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1.
Br J Cancer ; 126(7): 994-1003, 2022 04.
Artigo em Inglês | MEDLINE | ID: mdl-34773100

RESUMO

Protein kinase CSNK2 (CK2) is a pleiotropic serine/threonine kinase frequently dysregulated in solid and hematologic malignancies. To consolidate a wide range of biological and clinically oriented data from this unique kinase in cancer, this systematic review summarises existing knowledge from in vitro, in vivo and pre-clinical studies on CSNK2 across 24 different human cancer types. CSNK2 mRNA transcripts, protein levels and activity were found to be routinely upregulated in cancer, and commonly identified phosphotargets included AKT, STAT3, RELA, PTEN and TP53. Phenotypically, it frequently influenced evasion of apoptosis, enhancement of proliferation, cell invasion/metastasis and cell cycle control. Clinically, it held prognostic significance across 14 different cancers, and its inhibition in xenograft experiments resulted in a positive treatment response in 12. In conjunction with commentary on preliminary studies of CSNK2 inhibitors in humans, this review harmonises an extensive body of CSNK2 data in cancer and reinforces its emergence as an attractive target for cancer therapy. Continuing to investigate CSNK2 will be crucial to advancing our understanding of CSNK2 biology, and offers the promise of important new discoveries scientifically and clinically.


Assuntos
Caseína Quinase II , Neoplasias , Apoptose/genética , Pontos de Checagem do Ciclo Celular , Proliferação de Células/genética , Humanos , Neoplasias/tratamento farmacológico
2.
Proc Natl Acad Sci U S A ; 115(30): E7081-E7090, 2018 07 24.
Artigo em Inglês | MEDLINE | ID: mdl-29987005

RESUMO

The huntingtin N17 domain is a modulator of mutant huntingtin toxicity and is hypophosphorylated in Huntington's disease (HD). We conducted high-content analysis to find compounds that could restore N17 phosphorylation. One lead compound from this screen was N6-furfuryladenine (N6FFA). N6FFA was protective in HD model neurons, and N6FFA treatment of an HD mouse model corrects HD phenotypes and eliminates cortical mutant huntingtin inclusions. We show that N6FFA restores N17 phosphorylation levels by being salvaged to a triphosphate form by adenine phosphoribosyltransferase (APRT) and used as a phosphate donor by casein kinase 2 (CK2). N6FFA is a naturally occurring product of oxidative DNA damage. Phosphorylated huntingtin functionally redistributes and colocalizes with CK2, APRT, and N6FFA DNA adducts at sites of induced DNA damage. We present a model in which this natural product compound is salvaged to provide a triphosphate substrate to signal huntingtin phosphorylation via CK2 during low-ATP stress under conditions of DNA damage, with protective effects in HD model systems.


Assuntos
Adenina , Adutos de DNA/metabolismo , Dano ao DNA , Doença de Huntington/tratamento farmacológico , Neurônios/metabolismo , Transdução de Sinais/efeitos dos fármacos , Adenina/análogos & derivados , Adenina/farmacocinética , Adenina/farmacologia , Adenina Fosforribosiltransferase/genética , Adenina Fosforribosiltransferase/metabolismo , Animais , Caseína Quinase II/genética , Caseína Quinase II/metabolismo , Linhagem Celular Transformada , Adutos de DNA/genética , Modelos Animais de Doenças , Humanos , Doença de Huntington/genética , Doença de Huntington/metabolismo , Doença de Huntington/patologia , Camundongos , Camundongos Transgênicos , Neurônios/patologia , Fosforilação/efeitos dos fármacos , Fosforilação/genética , Transdução de Sinais/genética
3.
Blood ; 130(25): 2774-2785, 2017 12 21.
Artigo em Inglês | MEDLINE | ID: mdl-28928125

