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1.
Annu Rev Biochem ; 83: 641-69, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-24905788

RESUMO

The importance of PTEN in cellular function is underscored by the frequency of its deregulation in cancer. PTEN tumor-suppressor activity depends largely on its lipid phosphatase activity, which opposes PI3K/AKT activation. As such, PTEN regulates many cellular processes, including proliferation, survival, energy metabolism, cellular architecture, and motility. More than a decade of research has expanded our knowledge about how PTEN is controlled at the transcriptional level as well as by numerous posttranscriptional modifications that regulate its enzymatic activity, protein stability, and cellular location. Although the role of PTEN in cancers has long been appreciated, it is also emerging as an important factor in other diseases, such as diabetes and autism spectrum disorders. Our understanding of PTEN function and regulation will hopefully translate into improved prognosis and treatment for patients suffering from these ailments.


Assuntos
Regulação Enzimológica da Expressão Gênica , PTEN Fosfo-Hidrolase/fisiologia , Animais , Ciclo Celular , Movimento Celular , Núcleo Celular/metabolismo , Polaridade Celular , Proliferação de Células , Sobrevivência Celular , Ativação Enzimática , Humanos , Lipídeos/química , Neoplasias/metabolismo , Oxigênio/química , PTEN Fosfo-Hidrolase/química , Fosforilação , Prognóstico , Mapeamento de Interação de Proteínas , Multimerização Proteica , Estrutura Terciária de Proteína , Especificidade por Substrato , Ubiquitina/química
2.
Cell ; 158(6): 1221-1224, 2014 Sep 11.
Artigo em Inglês | MEDLINE | ID: mdl-25215479

RESUMO

This year, the Albert Lasker Basic Medical Research Award will be shared by Peter Walter and Kazutoshi Mori for discoveries revealing the molecular mechanism of the unfolded protein response, an intracellular quality control system that detects harmful misfolded proteins in the endoplasmic reticulum and then signals the nucleus to carry out corrective measures.


Assuntos
Distinções e Prêmios , Saccharomyces cerevisiae/metabolismo , Resposta a Proteínas não Dobradas , Animais , História do Século XX , História do Século XXI , Humanos , Japão , Proteínas/metabolismo , Saccharomyces cerevisiae/citologia , Estados Unidos
3.
Neurobiol Dis ; 181: 106119, 2023 06 01.
Artigo em Inglês | MEDLINE | ID: mdl-37059210

RESUMO

Lafora disease is a rare recessive form of progressive myoclonic epilepsy, usually diagnosed during adolescence. Patients present with myoclonus, neurological deterioration, and generalized tonic-clonic, myoclonic, or absence seizures. Symptoms worsen until death, usually within the first ten years of clinical onset. The primary histopathological hallmark is the formation of aberrant polyglucosan aggregates called Lafora bodies in the brain and other tissues. Lafora disease is caused by mutations in either the EPM2A gene, encoding laforin, or the EPM2B gene, coding for malin. The most frequent EPM2A mutation is R241X, which is also the most prevalent in Spain. The Epm2a-/- and Epm2b-/- mouse models of Lafora disease show neuropathological and behavioral abnormalities similar to those seen in patients, although with a milder phenotype. To obtain a more accurate animal model, we generated the Epm2aR240X knock-in mouse line with the R240X mutation in the Epm2a gene, using genetic engineering based on CRISPR-Cas9 technology. Epm2aR240X mice exhibit most of the alterations reported in patients, including the presence of LBs, neurodegeneration, neuroinflammation, interictal spikes, neuronal hyperexcitability, and cognitive decline, despite the absence of motor impairments. The Epm2aR240X knock-in mouse displays some symptoms that are more severe that those observed in the Epm2a-/- knock-out, including earlier and more pronounced memory loss, increased levels of neuroinflammation, more interictal spikes and increased neuronal hyperexcitability, symptoms that more precisely resemble those observed in patients. This new mouse model can therefore be specifically used to evaluate how new therapies affects these features with greater precision.


