentiation.


Assuntos
Adipócitos/citologia , Adipócitos/metabolismo , Diferenciação Celular , Núcleo Celular/metabolismo , PPAR gama/metabolismo , Fator de Processamento Associado a PTB/metabolismo , Transativadores/metabolismo , Fatores de Transcrição/metabolismo , Células 3T3-L1 , Animais , Regulação da Expressão Gênica , Células HeLa , Humanos , Gotículas Lipídicas/metabolismo , Camundongos , Fosforilação , Fosfotirosina/metabolismo , Ligação Proteica , RNA Interferente Pequeno/metabolismo
7.
Org Lett ; 23(11): 4244-4249, 2021 06 04.
Artigo em Inglês | MEDLINE | ID: mdl-34029466

RESUMO

Access to phosphotyrosine (pTyr) mimetics requires multistep syntheses, and therefore late stage incorporation of these mimetics into peptides is not feasible. Here, we develop and employ metallaphotoredox catalysis using 4-halogenated phenylalanine to afford a variety of protected pTyr mimetics in one step. This methodology was shown to be tolerant of common protecting groups and applicable to the late stage pTyr mimetic modification of protected and unprotected peptides, and peptides of biological relevance.


Assuntos
Peptídeos/metabolismo , Fenilalanina/química , Fosfotirosina/metabolismo , Catálise , Estrutura Molecular , Peptídeos/química , Fosfotirosina/química , Transdução de Sinais/fisiologia
8.
Sci Rep ; 11(1): 8675, 2021 04 21.
Artigo em Inglês | MEDLINE | ID: mdl-33883672

RESUMO

In epithelial cancers, the epidermal growth factor receptor (EGFR) and integrin α6ß4 are frequently overexpressed and found to synergistically activate intracellular signaling pathways that promote cell proliferation and migration. In cancer cells, the ß4 subunit is phosphorylated at tyrosine residues not normally recognized as kinase substrates; however, the function of these phosphotyrosine residues in cancer cells is a subject of much debate. In EGFR-overexpressing carcinoma cells, we found that the Src family kinase (SFK) inhibitor PP2 reduces ß4 tyrosine phosphorylation following the activation of EGFR. However, siRNA mediated knockdown of the SFKs Src, Fyn, Yes and Lyn, individually or in combination, did not affect the EGF-induced phosphorylation of ß4. Using phospho-peptide affinity chromatography and mass spectrometry, we found that PLCγ1 binds ß4 at the phosphorylated residues Y1422/Y1440, but were unable to verify this interaction in A431 carcinoma cells that overexpress the EGFR. Furthermore, using A431 cells devoid of ß4 or reconstituted with phenylalanine specific mutants of ß4, the activation of several downstream signaling pathways, including PLCγ/PKC, MAPK and PI3K/Akt, were not substantially affected. We conclude that tyrosine-phosphorylated ß4 does not enhance EGFR-mediated signaling in EGFR-overexpressing cells, despite the fact that this integrin subunit is highly tyrosine phosphorylated in these cells.


Assuntos
Regulação Neoplásica da Expressão Gênica/genética , Integrina beta4/metabolismo , Neoplasias Cutâneas/metabolismo , Tirosina/metabolismo , Animais , Linhagem Celular Tumoral , Humanos , Integrina beta4/fisiologia , Espectrometria de Massas , Fosforilação , Fosfotirosina/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transdução de Sinais , Neoplasias Cutâneas/genética
9.
Nat Commun ; 12(1): 2163, 2021 04 12.
Artigo em Inglês | MEDLINE | ID: mdl-33846331

RESUMO

γδ T cells are a distinct subgroup of T cells that bridge the innate and adaptive immune system and can attack cancer cells in an MHC-unrestricted manner. Trials of adoptive γδ T cell transfer in solid tumors have had limited success. Here, we show that DNA methyltransferase inhibitors (DNMTis) upregulate surface molecules on cancer cells related to γδ T cell activation using quantitative surface proteomics. DNMTi treatment of human lung cancer potentiates tumor lysis by ex vivo-expanded Vδ1-enriched γδ T cells. Mechanistically, DNMTi enhances immune synapse formation and mediates cytoskeletal reorganization via coordinated alterations of DNA methylation and chromatin accessibility. Genetic depletion of adhesion molecules or pharmacological inhibition of actin polymerization abolishes the potentiating effect of DNMTi. Clinically, the DNMTi-associated cytoskeleton signature stratifies lung cancer patients prognostically. These results support a combinatorial strategy of DNMTis and γδ T cell-based immunotherapy in lung cancer management.