RESUMO

Platelets, anucleated megakaryocyte (MK)-derived cells, play a major role in hemostasis and arterial thrombosis. Although protein kinase casein kinase 2 (CK2) is readily detected in MKs and platelets, the impact of CK2-dependent signaling on MK/platelet (patho-)physiology has remained elusive. The present study explored the impact of the CK2 regulatory ß-subunit on platelet biogenesis and activation. MK/platelet-specific genetic deletion of CK2ß (ck2ß-/- ) in mice resulted in a significant macrothrombocytopenia and an increased extramedullar megakaryopoiesis with an enhanced proportion of premature platelets. Although platelet life span was only mildly affected, ck2ß-/- MK displayed an abnormal microtubule structure with a drastically increased fragmentation within bone marrow and a significantly reduced proplatelet formation in vivo. In ck2ß-/- platelets, tubulin polymerization was disrupted, resulting in an impaired thrombopoiesis and an abrogated inositol 1,4,5-triphosphate receptor-dependent intracellular calcium (Ca2+) release. Presumably due to a blunted increase in the concentration of cytosolic Ca2+, activation-dependent increases of α and dense-granule secretion and integrin αIIbß3 activation, and aggregation were abrogated in ck2ß-/- platelets. Accordingly, thrombus formation and stabilization under high arterial shear rates were significantly diminished, and thrombotic vascular occlusion in vivo was significantly blunted in ck2ß-/- mice, accompanied by a slight prolongation of bleeding time. Following transient middle cerebral artery occlusion, ck2ß-/- mice displayed significantly reduced cerebral infarct volumes, developed significantly less neurological deficits, and showed significantly better outcomes after ischemic stroke than ck2ßfl/fl mice. The present observations reveal CK2ß as a novel powerful regulator of thrombopoiesis, Ca2+-dependent platelet activation, and arterial thrombosis in vivo.


Assuntos
Caseína Quinase II/fisiologia , Fragmentos de Peptídeos/fisiologia , Ativação Plaquetária , Trombopoese , Trombose/patologia , Animais , Plaquetas , Sinalização do Cálcio , Caseína Quinase II/deficiência , Megacariócitos/metabolismo , Megacariócitos/patologia , Megacariócitos/ultraestrutura , Camundongos , Camundongos Knockout , Fragmentos de Peptídeos/deficiência , Trombose/etiologia , Trombose/metabolismo
4.
Proc Natl Acad Sci U S A ; 112(38): E5246-52, 2015 Sep 22.
Artigo em Inglês | MEDLINE | ID: mdl-26372956

RESUMO

Compensatory growth and regeneration of skeletal muscle is dependent on the resident stem cell population, satellite cells (SCs). Self-renewal and maintenance of the SC niche is coordinated by the paired-box transcription factor Pax7, and yet continued expression of this protein inhibits the myoblast differentiation program. As such, the reduction or removal of Pax7 may denote a key prerequisite for SCs to abandon self-renewal and acquire differentiation competence. Here, we identify caspase 3 cleavage inactivation of Pax7 as a crucial step for terminating the self-renewal process. Inhibition of caspase 3 results in elevated Pax7 protein and SC self-renewal, whereas caspase activation leads to Pax7 cleavage and initiation of the myogenic differentiation program. Moreover, in vivo inhibition of caspase 3 activity leads to a profound disruption in skeletal muscle regeneration with an accumulation of SCs within the niche. We have also noted that casein kinase 2 (CK2)-directed phosphorylation of Pax7 attenuates caspase-directed cleavage. Together, these results demonstrate that SC fate is dependent on opposing posttranslational modifications of the Pax7 protein.


Assuntos
Caspase 3/metabolismo , Músculo Esquelético/metabolismo , Fator de Transcrição PAX7/metabolismo , Células Satélites de Músculo Esquelético/citologia , Sequência de Aminoácidos , Animais , Sítios de Ligação , Caseína Quinases/metabolismo , Diferenciação Celular , Linhagem da Célula , Células Cultivadas , Imuno-Histoquímica , Camundongos , Camundongos Endogâmicos C57BL , Dados de Sequência Molecular , Fosforilação , Proteínas Recombinantes/metabolismo , Regeneração , Homologia de Sequência de Aminoácidos , Células-Tronco/citologia
5.
Mol Cell ; 36(4): 547-59, 2009 Nov 25.
Artigo em Inglês | MEDLINE | ID: mdl-19941816

RESUMO

Increased transcriptional activity of beta-catenin resulting from Wnt/Wingless-dependent or -independent signaling has been detected in many types of human cancer, but the underlying mechanism of Wnt-independent regulation remains unclear. We demonstrate here that EGFR activation results in disruption of the complex of beta-catenin and alpha-catenin, thereby abrogating the inhibitory effect of alpha-catenin on beta-catenin transactivation via CK2alpha-dependent phosphorylation of alpha-catenin at S641. ERK2, which is activated by EGFR signaling, directly binds to CK2alpha via the ERK2 docking groove and phosphorylates CK2alpha primarily at T360/S362, subsequently enhancing CK2alpha activity toward alpha-catenin phosphorylation. In addition, levels of alpha-catenin S641 phosphorylation correlate with levels of ERK1/2 activity in human glioblastoma specimens and with grades of glioma malignancy. This EGFR-ERK-CK2-mediated phosphorylation of alpha-catenin promotes beta-catenin transactivation and tumor cell invasion. These findings highlight the importance of the crosstalk between EGFR and Wnt pathways in tumor development.


