RESUMO
Cystic Fibrosis (CF) is a genetic disease caused by mutations in CFTR gene expressing the anion selective channel CFTR located at the plasma membrane of different epithelial cells. The most commonly investigated variant causing CF is F508del. This mutation leads to structural defects in the CFTR protein, which are recognized by the endoplasmic reticulum (ER) quality control system. As a result, the protein is retained in the ER and degraded via the ubiquitin-proteasome pathway. Although blocking ubiquitination to stabilize the CFTR protein has long been considered a potential pharmacological approach in CF, progress in this area has been relatively slow. Currently, no compounds targeting this pathway have entered clinical trials for CF. On the other hand, the emergence of Orkambi initially, and notably the subsequent introduction of Trikafta/Kaftrio, have demonstrated the effectiveness of molecular chaperone-based therapies for patients carrying the F508del variant and even showed efficacy against other variants. These treatments directly target the CFTR variant protein without interfering with cell signaling pathways. This review discusses the limits and potential future of targeting protein ubiquitination in CF.
Assuntos
Regulador de Condutância Transmembrana em Fibrose Cística , Fibrose Cística , Ubiquitinação , Fibrose Cística/metabolismo , Fibrose Cística/genética , Fibrose Cística/tratamento farmacológico , Fibrose Cística/patologia , Humanos , Regulador de Condutância Transmembrana em Fibrose Cística/metabolismo , Regulador de Condutância Transmembrana em Fibrose Cística/genética , Retículo Endoplasmático/metabolismo , Animais , Mutação , Ubiquitina/metabolismoRESUMO
Cystic fibrosis (CF) is a genetic disorder caused by mutations in the gene encoding the cystic fibrosis transmembrane conductance regulator (CFTR), a selective anion channel expressed in the epithelium of various organs. The most frequent mutation is F508del. This mutation leads to a misfolded CFTR protein quickly degraded via ubiquitination in the endoplasmic reticulum. Although preventing ubiquitination stabilizes the protein, functionality is not restored due to impaired plasma membrane transport. However, inhibiting the ubiquitination process can improve the effectiveness of correctors which act as chemical chaperones, facilitating F508del CFTR trafficking to the plasma membrane. Previous studies indicate a crosstalk between SUMOylation and ubiquitination in the regulation of CFTR. In this study, we investigated the potential of inhibiting SUMOylation to increase the effects of correctors and enhance the rescue of the F508del mutant across various cell models. In the widely used CFBE41o-cell line expressing F508del-CFTR, inhibiting SUMOylation substantially boosted F508del expression, thereby increasing the efficacy of correctors. Interestingly, this outcome did not result from enhanced stability of the mutant channel, but rather from augmented cytomegalovirus (CMV) promoter-mediated gene expression of F508del-CFTR. Notably, CFTR regulated by endogenous promoters in multiple cell lines or patient cells was not influenced by SUMOylation inhibitors.
Assuntos
Regulador de Condutância Transmembrana em Fibrose Cística , Fibrose Cística , Sumoilação , Humanos , Fibrose Cística/tratamento farmacológico , Fibrose Cística/genética , Fibrose Cística/metabolismo , Regulador de Condutância Transmembrana em Fibrose Cística/genética , Regulador de Condutância Transmembrana em Fibrose Cística/metabolismo , Citomegalovirus , Mutação , Sumoilação/efeitos dos fármacos , Regiões Promotoras Genéticas/efeitos dos fármacosRESUMO
CK2 is a constitutively active protein kinase that assuring a constant level of phosphorylation to its numerous substrates supports many of the most important biological functions. Nevertheless, its activity has to be controlled and adjusted in order to cope with the varying needs of a cell, and several examples of a fine-tune regulation of its activity have been described. More importantly, aberrant regulation of this enzyme may have pathological consequences, e.g. in cancer, chronic inflammation, neurodegeneration, and viral infection. Our review aims at summarizing our current knowledge about CK2 regulation. In the first part, we have considered the most important stimuli shown to affect protein kinase CK2 activity/expression. In the second part, we focus on the molecular mechanisms by which CK2 can be regulated, discussing controversial aspects and future perspectives.