Assuntos
Citoesqueleto/metabolismo , Citotoxicidade Imunológica/genética , Epigênese Genética , Sinapses Imunológicas/genética , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/imunologia , Receptores de Antígenos de Linfócitos T gama-delta/metabolismo , Citoesqueleto de Actina/efeitos dos fármacos , Citoesqueleto de Actina/metabolismo , Animais , Linhagem Celular Tumoral , Citoesqueleto/efeitos dos fármacos , Citotoxicidade Imunológica/efeitos dos fármacos , DNA (Citosina-5-)-Metiltransferases/antagonistas & inibidores , DNA (Citosina-5-)-Metiltransferases/metabolismo , Decitabina/farmacologia , Inibidores Enzimáticos/farmacologia , Epigênese Genética/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Sinapses Imunológicas/efeitos dos fármacos , Marcação por Isótopo , Ativação Linfocitária/efeitos dos fármacos , Ativação Linfocitária/genética , Subpopulações de Linfócitos/efeitos dos fármacos , Subpopulações de Linfócitos/metabolismo , Masculino , Camundongos Endogâmicos NOD , Fosfotirosina/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Análise de Sobrevida , Proteína Supressora de Tumor p53/metabolismo , Regulação para Cima/efeitos dos fármacos , Ensaios Antitumorais Modelo de Xenoenxerto
10.
Elife ; 102021 03 23.
Artigo em Inglês | MEDLINE | ID: mdl-33755016

RESUMO

SHP2 is a protein tyrosine phosphatase that normally potentiates intracellular signaling by growth factors, antigen receptors, and some cytokines, yet is frequently mutated in human cancer. Here, we examine the role of SHP2 in the responses of breast cancer cells to EGF by monitoring phosphoproteome dynamics when SHP2 is allosterically inhibited by SHP099. The dynamics of phosphotyrosine abundance at more than 400 tyrosine residues reveal six distinct response signatures following SHP099 treatment and washout. Remarkably, in addition to newly identified substrate sites on proteins such as occludin, ARHGAP35, and PLCγ2, another class of sites shows reduced phosphotyrosine abundance upon SHP2 inhibition. Sites of decreased phospho-abundance are enriched on proteins with two nearby phosphotyrosine residues, which can be directly protected from dephosphorylation by the paired SH2 domains of SHP2 itself. These findings highlight the distinct roles of the scaffolding and catalytic activities of SHP2 in effecting a transmembrane signaling response.


Assuntos
Receptores ErbB/metabolismo , Proteína Tirosina Fosfatase não Receptora Tipo 11/metabolismo , Proteômica/métodos , Catálise , Linhagem Celular Tumoral , Fator de Crescimento Epidérmico/metabolismo , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Humanos , Ocludina/metabolismo , Fosfolipase C gama/metabolismo , Fosfoproteínas/metabolismo , Fosforilação , Fosfotirosina/metabolismo , Piperidinas/metabolismo , Piperidinas/farmacologia , Ligação Proteica , Pirimidinas/metabolismo , Pirimidinas/farmacologia , Proteínas Repressoras/metabolismo , Transdução de Sinais/efeitos dos fármacos , Domínios de Homologia de src
11.
Plant Signal Behav ; 16(5): 1899488, 2021 05 04.
Artigo em Inglês | MEDLINE | ID: mdl-33784940