Assuntos
Caseína Quinase II/metabolismo , Fator de Crescimento Epidérmico/farmacologia , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Ativação Transcricional/efeitos dos fármacos , alfa Catenina/metabolismo , beta Catenina/genética , Sequência de Aminoácidos , Sítios de Ligação , Ativação Enzimática/efeitos dos fármacos , Receptores ErbB/metabolismo , Glioblastoma/enzimologia , Glioblastoma/patologia , Humanos , Dados de Sequência Molecular , Invasividade Neoplásica , Fosforilação/efeitos dos fármacos , Fosfosserina/metabolismo , Ligação Proteica/efeitos dos fármacos , alfa Catenina/química , beta Catenina/metabolismo
6.
Biochim Biophys Acta ; 1850(10): 2077-86, 2015 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-25766872

RESUMO

BACKGROUND: Protein phosphorylation is a universal regulatory mechanism that involves an extensive network of protein kinases. The discovery of the phosphorylation-dependent peptidyl-prolyl isomerase Pin1 added an additional layer of complexity to these regulatory networks. SCOPE OF REVIEW: We have evaluated interactions between Pin1 and the regulatory kinome and proline-dependent phosphoproteome taking into consideration findings from targeted studies as well as data that has emerged from systematic phosphoproteomic workflows and from curated protein interaction databases. MAJOR CONCLUSIONS: The relationship between Pin1 and the regulatory protein kinase networks is not restricted simply to the recognition of proteins that are substrates for proline-directed kinases. In this respect, Pin1 itself is phosphorylated in cells by protein kinases that modulate its functional properties. Furthermore, the phosphorylation-dependent targets of Pin1 include a number of protein kinases as well as other enzymes such as phosphatases and regulatory subunits of kinases that modulate the actions of protein kinases. GENERAL SIGNIFICANCE: As a result of its interactions with numerous protein kinases and their substrates, as well as itself being a target for phosphorylation, Pin1 has an intricate relationship with the regulatory protein kinase and phosphoproteomic networks that orchestrate complex cellular processes and respond to environmental cues. This article is part of a Special Issue entitled Proline-directed Foldases: Cell Signaling Catalysts and Drug Targets.


Assuntos
Peptidilprolil Isomerase/metabolismo , Fosfoproteínas/metabolismo , Proteínas Quinases/metabolismo , Proteoma/metabolismo , Animais , Humanos , Peptidilprolil Isomerase de Interação com NIMA , Peptidilprolil Isomerase/genética , Fosforilação/fisiologia , Proteínas Quinases/genética , Proteoma/genética
7.
Biochim Biophys Acta ; 1852(5): 905-12, 2015 May.
Artigo em Inglês | MEDLINE | ID: mdl-25595659

RESUMO

Pin1 is a phosphorylation-dependent peptidyl-prolyl isomerase that plays a critical role in mediating protein conformational changes involved in signaling processes related to cell cycle control. Pin1 has also been implicated as being neuroprotective in aging-related neurodegenerative disorders including Alzheimer's disease where Pin1 activity is diminished. Notably, recent proteomic analysis of brain samples from patients with mild cognitive impairment revealed that Pin1 is oxidized and also displays reduced activity. Since the Pin1 active site contains a functionally critical cysteine residue (Cys113) with a low predicted pK(a), we hypothesized that Cys113 is sensitive to oxidation. Consistent with this hypothesis, we observed that treatment of Pin1 with hydrogen peroxide results in a 32Da mass increase, likely resulting from the oxidation of Cys113 to sulfinic acid (Cys-SO(2)H). This modification results in loss of peptidyl-prolyl isomerase activity. Notably, Pin1 with Cys113 substituted by aspartic acid retains activity and is no longer sensitive to oxidation. Structural studies by X-ray crystallography revealed increased electron density surrounding Cys113 following hydrogen peroxide treatment. At lower concentrations of hydrogen peroxide, oxidative inhibition of Pin1 can be partially reversed by treatment with dithiothreitol, suggesting that oxidation could be a reversible modification with a regulatory role. We conclude that the loss of Pin1 activity upon oxidation results from oxidative modification of the Cys113 sulfhydryl to sulfenic (Cys-SOH) or sulfinic acid (Cys-SO(2)H). Given the involvement of Pin1 in pathological processes related to neurodegenerative diseases and to cancer, these findings could have implications for the prevention or treatment of disease.