Assuntos
Disfunção Cognitiva , Doença de Lafora , Animais , Camundongos , Disfunção Cognitiva/genética , Doença de Lafora/genética , Doença de Lafora/patologia , Camundongos Knockout , Doenças Neuroinflamatórias , Proteínas Tirosina Fosfatases não Receptoras/genética , Ubiquitina-Proteína Ligases/genética
4.
J Biol Chem ; 296: 100267, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33759783

RESUMO

The study of extracellular phosphorylation was initiated in late 19th century when the secreted milk protein, casein, and egg-yolk protein, phosvitin, were shown to be phosphorylated. However, it took more than a century to identify Fam20C, which phosphorylates both casein and phosvitin under physiological conditions. This kinase, along with its family members Fam20A and Fam20B, defined a new family with altered amino acid sequences highly atypical from the canonical 540 kinases comprising the kinome. Fam20B is a glycan kinase that phosphorylates xylose residues and triggers peptidoglycan biosynthesis, a role conserved from sponges to human. The protein kinase, Fam20C, conserved from nematodes to humans, phosphorylates well over 100 substrates in the secretory pathway with overall functions postulated to encompass endoplasmic reticulum homeostasis, nutrition, cardiac function, coagulation, and biomineralization. The preferred phosphorylation motif of Fam20C is SxE/pS, and structural studies revealed that related member Fam20A allosterically activates Fam20C by forming a heterodimeric/tetrameric complex. Fam20A, a pseudokinase, is observed only in vertebrates. Loss-of-function genetic alterations in the Fam20 family lead to human diseases such as amelogenesis imperfecta, nephrocalcinosis, lethal and nonlethal forms of Raine syndrome with major skeletal defects, and altered phosphate homeostasis. Together, these three members of the Fam20 family modulate a diverse network of secretory pathway components playing crucial roles in health and disease. The overarching theme of this review is to highlight the progress that has been made in the emerging field of extracellular phosphorylation and the key roles secretory pathway kinases play in an ever-expanding number of cellular processes.


Assuntos
Caseína Quinase I/metabolismo , Proteínas da Matriz Extracelular/metabolismo , Caseína Quinase I/química , Retículo Endoplasmático/metabolismo , Proteínas da Matriz Extracelular/química , Homeostase , Humanos , Miocárdio/metabolismo , Fosforilação , Via Secretória , Transdução de Sinais , Relação Estrutura-Atividade , Especificidade por Substrato
5.
J Biol Chem ; 294(5): 1638-1642, 2019 02 01.
Artigo em Inglês | MEDLINE | ID: mdl-30710011

RESUMO

Herb Tabor was the Editor-in-Chief of the Journal of Biological Chemistry (JBC) spanning the years 1971-2010. This year, Herb turns 100. What do you give a person turning 100? Our answer to this question was to dedicate two of our favorite JBC papers to Herb. Both of these papers focus on reversible phosphorylation, which we briefly review. In addition, we delve into a new finding that centers around a novel family of secreted kinases, suggesting that there are many new and exciting discoveries yet to explore.


Assuntos
Bioquímica/história , Publicações Periódicas como Assunto , Proteínas Tirosina Fosfatases/metabolismo , Proteínas Tirosina Quinases/metabolismo , História do Século XX , História do Século XXI , Humanos , Fosforilação
6.
Epilepsy Behav ; 103(Pt A): 106839, 2020 02.
Artigo em Inglês | MEDLINE | ID: mdl-31932179

RESUMO

Lafora disease (LD) is both a fatal childhood epilepsy and a glycogen storage disease caused by recessive mutations in either the Epilepsy progressive myoclonus 2A (EPM2A) or EPM2B genes. Hallmarks of LD are aberrant, cytoplasmic carbohydrate aggregates called Lafora bodies (LBs) that are a disease driver. The 5th International Lafora Epilepsy Workshop was recently held in Alcala de Henares, Spain. The workshop brought together nearly 100 clinicians, academic and industry scientists, trainees, National Institutes of Health (NIH) representation, and friends and family members of patients with LD. The workshop covered aspects of LD ranging from defining basic scientific mechanisms to elucidating a LD therapy or cure and a recently launched LD natural history study.