Assuntos
Caseína Quinase II/metabolismo , Proteínas de Neoplasias/metabolismo , Neoplasias/enzimologia , Transdução de Sinais , Viroses/enzimologia , Animais , Humanos , Inflamação/enzimologiaRESUMO
The advent of Trikafta (Kaftrio in Europe) (a triple-combination therapy based on two correctors-elexacaftor/tezacaftor-and the potentiator ivacaftor) has represented a revolution for the treatment of patients with cystic fibrosis (CF) carrying the most common misfolding mutation, F508del-CFTR. This therapy has proved to be of great efficacy in people homozygous for F508del-CFTR and is also useful in individuals with a single F508del allele. Nevertheless, the efficacy of this therapy needs to be improved, especially in light of the extent of its use in patients with rare class II CFTR mutations. Using CFBE41o- cells expressing F508del-CFTR, we provide mechanistic evidence that targeting the E1 ubiquitin-activating enzyme (UBA1) by TAK-243, a small molecule in clinical trials for other diseases, boosts the rescue of F508del-CFTR induced by CFTR correctors. Moreover, TAK-243 significantly increases the F508del-CFTR short-circuit current induced by elexacaftor/tezacaftor/ivacaftor in differentiated human primary airway epithelial cells, a gold standard for the pre-clinical evaluation of patients' responsiveness to pharmacological treatments. This new combinatory approach also leads to an improvement in CFTR conductance on cells expressing other rare CF-causing mutations, including N1303K, for which Trikafta is not approved. These findings show that Trikafta therapy can be improved by the addition of a drug targeting the misfolding detection machinery at the beginning of the ubiquitination cascade and may pave the way for an extension of Trikafta to low/non-responding rare misfolded CFTR mutants.
Assuntos
Aminofenóis/administração & dosagem , Benzodioxóis/administração & dosagem , Regulador de Condutância Transmembrana em Fibrose Cística/genética , Fibrose Cística/tratamento farmacológico , Fibrose Cística/genética , Indóis/administração & dosagem , Pirazóis/administração & dosagem , Piridinas/administração & dosagem , Pirimidinas/administração & dosagem , Pirrolidinas/administração & dosagem , Quinolonas/administração & dosagem , Sulfetos/administração & dosagem , Sulfonamidas/administração & dosagem , Enzimas Ativadoras de Ubiquitina/antagonistas & inibidores , Células Cultivadas , Fibrose Cística/metabolismo , Regulador de Condutância Transmembrana em Fibrose Cística/química , Regulador de Condutância Transmembrana em Fibrose Cística/metabolismo , Sinergismo Farmacológico , Quimioterapia Combinada , Inibidores Enzimáticos/administração & dosagem , Humanos , Mutação , Dobramento de Proteína/efeitos dos fármacos , Deleção de SequênciaRESUMO
Cystic fibrosis (CF) is caused by mutations in the gene encoding of the cystic fibrosis transmembrane conductance regulator (CFTR), an anion-selective plasma membrane channel that mainly regulates chloride transport in a variety of epithelia. More than 2000 mutations, most of which presumed to be disease-relevant, have been identified in the CFTR gene. The single CFTR mutation F508del (deletion of phenylalanine in position 508) is present in about 90% of global CF patients in at least one allele. F508del is responsible for the defective folding and processing of CFTR, failing to traffic to the plasma membrane and undergoing premature degradation via the ubiquitin-proteasome system. CFTR is subjected to different post-translational modifications (PTMs), and the possibility to modulate these PTMs has been suggested as a potential therapeutic strategy for the functional recovery of the disease-associated mutants. Recently, the PTM mapping of CFTR has identified some lysine residues that may undergo methylation or ubiquitination, suggesting a competition between these two PTMs. Our work hypothesis moves from the idea that favors methylation over ubiquitination, e.g., inhibiting demethylation could be a successful strategy for preventing the premature degradation of unstable CFTR mutants. Here, by using a siRNA library against all the human demethylases, we identified the enzymes whose downregulation increases F508del-CFTR stability and channel function. Our results show that KDM2A and KDM3B downregulation increases the stability of F508del-CFTR and boosts the functional rescue of the channel induced by CFTR correctors.