Assuntos
Domínio Catalítico , Cisteína/metabolismo , Peróxido de Hidrogênio/farmacologia , Peptidilprolil Isomerase/metabolismo , Biocatálise/efeitos dos fármacos , Cristalografia por Raios X , Cisteína/química , Cisteína/genética , Ditiotreitol/farmacologia , Relação Dose-Resposta a Droga , Humanos , Cinética , Modelos Moleculares , Peptidilprolil Isomerase de Interação com NIMA , Oxidantes/farmacologia , Oxirredução/efeitos dos fármacos , Peptidilprolil Isomerase/química , Peptidilprolil Isomerase/genética , Estrutura Terciária de Proteína , Espectrometria de Massas por Ionização por Electrospray , Ácidos Sulfênicos/química , Ácidos Sulfênicos/metabolismo , Ácidos Sulfínicos/química , Ácidos Sulfínicos/metabolismo , Fatores de Tempo
8.
Mol Cell ; 32(1): 57-69, 2008 Oct 10.
Artigo em Inglês | MEDLINE | ID: mdl-18851833

RESUMO

ADP-ribosylation controls many processes, including transcription, DNA repair, and bacterial toxicity. ADP-ribosyltransferases and poly-ADP-ribose polymerases (PARPs) catalyze mono- and poly-ADP-ribosylation, respectively, and depend on a highly conserved glutamate residue in the active center for catalysis. However, there is an apparent absence of this glutamate for the recently described PARP6-PARP16, raising questions about how these enzymes function. We find that PARP10, in contrast to PARP1, lacks the catalytic glutamate and has transferase rather than polymerase activity. Despite this fundamental difference, PARP10 also modifies acidic residues. Consequently, we propose an alternative catalytic mechanism for PARP10 compared to PARP1 in which the acidic target residue of the substrate functionally substitutes for the catalytic glutamate by using substrate-assisted catalysis to transfer ADP-ribose. This mechanism explains why the novel PARPs are unable to function as polymerases. This discovery will help to illuminate the different biological functions of mono- versus poly-ADP-ribosylation in cells.


Assuntos
ADP Ribose Transferases/metabolismo , Poli(ADP-Ribose) Polimerases/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , ADP Ribose Transferases/química , ADP Ribose Transferases/genética , Sequência de Aminoácidos , Domínio Catalítico , Linhagem Celular , Sequência Conservada , Humanos , Técnicas In Vitro , Modelos Moleculares , Dados de Sequência Molecular , Poli(ADP-Ribose) Polimerase-1 , Poli(ADP-Ribose) Polimerases/química , Poli(ADP-Ribose) Polimerases/genética , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Proteínas Proto-Oncogênicas/química , Proteínas Proto-Oncogênicas/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Homologia de Sequência de Aminoácidos , Especificidade por Substrato
9.
Mol Cell Proteomics ; 13(5): 1184-97, 2014 May.
Artigo em Inglês | MEDLINE | ID: mdl-24556848

RESUMO

Post-translational modifications of proteins regulate diverse cellular functions, with mounting evidence suggesting that hierarchical cross-talk between distinct modifications may fine-tune cellular responses. For example, in apoptosis, caspases promote cell death via cleavage of key structural and enzymatic proteins that in some instances is inhibited by phosphorylation near the scissile bond. In this study, we systematically investigated how protein phosphorylation affects susceptibility to caspase cleavage using an N-terminomic strategy, namely, a modified terminal amino isotopic labeling of substrates (TAILS) workflow, to identify proteins for which caspase-catalyzed cleavage is modulated by phosphatase treatment. We validated the effects of phosphorylation on three of the identified proteins and found that Yap1 and Golgin-160 exhibit decreased cleavage when phosphorylated, whereas cleavage of MST3 was promoted by phosphorylation. Furthermore, using synthetic peptides we systematically examined the influence of phosphoserine throughout the entirety of caspase-3, -7, and -8 recognition motifs and observed a general inhibitory effect of phosphorylation even at residues considered outside the classical consensus motif. Overall, our work demonstrates a role for phosphorylation in controlling caspase-mediated cleavage and shows that N-terminomic strategies can be tailored to study cross-talk between phosphorylation and proteolysis.