RESUMO

Receptor for activated C kinase 1 (RACK1) is WD-40 type scaffold protein, conserved in all eukaryote organisms. Many reports implicated RACK1 in plant hormone signal transduction pathways including in auxin and diverse stress signaling pathways; however, the precise molecular mechanism of its role is not understood. Previously, a group of small compounds targeting the Arabidopsis RACK1A functional site-Tyr248 have been developed. Here, the three different small compounds are used to elucidate the role of RACK1A in auxin mediated lateral root development. Through monitoring the auxin response in the architecture of lateral roots and auxin reporter assays, a small molecule- SD29-12 was found to stabilize the auxin induced RACK1A Tyr248 phosphorylation, thereby stimulating auxin signaling and inducing lateral roots formation. In contrast, two other compounds, SD29 and SD29-14, inhibited auxin induced RACK1A Tyr248 phosphorylation resulting in the inhibition of auxin sensitivity and alternation in the lateral roots formation. Taken together, auxin induced RACK1A Tyr248 phosphorylation is found to be the critical regulatory mechanism for auxin-mediated lateral root development. This work leads to the molecular understanding of the role RACK1A plays in the auxin induced lateral root development signaling pathways. The auxin signal stimulating compound has the potential to be used as auxin-based root inducing bio-stimulant.


Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Ácidos Indolacéticos/farmacologia , Fosfotirosina/metabolismo , Raízes de Plantas/crescimento & desenvolvimento , Receptores de Quinase C Ativada/metabolismo , Bibliotecas de Moléculas Pequenas/farmacologia , Arabidopsis/efeitos dos fármacos , Escuridão , Genes Reporter , Hipocótilo/crescimento & desenvolvimento , Fosforilação/efeitos dos fármacos , Raízes de Plantas/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos
12.
J Proteomics ; 238: 104134, 2021 04 30.
Artigo em Inglês | MEDLINE | ID: mdl-33561558

RESUMO

Mass-spectrometry (MS) based phosphoproteomics is increasingly used to explore aberrant cellular signaling and kinase driver activity, aiming to improve kinase inhibitor (KI) treatment selection in malignancies. Phosphorylation is a dynamic, highly regulated post-translational modification that may be affected by variation in pre-analytical sample handling, hampering the translational value of phosphoproteomics-based analyses. Here, we investigate the effect of delay in mononuclear cell isolation on acute myeloid leukemia (AML) phosphorylation profiles. We performed MS on immuno-precipitated phosphotyrosine (pY)-containing peptides isolated from AML samples after seven pre-defined delays before sample processing (direct processing, thirty minutes, one hour, two hours, three hours, four hours and 24 h delay). Up to four hours, pY phosphoproteomics profiles show limited variation. However, in samples processed with a delay of 24 h, we observed significant change in these phosphorylation profiles, with differential phosphorylation of 22 pY phosphopeptides (p < 0.01). This includes increased phosphorylation of pY phosphopeptides of JNK and p38 kinases indicative of stress response activation. Based on these results, we conclude that processing of AML samples should be standardized at all times and should occur within four hours after sample collection. SIGNIFICANCE: Our study provides a practical time-frame in which fresh peripheral blood samples from acute myeloid patients should be processed for phosphoproteomics, in order to warrant correct interpretation of in vivo biology. We show that up to four hours of delayed processing after sample collection, pY phosphoproteomic profiles remain stable. Extended delays are associated with perturbation of phosphorylation profiles. After a delay of 24 h, JNK activation loop phosphorylation is markedly increased and may serve as a biomarker for delayed processing. These findings are relevant for biomedical acute myeloid leukemia research, as phosphoproteomic techniques are of particular interest to investigate aberrant kinase signaling in relation to disease emergence and kinase inhibitor response. With these data, we aim to contribute to reproducible research with meaningful outcomes.