Assuntos
Fibrose Cística , Proteínas F-Box , Membrana Celular/metabolismo , Fibrose Cística/metabolismo , Regulador de Condutância Transmembrana em Fibrose Cística/genética , Regulador de Condutância Transmembrana em Fibrose Cística/metabolismo , Proteínas F-Box/genética , Humanos , Transporte de Íons , Histona Desmetilases com o Domínio Jumonji/metabolismo , MutaçãoRESUMO
Inhibition of human papillomavirus (HPV) replication is a promising therapeutic approach for intervening with HPV-related pathologies. Primary targets for interference are two viral proteins, E1 and E2, which are required for HPV replication. Both E1 and E2 are phosphoproteins; thus, the protein kinases that phosphorylate them might represent secondary targets to achieve inhibition of HPV replication. In the present study, we show that CX4945, an ATP-competitive small molecule inhibitor of casein kinase 2 (CK2) catalytic activity, suppresses replication of different HPV types, including novel HPV5NLuc, HPV11NLuc and HPV18NLuc marker genomes, but enhances the replication of HPV16 and HPV31. We further corroborate our findings using short interfering RNA (siRNA)-mediated knockdown of CK2 α and α' subunits in U2OS and CIN612 cells; we show that while both subunits are expressed in these cell lines, CK2α is required for HPV replication, but CK2α' is not. Furthermore, we demonstrate that CK2α acts in a kinase activity-dependent manner and regulates the stability and nuclear retention of endogenous E1 proteins of HPV11 and HPV18. This unique feature of CK2α makes it an attractive target for developing antiviral agents.
Assuntos
Papillomaviridae/fisiologia , Infecções por Papillomavirus/virologia , Fosfoproteínas/metabolismo , Proteínas Virais/metabolismo , Neoplasias Ósseas/metabolismo , Neoplasias Ósseas/patologia , Neoplasias Ósseas/virologia , Caseína Quinase II/genética , Caseína Quinase II/metabolismo , Humanos , Osteossarcoma/metabolismo , Osteossarcoma/patologia , Osteossarcoma/virologia , Infecções por Papillomavirus/genética , Infecções por Papillomavirus/metabolismo , Fosfoproteínas/genética , Fosforilação , Células Tumorais Cultivadas , Proteínas Virais/genéticaRESUMO
The making of a protein is based on the combination of 20 different monomers (22 considering selenocysteine and pyrrolysine, the latest present only in some archaea and bacteria) giving the possibility of building a variety of structures from the simplest to the most complex, rigid or highly dynamic, and suited to carry out a wide range of structural and functional roles [...].
Assuntos
Doença , Biossíntese de Proteínas , Processamento de Proteína Pós-Traducional , Proteínas/química , Proteínas/metabolismo , Animais , Humanos , Transdução de SinaisRESUMO
Viable clones of C2C12 myoblasts where both catalytic subunits of protein kinase CK2 had been knocked out by the CRISPR/Cas9 methodology have recently been generated, thus challenging the concept that CK2 is essential for cell viability. Here we present evidence that these cells are still endowed with a residual "CK2-like" activity that is able to phosphorylate Ser-13 of endogenous CDC37. Searching for a molecular entity accounting for such an activity we have identified a band running slightly ahead of CK2α' on SDS-PAGE. This band is not detectable by in-gel casein kinase assay but it co-immuno-precipitates with the ß-subunit being downregulated by specific CK2α' targeting siRNA treatment. Its size and biochemical properties are consistent with those of CK2α' mutants deleted upstream of Glu-15 generated during the knockout process. This mutant sheds light on the role of the CK2 N-terminal segment as a regulator of activity and stability. Comparable cytotoxic efficacy of two selective and structurally unrelated CK2 inhibitors support the view that survival of CK2α/α'-/- cells relies on this deleted form of CK2α', whose discovery provides novel perspectives about the biological role of CK2.
Assuntos
Caseína Quinase II/química , Caseína Quinase II/metabolismo , Domínio Catalítico , Deleção de Sequência , Sequência de Aminoácidos , Animais , Caseína Quinase II/deficiência , Linhagem Celular , Sobrevivência Celular , Camundongos Knockout , Peptídeos/metabolismo , Fosforilação , Fosfosserina/metabolismo , Estabilidade Proteica , Especificidade por SubstratoRESUMO
Casein kinase 2 (CK2) is a tetrameric protein kinase composed of 2 catalytic (α and α') and 2 regulatory ß subunits. Our study provides the first molecular and cellular characterization of the different CK2 subunits, highlighting their individual roles in skeletal muscle specification and differentiation. Analysis of C2C12 cell knockout for each CK2 subunit reveals that: 1) CK2ß is mandatory for the expression of the muscle master regulator myogenic differentiation 1 in proliferating myoblasts, thus controlling both myogenic commitment and subsequent muscle-specific gene expression and myotube formation; 2) CK2α is involved in the activation of the muscle-specific gene program; and 3) CK2α' activity regulates myoblast fusion by mediating plasma membrane translocation of fusogenic proteins essential for membrane coalescence, like myomixer. Accordingly, CK2α' overexpression in C2C12 cells and in mouse regenerating muscle is sufficient to increase myofiber size and myonuclei content via enhanced satellite cell fusion. Consistent with these results, pharmacological inhibition of CK2 activity substantially blocks the expression of myogenic markers and muscle cell fusion both in vitro in C2C12 and primary myoblasts and in vivo in mouse regenerating muscle and zebrafish development. Overall, our work describes the specific and coordinated functions of CK2 subunits in orchestrating muscle differentiation and fusogenic activity, highlighting CK2 relevance in the physiopathology of skeletal muscle tissue.-Salizzato, V., Zanin, S., Borgo, C., Lidron, E., Salvi, M., Rizzuto, R., Pallafacchina, G., Donella-Deana, A. Protein kinase CK2 subunits exert specific and coordinated functions in skeletal muscle differentiation and fusogenic activity.