Assuntos
Caspases/química , Caspases/metabolismo , Peptídeos/metabolismo , Proteômica/métodos , Células HeLa , Humanos , Marcação por Isótopo , Proteínas de Membrana/metabolismo , Modelos Moleculares , Fosfoproteínas/metabolismo , Fosforilação , Processamento de Proteína Pós-Traducional , Proteínas Serina-Treonina Quinases/metabolismo , Proteólise
10.
J Biol Chem ; 288(42): 30075-30086, 2013 Oct 18.
Artigo em Inglês | MEDLINE | ID: mdl-23990466

RESUMO

Precise cell division is essential for multicellular development, and defects in this process have been linked to cancer. Septins are a family of proteins that are required for mammalian cell division, but their function and mode of regulation during this process are poorly understood. Here, we demonstrate that cyclin-dependent kinase 1 (Cdk1) phosphorylates septin 9 (SEPT9) upon mitotic entry, and this phosphorylation controls association with the proline isomerase, Pin1. Both SEPT9 and Pin1 are critical for mediating the final separation of daughter cells. Expression of mutant SEPT9 that is defective in Pin1 binding was unable to rescue cytokinesis defects caused by SEPT9 depletion but rather induced dominant-negative defects in cytokinesis. However, unlike SEPT9 depletion, Pin1 was not required for the accumulation of the exocyst complex at the midbody. These results suggest that SEPT9 plays multiple roles in abscission, one of which is regulated by the action of Cdk1 and Pin1.


Assuntos
Proteína Quinase CDC2/metabolismo , Citocinese/fisiologia , Peptidilprolil Isomerase/metabolismo , Septinas/metabolismo , Proteína Quinase CDC2/genética , Regulação da Expressão Gênica/fisiologia , Células HeLa , Humanos , Mutação , Peptidilprolil Isomerase de Interação com NIMA , Peptidilprolil Isomerase/genética , Fosforilação/fisiologia , Ligação Proteica , Septinas/genética
11.
Biochim Biophys Acta ; 1834(7): 1352-8, 2013 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-23416530

RESUMO

Since protein kinases have been implicated in numerous human diseases, kinase inhibitors have emerged as promising therapeutic agents. Despite this promise, there has been a relative lag in the development of unbiased strategies to validate both inhibitor specificity and the ability to inhibit target activity within living cells. To overcome these limitations, our efforts have been focused on the development of systematic strategies that employ chemical and functional proteomics. We utilized these strategies to evaluate small molecule inhibitors of protein kinase CK2, a constitutively active kinase that has recently emerged as target for anti-cancer therapy in clinical trials. Our chemical proteomics strategies used ATP or CK2 inhibitors immobilized on sepharose beads together with mass spectrometry to capture and identify binding partners from cell extracts. These studies have verified that interactions between CK2 and its inhibitors occur in complex mixtures. However, in the case of CK2 inhibitors related to 4,5,6,7-tetrabromo-1H-benzotriazole (TBB), our work has also revealed off-targets for the inhibitors. To complement these studies, we devised functional proteomics approaches to identify proteins that exhibit decreases in phosphorylation when cells are treated with CK2 inhibitors. To identify and validate those proteins that are direct substrates for CK2, we have also employed mutants of CK2 with decreased inhibitor sensitivity. Overall, our studies have yielded systematic platforms for studying CK2 inhibitors which we believe will foster efforts to define the biological functions of CK2 and to rigorously investigate its potential as a candidate for molecular-targeted therapy. This article is part of a Special Issue entitled: Inhibitors of Protein Kinases (2012).


Assuntos
Trifosfato de Adenosina/metabolismo , Inibidores de Proteínas Quinases/farmacologia , Proteínas Quinases/metabolismo , Proteômica/métodos , Caseína Quinase II/antagonistas & inibidores , Caseína Quinase II/química , Caseína Quinase II/metabolismo , Humanos , Modelos Moleculares , Ligação Proteica , Inibidores de Proteínas Quinases/química , Inibidores de Proteínas Quinases/metabolismo , Proteínas Quinases/química , Especificidade por Substrato , Triazóis/química , Triazóis/farmacologia
12.
bioRxiv ; 2024 Sep 11.
Artigo em Inglês | MEDLINE | ID: mdl-39314291

RESUMO

Pannexin 1 (PANX1) is upregulated in many cancers, where its activity and signalling promote tumorigenic properties. Here, we report a novel ∼25 kDa isoform of human PANX1 (hPANX1-25K) which lacks the N-terminus and was detected in several human cancer cell lines including melanoma, osteosarcoma, breast cancer and glioblastoma multiforme. This isoform was increased upon hPANX1 CRISPR/Cas9 deletion targeting the first exon near M1, suggesting a potential alternative translation initiation (ATI) site. hPANX1-25K was confirmed to be a hPANX1 isoform via mass spectrometry, can be N-linked glycosylated at N254, and can interact with both ß-catenin and full length hPANX1. A double deletion of hPANX1 and hPANX1-25K reduces cell growth and viability in cancer cells. hPANX1-25K is prevalent throughout melanoma progression, and its levels are increased in squamous cell carcinoma cells and patient-derived tumours, compared to keratinocytes and normal skin, indicating that it may be differentially regulated in normal and cancer cells.