Assuntos
Leucemia Mieloide Aguda , Separação Celular , Humanos , Fosforilação , Fosfotirosina/metabolismo , Proteômica
13.
J Cell Biol ; 220(2)2021 02 01.
Artigo em Inglês | MEDLINE | ID: mdl-33411917

RESUMO

Receptor degradation terminates signaling by activated receptor tyrosine kinases. Degradation of EGFR occurs in lysosomes and requires the switching of RAB5 for RAB7 on late endosomes to enable their fusion with the lysosome, but what controls this critical switching is poorly understood. We show that the tyrosine kinase FER alters PKCδ function by phosphorylating it on Y374, and that phospho-Y374-PKCδ prevents RAB5 release from nascent late endosomes, thereby inhibiting EGFR degradation and promoting the recycling of endosomal EGFR to the cell surface. The rapid association of phospho-Y374-PKCδ with EGFR-containing endosomes is diminished by PTPN14, which dephosphorylates phospho-Y374-PKCδ. In triple-negative breast cancer cells, the FER-dependent phosphorylation of PKCδ enhances EGFR signaling and promotes anchorage-independent cell growth. Importantly, increased Y374-PKCδ phosphorylation correlating with arrested late endosome maturation was identified in ∼25% of triple-negative breast cancer patients, suggesting that dysregulation of this pathway may contribute to their pathology.


Assuntos
Endocitose , Proteína Quinase C-delta/metabolismo , Proteínas Tirosina Quinases/metabolismo , Proteólise , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Endocitose/efeitos dos fármacos , Endossomos/efeitos dos fármacos , Endossomos/metabolismo , Ativação Enzimática/efeitos dos fármacos , Fator de Crescimento Epidérmico/farmacologia , Receptores ErbB/genética , Receptores ErbB/metabolismo , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Feminino , Humanos , Mitógenos/farmacologia , Fosforilação/efeitos dos fármacos , Fosfotirosina/metabolismo , Estabilidade Proteica/efeitos dos fármacos , Transporte Proteico/efeitos dos fármacos , Proteínas Tirosina Fosfatases não Receptoras/deficiência , Proteínas Tirosina Fosfatases não Receptoras/metabolismo , Proteólise/efeitos dos fármacos , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Fatores de Tempo , Ubiquitinação/efeitos dos fármacos , Proteínas rab de Ligação ao GTP/metabolismo
14.
J Cell Mol Med ; 25(1): 132-146, 2021 01.
Artigo em Inglês | MEDLINE | ID: mdl-33191645

RESUMO

It has been reported that chemokine CX3 CL1 can regulate various tumours by binding to its unique receptor CX3 CR1. However, the effect of CX3 CL1-CX3 CR1 on the lung adenocarcinoma and lung squamous cell carcinoma is still unclear. Here, we showed that CX3 CL1 can further invasion and migration of lung adenocarcinoma A549 and lung squamous cell carcinoma H520. In addition, Western blot and immunofluorescence test indicated CX3 CL1 up-regulated the phosphorylation level of cortactin, which is a marker of cell pseudopodium. Meanwhile, the phosphorylation levels of c-Src and c-Abl, which are closely related to the regulation of cortactin phosphorylation, are elevated. Nevertheless, the src/abl inhibitor bosutinib and mutations of cortactin phosphorylation site could inhibit the promotion effect of CX3 CL1 on invasion and migration of A549 and H520. Moreover, these results of MTT, Hoechst staining and Western blot suggested that CX3 CL1 had no effect on the proliferation and apoptosis of A549 and H520 in vitro. The effects of CX3 CL1 were also verified by the subcutaneous tumour formation in nude mice, which showed that it could promote proliferation and invasion of A549 in vivo. In summary, our results indicated that CX3 CL1 furthered invasion and migration in lung cancer cells partly via activating cortactin, and CX3 CL1 may be a potential molecule in regulating the migration and invasion of lung cancer.