Assuntos
Caseína Quinase II/fisiologia , Músculo Esquelético/citologia , Músculo Esquelético/enzimologia , Animais , Caseína Quinase II/antagonistas & inibidores , Caseína Quinase II/genética , Diferenciação Celular/genética , Diferenciação Celular/fisiologia , Fusão Celular , Linhagem Celular , Técnicas de Inativação de Genes , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Modelos Biológicos , Desenvolvimento Muscular/genética , Desenvolvimento Muscular/fisiologia , Proteína MyoD/genética , Proteína MyoD/metabolismo , Mioblastos Esqueléticos/citologia , Mioblastos Esqueléticos/enzimologia , Subunidades Proteicas , Células Satélites de Músculo Esquelético/citologia , Células Satélites de Músculo Esquelético/enzimologia , Peixe-Zebra , Proteínas de Peixe-Zebra/antagonistas & inibidores , Proteínas de Peixe-Zebra/genética , Proteínas de Peixe-Zebra/fisiologiaRESUMO
CK2 denotes a ubiquitous and pleiotropic protein kinase whose holoenzyme is composed of two catalytic (α and/or α') and two regulatory ß subunits. The CK2 consensus sequence, S/T-x-x-D/E/pS/pT is present in numerous phosphosites, but it is not clear how many of these are really generated by CK2. To gain information about this issue, advantage has been taken of C2C12 cells entirely deprived of both CK2 catalytic subunits by the CRISPR/Cas9 methodology. A comparative SILAC phosphoproteomics analysis reveals that, although about 30% of the quantified phosphosites do conform to the CK2 consensus, only one-third of these are substantially reduced in the CK2α/α'(-/-) cells, consistent with their generation by CK2. A parallel study with C2C12 cells deprived of the regulatory ß subunit discloses a role of this subunit in determining CK2 targeting. We also find that phosphosites notoriously generated by CK2 are not fully abrogated in CK2α/α'(-/-) cells, while some phosphosites unrelated to CK2 are significantly altered. Collectively taken our data allow to conclude that the phosphoproteome generated by CK2 is not as ample and rigidly pre-determined as it was believed before. They also show that the lack of CK2 promotes phosphoproteomics perturbations attributable to kinases other than CK2.
Assuntos
Caseína Quinase II/metabolismo , Fosfopeptídeos/metabolismo , Animais , Caseína Quinase II/genética , Linhagem Celular , Deleção de Genes , Técnicas de Inativação de Genes , Camundongos , Fosfopeptídeos/análise , Fosforilação , Proteômica/métodosRESUMO
Protein kinase CK2 (CK2) is a highly conserved and ubiquitous kinase is involved in crucial biological processes, including proliferation, migration, and differentiation. CK2 holoenzyme is a tetramer composed by two catalytically active (α/α') and two regulatory (ß) subunits and exerts its function on a broad range of targets. In the brain, it regulates different steps of neurodevelopment, such as neural differentiation, neuritogenesis, and synaptic plasticity. Interestingly, CK2 mutations have been recently linked to neurodevelopmental disorders; however, the functional requirements of the individual CK2 subunits in neurodevelopment have not been yet investigated. Here, we disclose the role of CK2 on the migration and adhesion properties of GN11 cells, an established model of mouse immortalized neurons, by different in vitro experimental approaches. Specifically, the cellular requirement of this kinase has been assessed pharmacologically and genetically by exploiting CK2 inhibitors and by generating subunit-specific CK2 knockout GN11 cells (with a CRISPR/Cas9-based approach). We show that CK2α' subunit has a primary role in increasing cell adhesion and reducing migration properties of GN11 cells by activating the Akt-GSK3ß axis, whereas CK2α subunit is dispensable. Further, the knockout of the CK2ß regulatory subunits counteracts cell migration, inducing dramatic alterations in the cytoskeleton not observed in CK2α' knockout cells. Collectively taken, our data support the view that the individual subunits of CK2 play different roles in cell migration and adhesion properties of GN11 cells, supporting independent roles of the different subunits in these processes.