13.
J Biol Chem ; 287(34): 29184-93, 2012 Aug 17.
Artigo em Inglês | MEDLINE | ID: mdl-22753409

RESUMO

Pannexin 1 (Panx1) is a channel-forming glycoprotein expressed in different cell types of mammalian skin. We examined the role of Panx1 in melanoma tumorigenesis and metastasis since qPCR and Western blots revealed that mouse melanocytes exhibited low levels of Panx1 while increased Panx1 expression was correlated with tumor cell aggressiveness in the isogenic melanoma cell lines (B16-F0, -F10, and -BL6). Panx1 shRNA knockdown (Panx1-KD) generated stable BL6 cell lines, with reduced dye uptake, that showed a marked increase in melanocyte-like cell characteristics including higher melanin production, decreased cell migration and enhanced formation of cellular projections. Western blotting and proteomic analyses using 2D-gel/mass spectroscopy identified vimentin and ß-catenin as two of the markers of malignant melanoma that were down-regulated in Panx1-KD cells. Xenograft Panx1-KD cells grown within the chorioallantoic membrane of avian embryos developed tumors that were significantly smaller than controls. Mouse-Alu qPCR of the excised avian embryonic organs revealed that tumor metastasis to the liver was significantly reduced upon Panx1 knockdown. These data suggest that while Panx1 is present in skin melanocytes it is up-regulated during melanoma tumor progression, and tumorigenesis can be inhibited by the knockdown of Panx1 raising the possibility that Panx1 may be a viable target for the treatment of melanoma.


Assuntos
Conexinas/biossíntese , Regulação Neoplásica da Expressão Gênica , Melaninas/metabolismo , Melanócitos/metabolismo , Melanoma/metabolismo , Proteínas de Neoplasias/metabolismo , Proteínas do Tecido Nervoso/biossíntese , Animais , Linhagem Celular Tumoral , Movimento Celular/genética , Conexinas/genética , Técnicas de Silenciamento de Genes , Células HEK293 , Humanos , Melaninas/genética , Melanócitos/patologia , Melanoma/genética , Melanoma/patologia , Melanoma/terapia , Camundongos , Proteínas de Neoplasias/genética , Proteínas do Tecido Nervoso/genética , Regulação para Cima/genética , Vimentina/genética , Vimentina/metabolismo , beta Catenina/genética , beta Catenina/metabolismo
14.
J Cell Sci ; 124(Pt 14): 2341-8, 2011 Jul 15.
Artigo em Inglês | MEDLINE | ID: mdl-21693590

RESUMO

CK2 is a serine/threonine kinase with many substrates, largely unknown modes of regulation and essential roles in mitotic progression. CK2α, a catalytic subunit of CK2, is phosphorylated in mitosis, and here we examine the effect of phosphorylation on CK2α localization. Using phosphospecific antibodies, we show that CK2α localizes to the mitotic spindle in a phosphorylation-dependent manner. Mitotic spindle localization requires the unique C-terminus of CK2α, and involves a novel regulatory mechanism in which phosphorylation of CK2α facilitates binding to the peptidyl-prolyl isomerase Pin1, which is required for CK2α mitotic spindle localization. This could explain how the constitutive activity of CK2α might be targeted towards mitotic substrates. Furthermore, because Pin1 has many important spindle substrates, this might represent a general mechanism for localization of mitotic signalling proteins.


Assuntos
Caseína Quinase II/metabolismo , Peptidilprolil Isomerase/metabolismo , Fuso Acromático/enzimologia , Linhagem Celular Tumoral , Células HeLa , Humanos , Isoenzimas , Mitose/fisiologia , Peptidilprolil Isomerase de Interação com NIMA , Fosforilação , Ligação Proteica , Estrutura Terciária de Proteína
15.
Blood ; 118(1): 156-66, 2011 Jul 07.
Artigo em Inglês | MEDLINE | ID: mdl-21527517

RESUMO

JAK-STAT signaling is involved in the regulation of cell survival, proliferation, and differentiation. JAK tyrosine kinases can be transiently activated by cytokines or growth factors in normal cells, whereas they become constitutively activated as a result of mutations that affect their function in tumors. Specifically, the JAK2V617F mutation is present in the majority of patients with myeloproliferative disorders (MPDs) and is implicated in the pathogenesis of these diseases. In the present study, we report that the kinase CK2 is a novel interaction partner of JAKs and is essential for JAK-STAT activation. We demonstrate that cytokine-induced activation of JAKs and STATs and the expression of suppressor of cytokine signaling 3 (SOCS-3), a downstream target, are inhibited by CK2 small interfering RNAs or pharmacologic inhibitors. Endogenous CK2 is associated with JAK2 and JAK1 and phosphorylates JAK2 in vitro. To extend these findings, we demonstrate that CK2 interacts with JAK2V617F and that CK2 inhibitors suppress JAK2V617F autophosphorylation and downstream signaling in HEL92.1.7 cells (HEL) and primary cells from polycythemia vera (PV) patients. Furthermore, CK2 inhibitors potently induce apoptosis of HEL cells and PV cells. Our data provide evidence for novel cross-talk between CK2 and JAK-STAT signaling, with implications for therapeutic intervention in JAK2V617F-positive MPDs.