Assuntos
Quimiocina CXCL1/metabolismo , Cortactina/metabolismo , Neoplasias Pulmonares/metabolismo , Fosfotirosina/metabolismo , Animais , Apoptose , Receptor 1 de Quimiocina CX3C/metabolismo , Linhagem Celular Tumoral , Movimento Celular , Proliferação de Células , Humanos , Neoplasias Pulmonares/patologia , Masculino , Camundongos Nus , Invasividade Neoplásica , Fosforilação , Proteínas Proto-Oncogênicas c-abl/metabolismo , Transdução de Sinais/efeitos dos fármacos , Quinases da Família src/metabolismo
15.
Protein Sci ; 30(3): 558-570, 2021 03.
Artigo em Inglês | MEDLINE | ID: mdl-33314411

RESUMO

Protein engineering through directed evolution is an effective way to obtain proteins with novel functions with the potential applications as tools for diagnosis or therapeutics. Many natural proteins have undergone directed evolution in vitro in the test tubes in the laboratories worldwide, resulting in the numerous protein variants with novel or enhanced functions. we constructed here an SH2 variant library by randomizing 8 variable residues in its phosphotyrosine (pTyr) binding pocket. Selection of this library by a pTyr peptide led to the identification of SH2 variants with enhanced affinities measured by EC50. Fluorescent polarization was then applied to quantify the binding affinities of the newly identified SH2 variants. As a result, three SH2 variants, named V3, V13 and V24, have comparable binding affinities with the previously identified SH2 triple-mutant superbinder. Biolayer Interferometry assay was employed to disclose the kinetics of the binding of these SH2 superbinders to the phosphotyrosine peptide. The results indicated that all the SH2 superbinders have two-orders increase of the dissociation rate when binding the pTyr peptide while there was no significant change in their associate rates. Intriguingly, though binding the pTyr peptide with comparable affinity with other SH2 superbinders, the V3 does not bind to the sTyr peptide. However, variant V13 and V24 have cross-reactivity with both pTyr and sTyr peptides. The newly identified superbinders could be utilized as tools for the identification of pTyr-containing proteins from tissues under different physiological or pathophysiological conditions and may have the potential in the therapeutics.


Assuntos
Evolução Molecular Direcionada/métodos , Fosfotirosina , Proteínas Proto-Oncogênicas c-fyn , Proteínas Recombinantes , Domínios de Homologia de src/genética , Sítios de Ligação/genética , Técnicas de Visualização da Superfície Celular , Escherichia coli/genética , Humanos , Biblioteca de Peptídeos , Fosfotirosina/química , Fosfotirosina/metabolismo , Ligação Proteica/genética , Engenharia de Proteínas , Proteínas Proto-Oncogênicas c-fyn/química , Proteínas Proto-Oncogênicas c-fyn/genética , Proteínas Proto-Oncogênicas c-fyn/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo
16.
Mol Syst Biol ; 16(12): e9819, 2020 12.
Artigo em Inglês | MEDLINE | ID: mdl-33289969

RESUMO

Alzheimer's disease (AD) is characterized by the appearance of amyloid-ß plaques, neurofibrillary tangles, and inflammation in brain regions involved in memory. Using mass spectrometry, we have quantified the phosphoproteome of the CK-p25, 5XFAD, and Tau P301S mouse models of neurodegeneration. We identified a shared response involving Siglec-F which was upregulated on a subset of reactive microglia. The human paralog Siglec-8 was also upregulated on microglia in AD. Siglec-F and Siglec-8 were upregulated following microglial activation with interferon gamma (IFNγ) in BV-2 cell line and human stem cell-derived microglia models. Siglec-F overexpression activates an endocytic and pyroptotic inflammatory response in BV-2 cells, dependent on its sialic acid substrates and immunoreceptor tyrosine-based inhibition motif (ITIM) phosphorylation sites. Related human Siglecs induced a similar response in BV-2 cells. Collectively, our results point to an important role for mouse Siglec-F and human Siglec-8 in regulating microglial activation during neurodegeneration.