Assuntos
Caseína Quinase II/genética , Neurônios/citologia , Animais , Caseína Quinase II/metabolismo , Adesão Celular , Linhagem Celular , Movimento Celular , Técnicas de Silenciamento de Genes , Glicogênio Sintase Quinase 3 beta/metabolismo , Camundongos , Mutação , Neurônios/metabolismo , Fosforilação , Proteínas Proto-Oncogênicas c-akt/metabolismo , Transdução de SinaisRESUMO
Transepithelial bicarbonate secretion by human airway submucosal glands and surface epithelial cells is crucial to maintain the pH-sensitive innate defence mechanisms of the lung. cAMP agonists stimulate HCO3- secretion via coordinated increases in basolateral HCO3- influx and accumulation, as well as CFTR-dependent HCO3- efflux at the luminal membrane of airway epithelial cells. Here, we investigated the regulation of a basolateral located, DIDS-sensitive, Cl-/HCO3- exchanger, anion exchanger 2 (AE2; SLC4A2) which is postulated to act as an acid loader, and therefore potential regulator of HCO3- secretion, in human airway epithelial cells. Using intracellular pH measurements performed on Calu-3 cells, we demonstrate that the activity of the basolateral Cl-/HCO3- exchanger was significantly downregulated by cAMP agonists, via a PKA-independent mechanism and also required Ca2+ and calmodulin under resting conditions. AE2 contains potential phosphorylation sites by a calmodulin substrate, protein kinase CK2, and we demonstrated that AE2 activity was reduced in the presence of CK2 inhibition. Moreover, CK2 inhibition abolished the activity of AE2 in primary human nasal epithelia. Studies performed on mouse AE2 transfected into HEK-293T cells confirmed almost identical Ca2+/calmodulin and CK2 regulation to that observed in Calu-3 and primary human nasal cells. Furthermore, mouse AE2 activity was reduced by genetic knockout of CK2, an effect which was rescued by exogenous CK2 expression. Together, these findings are the first to demonstrate that CK2 is a key regulator of Cl--dependent HCO3- export at the serosal membrane of human airway epithelial cells.
Assuntos
Bicarbonatos/metabolismo , Caseína Quinase II/metabolismo , Antiportadores de Cloreto-Bicarbonato/metabolismo , Cloretos/metabolismo , Mucosa Nasal/metabolismo , Animais , Linhagem Celular , Linhagem Celular Tumoral , Membrana Celular/metabolismo , AMP Cíclico/metabolismo , Células Epiteliais/metabolismo , Células HEK293 , Humanos , Concentração de Íons de Hidrogênio , CamundongosRESUMO
Variations in the α-synuclein-encoding SNCA gene represent the greatest genetic risk factor for Parkinson's disease (PD), and duplications/triplications of SNCA cause autosomal dominant familial PD. These facts closely link brain levels of α-synuclein with the risk of PD, and make lowering α-synuclein levels a therapeutic strategy for the treatment of PD and related synucleinopathies. In this paper, we corroborate previous findings on the ability of overexpressed Polo-like kinase 2 (PLK-2) to decrease cellular α-synuclein, but demonstrate that the process is independent of PLK-2 phosphorylating S129 in α-synuclein because a similar reduction is achieved with the non-phosphorable S129A mutant α-synuclein. Using a specific PLK-2 inhibitor (compound 37), we demonstrate that endogenous PLK-2 phosphorylates S129 only in some cells, but increases α-synuclein protein levels in all tested cell cultures and brain slices. PLK-2 is found to regulate the transcription of α-synuclein mRNA from both the endogenous mouse SNCA gene and transgenic vectors that only contain the open reading frame. Moreover, we are the first to show that regulation of α-synuclein by PLK-2 is of physiological importance since 10days' inhibition of endogenous PLK-2 in wt C57BL/6 mice increases endogenous α-synuclein protein levels. Our findings collectively demonstrate that PLK-2 regulates α-synuclein levels by a previously undescribed transcription-based mechanism. This mechanism is active in cells and brain tissue, opening up for alternative strategies for modulating α-synuclein levels and thereby for the possibility of modifying disease progression in synucleinopaties.