Assuntos
Caseína Quinase II/metabolismo , Neoplasias Hematológicas/metabolismo , Proteínas Quinases JNK Ativadas por Mitógeno/metabolismo , Policitemia Vera/metabolismo , Fatores de Transcrição STAT/metabolismo , Transdução de Sinais/fisiologia , Animais , Apoptose/fisiologia , Caseína Quinase II/antagonistas & inibidores , Caseína Quinase II/genética , Linhagem Celular Transformada , Linhagem Celular Tumoral , Sobrevivência Celular/fisiologia , Fibroblastos/citologia , Fibroblastos/metabolismo , Neoplasias Hematológicas/tratamento farmacológico , Neoplasias Hematológicas/patologia , Humanos , Janus Quinase 1/metabolismo , Janus Quinase 2/metabolismo , Camundongos , Fosforilase a/fisiologia , Policitemia Vera/tratamento farmacológico , Policitemia Vera/patologia
16.
Biochim Biophys Acta Gen Subj ; 1867(10): 130441, 2023 10.
Artigo em Inglês | MEDLINE | ID: mdl-37543358

RESUMO

Dramatic advances in phosphoproteomics and the development of a selective chemical probe have presented new opportunities for revealing the cellular landscape of substrates for CSNK2 (formerly known as CK2 or casein kinase II). In addition to deciphering the role(s) of CSNK2 in physiology and pathophysiology, the CSNK2 phosphoproteome offers the promise of instructing the development of CSNK2-targeted therapy.


Assuntos
COVID-19 , Humanos , Pandemias , Caseína Quinase II
17.
Biomedicines ; 11(9)2023 Aug 28.
Artigo em Inglês | MEDLINE | ID: mdl-37760847

RESUMO

Resistance to protein tyrosine kinase inhibitors (TKIs) presents a significant challenge in therapeutic target development for cancers such as triple-negative breast cancer (TNBC), where conventional therapies are ineffective at combatting systemic disease. Due to increased expression, the receptor tyrosine kinases EGFR (epidermal growth factor receptor) and c-Met are potential targets for treatment. However, targeted anti-EGFR and anti-c-Met therapies have faced mixed results in clinical trials due to acquired resistance. We hypothesize that adaptive responses in regulatory kinase networks within the EGFR and c-Met signaling axes contribute to the development of acquired erlotinib and cabozantinib resistance. To test this, we developed two separate models for cabozantinib and erlotinib resistance using the MDA-MB-231 and MDA-MB-468 cell lines, respectively. We observed that erlotinib- or cabozantinib-resistant cell lines demonstrate enhanced cell proliferation, migration, invasion, and activation of EGFR or c-Met downstream signaling (respectively). Using a SILAC (Stable Isotope Labeling of Amino acids in Cell Culture)-labeled quantitative mass spectrometry proteomics approach, we assessed the effects of erlotinib or cabozantinib resistance on the phosphoproteome, proteome, and kinome. Using this integrated proteomics approach, we identified several potential kinase mediators of cabozantinib resistance and confirmed the contribution of AKT1 to erlotinib resistance in TNBC-resistant cell lines.