Assuntos
Inflamação/patologia , Microglia/metabolismo , Degeneração Neural/patologia , Fosfoproteínas/metabolismo , Proteômica , Lectinas Semelhantes a Imunoglobulina de Ligação ao Ácido Siálico/metabolismo , Doença de Alzheimer/metabolismo , Doença de Alzheimer/patologia , Sequência de Aminoácidos , Animais , Anticorpos/metabolismo , Morte Celular , Linhagem Celular , Humanos , Inflamação/metabolismo , Interferon gama/metabolismo , Camundongos Transgênicos , Microglia/patologia , Degeneração Neural/metabolismo , Peptídeos/metabolismo , Fagocitose , Fosfotirosina/metabolismo , Proteoma/metabolismo , Lectinas Semelhantes a Imunoglobulina de Ligação ao Ácido Siálico/química , Transdução de Sinais , Regulação para Cima
17.
J Biol Chem ; 295(52): 18105-18121, 2020 12 25.
Artigo em Inglês | MEDLINE | ID: mdl-33087447

RESUMO

IQGAP1 is a key scaffold protein that regulates numerous cellular processes and signaling pathways. Analogous to many other cellular proteins, IQGAP1 undergoes post-translational modifications, including phosphorylation. Nevertheless, very little is known about the specific sites of phosphorylation or the effects on IQGAP1 function. Here, using several approaches, including MS, site-directed mutagenesis, siRNA-mediated gene silencing, and chemical inhibitors, we identified the specific tyrosine residues that are phosphorylated on IQGAP1 and evaluated the effect on function. Tyr-172, Tyr-654, Tyr-855, and Tyr-1510 were phosphorylated on IQGAP1 when phosphotyrosine phosphatase activity was inhibited in cells. IQGAP1 was phosphorylated exclusively on Tyr-1510 under conditions with enhanced MET or c-Src signaling, including in human lung cancer cell lines. This phosphorylation was significantly reduced by chemical inhibitors of MET or c-Src or by siRNA-mediated knockdown of MET. To investigate the biological sequelae of phosphorylation, we generated a nonphosphorylatable IQGAP1 construct by replacing Tyr-1510 with alanine. The ability of hepatocyte growth factor, the ligand for MET, to promote AKT activation and cell migration was significantly greater when IQGAP1-null cells were reconstituted with IQGAP1 Y1510A than when cells were reconstituted with WT IQGAP1. Collectively, our data suggest that phosphorylation of Tyr-1510 of IQGAP1 alters cell function. Because increased MET signaling is implicated in the development and progression of several types of carcinoma, IQGAP1 may be a potential therapeutic target in selected malignancies.


Assuntos
Movimento Celular , Fibroblastos/metabolismo , Fosfotirosina/metabolismo , Processamento de Proteína Pós-Traducional , Proteínas Proto-Oncogênicas c-met/metabolismo , Proteínas Ativadoras de ras GTPase/metabolismo , Animais , Células Cultivadas , Fibroblastos/citologia , Humanos , Camundongos , Proteínas Proto-Oncogênicas c-met/genética , Transdução de Sinais , Proteínas Ativadoras de ras GTPase/genética
18.
Front Endocrinol (Lausanne) ; 11: 575220, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-33042028

RESUMO

The Src homology 2 (SH2) domain has a special role as one of the cornerstone examples of a "modular" domain. The interactions of this domain are very well-conserved, and have long been described as a bidentate, or "two-pronged plug" interaction between the domain and a phosphotyrosine (pTyr) peptide. Recent work has, however, highlighted unusual features of the SH2 domain that illustrate a greater diversity than was previously appreciated. In this review we discuss some of the novel and unusual characteristics across the SH2 family, including unusual peptide binding pockets, multiple pTyr recognition sites, recognition sites for unphosphorylated peptides, and recently identified variability in the conserved FLVR motif.


Assuntos
Fosfopeptídeos/metabolismo , Fosfotirosina/metabolismo , Domínios de Homologia de src , Animais , Sítios de Ligação , Humanos , Modelos Moleculares , Fosfopeptídeos/química , Fosforilação , Fosfotirosina/química , Ligação Proteica
19.
J Cell Mol Med ; 24(20): 11960-11971, 2020 10.
Artigo em Inglês | MEDLINE | ID: mdl-32914567