Assuntos
Proteínas Serina-Treonina Quinases/metabolismo , RNA Mensageiro/biossíntese , alfa-Sinucleína/metabolismo , Animais , Encéfalo/metabolismo , Linhagem Celular Tumoral , Células HEK293 , Humanos , Camundongos Endogâmicos C57BL , Neurônios/metabolismo , Fases de Leitura Aberta , Fosforilação , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Proteínas Serina-Treonina Quinases/genética , Proteínas Recombinantes/metabolismo , Transcrição Gênica/fisiologia , alfa-Sinucleína/genéticaRESUMO
A SILAC analysis performed on HEK-293T cells either treated or not for 3h with the CK2 inhibitor quinalizarin (QZ) led to the quantification of 53 phosphopeptides whose amount was reduced by 50% or more by QZ. These phosphopeptides include altogether 69 phosphoresidues, a large proportion of which (almost 50%) are generated by CK2, while the others do not conform to the CK2 consensus. Intriguingly QZ treatment also promotes a 50% or more increase of 108 phosphopeptides including altogether 117 phosphoresidues, 30% of which conform to the CK2 consensus. Here we disclose two mechanisms by which the level of certain phosphosites can be increased rather than decreased by QZ: one relies on the uneven dephosphorylation rate of phosphoresidues close to each other, causing, upon CK2 blockage, the decrease/disappearance of bis-phosphorylated peptides paralleled by the rise of one of its two singly phosphorylated derivatives; the other reflects the increased cellular concentration of a subset of proteins whose expression is substantially up-regulated by QZ treatment. These proteins do not include CK2 itself, whose subunit level is unaffected by QZ. They do include instead a number of substrates whose phosphorylation is increased upon QZ treatment, as well as several kinase/phosphatase regulators and a large number of components of the ribosomal and proteasomal machinery, a circumstance that may partially account for side effects of QZ not directly executed by CK2. Especially remarkable is the finding that all the components of the proteasomal catalytic core and of the PA28 complex committed to the degradation of the non-ubiquitinated proteins are increased, while those of the regulatory complex 19S conferring the ability to degrade the ubiquitinated proteins are unaffected. This article is part of a Special Issue entitled: Inhibitors of Protein Kinases.
Assuntos
Antraquinonas/farmacologia , Caseína Quinase II/metabolismo , Fosfopeptídeos/biossíntese , Inibidores de Proteínas Quinases/farmacologia , Caseína Quinase II/antagonistas & inibidores , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Humanos , Fosfopeptídeos/metabolismo , ProteômicaRESUMO
Fam20C is an atypical kinase implicated in bio-mineralization and phosphate homeostasis disorders, and has recently been shown to account for the activity of an orphan enzyme ("genuine casein kinase", G-CK) previously characterized for its ability to phosphorylate casein and a plethora of secreted proteins at serine residues specified by the S-x-E/pS motif. Fam20C/G-CK activity is only appreciable in the presence of high Mn2+ concentration (>1 mM), and is negligible if Mn2+ is replaced by physiological Mg2+ concentrations. Here we show that sphingosine (but not its biological precursor ceramide) not only stimulates several-fold Fam20C activity in the presence of Mn2+, but also confers a comparable activity to Fam20C assayed with Mg2+. Activation by sphingosine is evident using a variety of substrates, and is accounted for by both higher Vmax and decreased KmATP, as judged from kinetics run with the ß(28-40) substrate peptide and a physiological substrate, BMP-15. Sphingosine also protects Fam20C from thermal inactivation. Consistent with the in vitro results, by treating Fam20C expressing HEK293T cells with myriocin, a potent inhibitor of the sphingosine biosynthetic pathway, the activity of Fam20C released into the conditioned medium is substantially decreased corroborating the concept that sphingosine (or related metabolite(s)) is a co-factor required by Fam20C to optimally display its biological functions. None of the small molecule kinase inhibitors tested so far were able to inhibit Fam20C. Interestingly however fingolimod, an immunosuppressive drug structurally related to sphingosine, used for the treatment of multiple sclerosis, is a powerful activator of Fam20C, both wild type and its pathogenic, loss of function, T268M mutant. This article is part of a Special Issue entitled: Inhibitors of Protein Kinases.