18.
J Biol Chem ; 286(13): 11456-68, 2011 Apr 01.
Artigo em Inglês | MEDLINE | ID: mdl-21296876

RESUMO

Internalization of the Na(+)/H(+) exchanger NHE5 into recycling endosomes is enhanced by the endocytic adaptor proteins ß-arrestin1 and -2, best known for their preferential recognition of ligand-activated G protein-coupled receptors (GPCRs). However, the mechanism underlying their atypical association with non-GPCRs, such as NHE5, is unknown. In this study, we identified a highly acidic, serine/threonine-rich, di-isoleucine motif (amino acids 697-723) in the cytoplasmic C terminus of NHE5 that is recognized by ß-arrestin2. Gross deletions of this site decreased the state of phosphorylation of NHE5 as well as its binding and responsiveness to ß-arrestin2 in intact cells. More refined in vitro analyses showed that this site was robustly phosphorylated by the acidotropic protein kinase CK2, whereas other kinases, such as CK1 or the GPCR kinase GRK2, were considerably less potent. Simultaneous mutation of five Ser/Thr residues within 702-714 to Ala ((702)ST/AA(714)) abolished phosphorylation and binding of ß-arrestin2. In transfected cells, the CK2 catalytic α subunit formed a complex with NHE5 and decreased wild-type but not (702)ST/AA(714) NHE5 activity, further supporting a regulatory role for this kinase. The rate of internalization of (702)ST/AA(714) was also diminished and relatively insensitive to overexpression of ß-arrestin2. However, unlike in vitro, this mutant retained its ability to form a complex with ß-arrestin2 despite its lack of responsiveness. Additional mutations of two di-isoleucine-based motifs (I697A/L698A and I722A/I723A) that immediately flank the acidic cluster, either separately or together, were required to disrupt their association. These data demonstrate that discrete elements of an elaborate sorting signal in NHE5 contribute to ß-arrestin2 binding and trafficking along the recycling endosomal pathway.


Assuntos
Arrestinas/metabolismo , Endocitose/fisiologia , Endossomos/metabolismo , Sinais Direcionadores de Proteínas/fisiologia , Trocadores de Sódio-Hidrogênio/metabolismo , Motivos de Aminoácidos , Substituição de Aminoácidos , Arrestinas/genética , Caseína Quinase I/genética , Caseína Quinase I/metabolismo , Caseína Quinase II/genética , Caseína Quinase II/metabolismo , Linhagem Celular , Endossomos/genética , Quinase 2 de Receptor Acoplado a Proteína G/genética , Quinase 2 de Receptor Acoplado a Proteína G/metabolismo , Humanos , Mutação de Sentido Incorreto , Ligação Proteica/fisiologia , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Estrutura Terciária de Proteína , Transporte Proteico/fisiologia , Trocadores de Sódio-Hidrogênio/genética , beta-Arrestinas
19.
J Am Chem Soc ; 134(41): 17036-45, 2012 Oct 17.
Artigo em Inglês | MEDLINE | ID: mdl-22764889

RESUMO

Protein kinases catalyze the phosphorylation of cellular proteins involved in the regulation of many cellular processes and have emerged as promising targets for the treatment of several diseases. Conventional assays to monitor protein kinase activity are limited because they typically rely on transfer of radioactive phosphate or phospho-specific antibodies that recognize specific substrates or sequence motifs. To overcome the limitations of conventional assays, we have developed a versatile approach based on transfer of ferrocene-phosphate that can be readily monitored using electrochemical detection or detection with antiferrocene antibodies in an immunoarray format. This assay is readily adapted to multiplex arrays and can be employed for monitoring kinase activity in complex mixtures and for kinase inhibitor profiling.


Assuntos
Técnicas Eletroquímicas , Imunoensaio , Proteínas Quinases/metabolismo , Animais , Anticorpos/metabolismo , Biocatálise , Fosforilação , Proteínas Quinases/química , Coelhos
20.
Am J Hum Genet ; 84(2): 134-47, 2009 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-19185282

RESUMO

Six infants in an Old Order Amish pedigree were observed to be affected with endocrine-cerebro-osteodysplasia (ECO). ECO is a previously unidentified neonatal lethal recessive disorder with multiple anomalies involving the endocrine, cerebral, and skeletal systems. Autozygosity mapping and sequencing identified a previously unknown missense mutation, R272Q, in ICK, encoding intestinal cell kinase (ICK). Our results established that R272 is conserved across species and among ethnicities, and three-dimensional analysis of the protein structure suggests protein instability due to the R272Q mutation. We also demonstrate that the R272Q mutant fails to localize at the nucleus and has diminished kinase activity. These findings suggest that ICK plays a key role in the development of multiple organ systems.


Assuntos
Doenças Ósseas/genética , Doenças do Sistema Nervoso Central/genética , Doenças do Sistema Endócrino/genética , Etnicidade/genética , Mutação , Proteínas Serina-Treonina Quinases/genética , Animais , Autopsia , Doenças Ósseas/sangue , Doenças Ósseas/patologia , Encéfalo/enzimologia , Doenças do Sistema Nervoso Central/patologia , Sequência Conservada , Doenças do Sistema Endócrino/sangue , Doenças do Sistema Endócrino/patologia , Éxons , Feminino , Genes Recessivos , Humanos , Rim/enzimologia , Fígado/enzimologia , Masculino , Linhagem , Proteínas Serina-Treonina Quinases/sangue , Irmãos , Especificidade da Espécie , Síndrome
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