RESUMO

Osteosarcoma (OS) is the most common primary malignant bone tumour in children and adolescents. The long-term survival rate of OS patients is stubbornly low mainly due to the chemotherapy resistance. We therefore aimed to investigate the antitumoral effects and underlying mechanisms of proanthocyanidin B2 (PB2) on OS cells in the current study. The effect of PB2 on the proliferation and apoptosis of OS cell lines was assessed by CCK-8, colony formation, and flow cytometry assays. The target gene and protein expression levels were measured by qRT-PCR and Western blotting. A xenograft mouse model was established to assess the effects of PB2 on OS proliferation and apoptosis in vivo. Results from in vitro experiments showed that PB2 inhibited the proliferation and induced apoptosis of OS cells, and also increased the expression levels of apoptosis-related proteins. Moreover, PB2 induced OS cell apoptosis through suppressing the PI3K/AKT signalling pathway. The in vivo experiments further confirmed that PB2 could inhibit OS tumour growth and induce its apoptosis. Taken together, these results suggested that PB2 inhibited the proliferation and induced apoptosis of OS cells through the suppression of the PI3K/AKT signalling pathway.


Assuntos
Apoptose/efeitos dos fármacos , Osteossarcoma/enzimologia , Osteossarcoma/patologia , Fosfatidilinositol 3-Quinases/metabolismo , Proantocianidinas/farmacologia , Proteínas Proto-Oncogênicas c-akt/metabolismo , Transdução de Sinais , Animais , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Humanos , Masculino , Camundongos Endogâmicos BALB C , Camundongos Nus , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Membranas Mitocondriais/efeitos dos fármacos , Membranas Mitocondriais/metabolismo , Permeabilidade , Fosfotirosina/metabolismo , Transdução de Sinais/efeitos dos fármacos , Ensaios Antitumorais Modelo de Xenoenxerto
20.
Int J Mol Sci ; 21(16)2020 Aug 12.
Artigo em Inglês | MEDLINE | ID: mdl-32806748

RESUMO

Focal adhesion kinase (FAK) is over-expressed and is correlated with aggressiveness in adult hepatocellular carcinoma (HCC). Inhibition of FAK decreases HCC invasiveness by down-regulating Enhancer of Zeste homolog 2 (EZH2), an epigenetic controller, that acts in transcriptional repression of a large number of genes via histone 3 methylation of lysine 27 (H3K27me3). Here, we investigated the hepatic expression of total FAK, EZH2, H3K27me3, and proliferating cell nuclear antigen (PCNA) in 17 pediatric HCCs and 8 healthy livers (CTRL). Quantitative imaging analysis showed that FAK gene/protein expression is up-regulated in HCCs compared to CTRL and, among tumor samples the levels of this gene/protein are significantly higher in cirrhotic HCCs than in a healthy milieu. Accordingly, the protein levels of EZH2 were also significantly increased in HCCs from a cirrhotic background. Intriguingly, the protein expression of FAK, EZH2, and PCNA significantly inversely correlated with tumor size. Finally, in HCC samples, mainly in cirrhotic background, the up-regulation of FAK gene positively correlated with that observed in ß-Catenin gene. Conclusion: FAK gene/protein is over-expressed in pediatric HCCs concomitantly to EZH2 protein and ß-Catenin gene, with a more significant up-regulation in a cirrhotic background. This triad of interactors deserves further investigations for translational application.


Assuntos
Carcinoma Hepatocelular/diagnóstico , Carcinoma Hepatocelular/enzimologia , Proteína-Tirosina Quinases de Adesão Focal/metabolismo , Neoplasias Hepáticas/diagnóstico , Neoplasias Hepáticas/enzimologia , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/patologia , Núcleo Celular/patologia , Criança , Proteína Potenciadora do Homólogo 2 de Zeste/metabolismo , Feminino , Regulação Neoplásica da Expressão Gênica , Histonas/metabolismo , Humanos , Cirrose Hepática/patologia , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/patologia , Lisina/metabolismo , Masculino , Metilação , Fosforilação , Fosfotirosina/metabolismo , Prognóstico , Antígeno Nuclear de Célula em Proliferação/metabolismo , Carga Tumoral , Regulação para Cima/genética , beta Catenina/genética , beta Catenina/metabolismo
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