Assuntos
Caseína Quinase I/genética , Proteínas da Matriz Extracelular/genética , Esclerose Múltipla/genética , Esfingosina/biossíntese , Sequência de Aminoácidos , Caseína Quinase I/química , Proteínas da Matriz Extracelular/química , Ácidos Graxos Monoinsaturados/farmacologia , Regulação da Expressão Gênica/efeitos dos fármacos , Células HEK293 , Humanos , Esclerose Múltipla/enzimologia , Fosforilação , Esfingosina/antagonistas & inibidores , Esfingosina/metabolismo , Ativação Transcricional/efeitos dos fármacosRESUMO
CK2 is an extremely pleiotropic Ser/Thr protein kinase, responsible for the generation of a large proportion of the human phosphoproteome and implicated in a wide variety of biological functions. CK2 plays a global role as an anti-apoptotic agent, a property which is believed to partially account for the addiction of many cancer cells to high CK2 levels. To gain information about the CK2 targets whose phosphorylation is primarily implicated in its pro-survival signaling advantage has been taken of quinalizarin (QZ) a cell permeable fairly specific CK2 inhibitor, previously shown to be able to block endogenous CK2 triggering an apoptotic response. HEK-293T cells either treated or not for 3h with 50µM QZ were exploited to perform a quantitative SILAC phosphoproteomic analysis of phosphosites readily responsive to QZ treatment. Our analysis led to the identification of 4883 phosphosites, belonging to 1693 phosphoproteins. 71 phosphosites (belonging to 47 proteins) underwent a 50% or more decreased occupancy upon QZ treatment. Almost 50% of these fulfilled the typical consensus sequence recognized by CK2 (S/T-x-x-E/D/pS) and in several cases were validated as bona fide substrates of CK2 either based on data in the literature or by performing in vitro phosphorylation experiments with purified proteins. The majority of the remaining phosphosites drastically decreased upon QZ treatment display the pS/T-P motif typical of proline directed protein kinases and a web logo extracted from them differentiates from the web logo extracted from all the proline directed phosphosites quantified during our analysis (1151 altogether). A paradoxical outcome of our study was the detection of 116 phosphosites (belonging to 92 proteins altogether) whose occupancy is substantially increased (50% or more), rather than decreased by QZ treatment: 40% of these display the typical motif recognized by proline directed kinases, while about 25% fulfill the CK2 consensus. Collectively taken our data on one side have led to the disclosure of a subset of CK2 targets which are likely to be implicated in the early steps of CK2 signaling counteracting apoptosis, on the other they provide evidence for the existence of side and off-target effects of the CK2 inhibitor quinalizarin, paving the road toward the detection of other kinases susceptible to this compound. This article is part of a Special Issue entitled: Medical Proteomics.
Assuntos
Antraquinonas/farmacologia , Caseína Quinase II/antagonistas & inibidores , Fosfoproteínas/metabolismo , Inibidores de Proteínas Quinases/farmacologia , Proteoma/metabolismo , Células HEK293 , HumanosRESUMO
By derivatizing the purely competitive CK2 inhibitor N1-(4,5,6,7-tetrabromo-1H-benzimidazol-2-yl)-propane-1,3-diamine (K137) at its 3-amino position with a peptidic fragment composed of three or four glutamic or aspartic acid residues, a new family of bisubstrate inhibitors has been generated whose ability to simultaneously interact with both the ATP and the phosphoacceptor substrate-binding sites has been probed by running mixed competition kinetics and by mutational mapping of the kinase residues implicated in substrate recognition. The most effective bisubstrate inhibitor, K137-E4, interacts with three functional regions of the kinase: the hydrophobic pocket close to the ATP-binding site, the basic residues of the p+1 loop that recognizes the acidic determinant at position n+1 and the basic residues of α-helixC that recognize the acidic determinant at position n+3. Compared with the parent inhibitor (K137), K137-E4 is severalfold more potent (IC50 25 compared with 130 nM) and more selective, failing to inhibit any other kinase as drastically as CK2 out of 140 enzymes, whereas 35 kinases are inhibited more potently than CK2 by K137. K137-E4 is unable to penetrate the cell and to inhibit endogenous CK2, its pro-apoptotic efficacy being negligible compared with cell-permeant inhibitors; however, it readily inhibits ecto-CK2 on the outer cell surface, reducing the phosphorylation of several external phosphoproteins. Inhibition of ecto-CK2 by K137-E4 is accompanied by a slower migration of cancer cells as judged by wound healing assays. On the basis of the cellular responses to K137-E4, we conclude that ecto-CK2 is implicated in cell motility, whereas its contribution to the pro-survival role of CK2 is negligible.
Assuntos
Benzimidazóis/química , Inibidores de Proteínas Quinases/química , Proteínas Quinases/química , Estrutura Secundária de Proteína/efeitos dos fármacos , Trifosfato de Adenosina/química , Trifosfato de Adenosina/metabolismo , Benzimidazóis/farmacologia , Sítios de Ligação , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Humanos , Cinética , Fosforilação/efeitos dos fármacos , Inibidores de Proteínas Quinases/farmacologia , Proteínas Quinases/efeitos dos fármacos , Proteínas Quinases/metabolismoRESUMO
Post-translational modification is the most common mechanism of regulating protein function. If phosphorylation is considered a key event in many signal transduction pathways, other modifications must be considered as well. In particular the side chain of lysine residues is a target of different modifications; notably acetylation, methylation, ubiquitylation, sumoylation, neddylation, etc. Mass spectrometry approaches combining highly sensitive instruments and specific enrichment strategies have enabled the identification of modified sites on a large scale. Here we make a comparative analysis of the most representative lysine modifications (ubiquitylation, acetylation, sumoylation and methylation) identified in the human proteome. This review focuses on conserved amino acids, secondary structures preference, subcellular localization of modified proteins, and signaling pathways where these modifications are implicated. We discuss specific differences and similarities between these modifications, characteristics of the crosstalk among lysine post translational modifications, and single nucleotide polymorphisms that could influence lysine post-translational modifications in humans.
RESUMO
The multivesicular body (MVB) sorting pathway is a mechanism for delivering transmembrane proteins into the lumen of the lysosome for degradation. ESCRT-III is the final complex in the pathway that assembles on endosomes and executes membrane scission of intraluminal vesicles. In addition, proteins of this complex are involved in other topologically similar processes such as cytokinesis, virus egress and autophagy. Here we show that protein kinase CK2α is involved in the phosphorylation of the ESCRT-III subunits CHMP3 and CHMP2B, as well as of VPS4B/SKD1, an ATPase that mediates ESCRT-III disassembly. This phosphorylation is observed both in vitro and in cells. While we do not observe recruitment of CK2α to endosomes, we demonstrate the localization of CK2α to midbodies during cytokinesis. Phosphomimetic and non-phosphorylatable mutants of ESCRT-III proteins can still bind endosomes and localize to midbodies, indicating that CK2α does not regulate ESCRT-III localization. Finally, we analyzed two cellular functions where CHMP3, CHMP2B and VPS4 are known to be involved, epidermal growth factor degradation and cytokinetic abscission. We demonstrate that the former is impaired by CK2α downregulation whereas the latter is not affected. Taken together, our results indicate that CK2α regulates the function of ESCRT-III proteins in MVB sorting.
Assuntos
Adenosina Trifosfatases/metabolismo , Caseína Quinase II/metabolismo , Complexos Endossomais de Distribuição Requeridos para Transporte/metabolismo , ATPases Associadas a Diversas Atividades Celulares , Adenosina Trifosfatases/análise , Caseína Quinase II/análise , Caseína Quinase II/genética , Regulação para Baixo , Complexos Endossomais de Distribuição Requeridos para Transporte/análise , Endossomos/metabolismo , Fator de Crescimento Epidérmico/metabolismo , Células HEK293 , Células HeLa , Humanos , FosforilaçãoRESUMO
CK2 is a Ser/Thr protein kinase composed of two catalytic (α/α') subunits and a non-catalytic ß-subunit dimer, whose activity is often abnormally high in cancer cells. The concept that CK2 may be dispensable for cell survival has been challenged by the finding that viable CK2α/α' knock-out myoblast clones still express small amounts of an N-terminally deleted α' subunit generated during the CRISPR/Cas9 procedure. Here we show that, although the overall CK2 activity of these CK2α(-/-)/Δα' (KO) cells is less than 10% compared to wild-type (WT) cells, the number of phosphosites with the CK2 consensus is comparable to that of WT cells. A more in-depth analysis, however, reveals that the two phosphoproteomes are not superimposable according to a number of criteria, notably a functional analysis of the phosphoproteome found in the two types of cells, and variable sensitivity of the phosphosites to two structurally unrelated CK2 inhibitors. These data support the idea that a minimal CK2 activity, as in KO cells, is sufficient to perform basic housekeeping functions essential for cell survival, but not to accomplish several specialized tasks required upon cell differentiation and transformation. From this standpoint, a controlled downregulation of CK2 would represent a safe and valuable anti-cancer strategy.