Assuntos
Congressos como Assunto/tendências , Educação/tendências , Internacionalidade , Doença de Lafora/terapia , Animais , Humanos , Doença de Lafora/epidemiologia , Doença de Lafora/genética , Mutação/genética , Proteínas Tirosina Fosfatases não Receptoras/genética , Espanha/epidemiologia
7.
Mol Cell ; 34(1): 93-103, 2009 Apr 10.
Artigo em Inglês | MEDLINE | ID: mdl-19362538

RESUMO

We show that the secreted antigen, IbpA, of the respiratory pathogen Histophilus somni induces cytotoxicity in mammalian cells via its Fic domains. Fic domains are defined by a core HPFxxGNGR motif and are conserved from bacteria to humans. We demonstrate that the Fic domains of IbpA catalyze a unique reversible adenylylation event that uses ATP to add an adenosine monophosphate (AMP) moiety to a conserved tyrosine residue in the switch I region of Rho GTPases. This modification requires the conserved histidine of the Fic core motif and renders Rho GTPases inactive. We further demonstrate that the only human protein containing a Fic domain, huntingtin yeast-interacting protein E (HYPE), also adenylylates Rho GTPases in vitro. Thus, we classify Fic domain-containing proteins as a class of enzymes that mediate bacterial pathogenesis as well as a previously unrecognized eukaryotic posttranslational modification that may regulate key signaling events.


Assuntos
Antígenos de Bactérias/fisiologia , Proteínas de Bactérias/fisiologia , Pasteurellaceae/imunologia , Transdução de Sinais , Fatores de Virulência/fisiologia , Citoesqueleto de Actina/metabolismo , Monofosfato de Adenosina/metabolismo , Trifosfato de Adenosina/metabolismo , Motivos de Aminoácidos , Antígenos de Bactérias/química , Proteínas de Bactérias/química , Proteínas de Transporte/química , Proteínas de Transporte/metabolismo , Proteínas de Transporte/fisiologia , Cisteína Endopeptidases/química , Células HeLa , Histidina/química , Histidina/metabolismo , Humanos , Proteínas de Membrana/química , Proteínas de Membrana/metabolismo , Proteínas de Membrana/fisiologia , Dados de Sequência Molecular , Nucleotidiltransferases , Pasteurellaceae/patogenicidade , Diester Fosfórico Hidrolases/farmacologia , Alinhamento de Sequência , Especificidade por Substrato , Tirosina/metabolismo , Fatores de Virulência/química , Proteínas rac de Ligação ao GTP/metabolismo , Proteínas rho de Ligação ao GTP/química , Proteínas rho de Ligação ao GTP/metabolismo
8.
J Biol Chem ; 290(13): 8482-99, 2015 Mar 27.
Artigo em Inglês | MEDLINE | ID: mdl-25601083

RESUMO

The maintenance of endoplasmic reticulum (ER) homeostasis is a critical aspect of determining cell fate and requires a properly functioning unfolded protein response (UPR). We have discovered a previously unknown role of a post-translational modification termed adenylylation/AMPylation in regulating signal transduction events during UPR induction. A family of enzymes, defined by the presence of a Fic (filamentation induced by cAMP) domain, catalyzes this adenylylation reaction. The human genome encodes a single Fic protein, called HYPE (Huntingtin yeast interacting protein E), with adenylyltransferase activity but unknown physiological target(s). Here, we demonstrate that HYPE localizes to the lumen of the endoplasmic reticulum via its hydrophobic N terminus and adenylylates the ER molecular chaperone, BiP, at Ser-365 and Thr-366. BiP functions as a sentinel for protein misfolding and maintains ER homeostasis. We found that adenylylation enhances BiP's ATPase activity, which is required for refolding misfolded proteins while coping with ER stress. Accordingly, HYPE expression levels increase upon stress. Furthermore, siRNA-mediated knockdown of HYPE prevents the induction of an unfolded protein response. Thus, we identify HYPE as a new UPR regulator and provide the first functional data for Fic-mediated adenylylation in mammalian signaling.


Assuntos
Proteínas de Transporte/fisiologia , Proteínas de Membrana/fisiologia , Nucleotidiltransferases/fisiologia , Processamento de Proteína Pós-Traducional , Resposta a Proteínas não Dobradas , Fator 6 Ativador da Transcrição/metabolismo , Apoptose , Sobrevivência Celular , Retículo Endoplasmático/metabolismo , Chaperona BiP do Retículo Endoplasmático , Glicosilação , Células HEK293 , Células HeLa , Proteínas de Choque Térmico/metabolismo , Humanos , Interações Hidrofóbicas e Hidrofílicas , Células MCF-7 , Estrutura Terciária de Proteína , Transporte Proteico , Transdução de Sinais , Regulação para Cima , eIF-2 Quinase/metabolismo
9.
J Biol Chem ; 288(7): 5176-85, 2013 Feb 15.
Artigo em Inglês | MEDLINE | ID: mdl-23293031

RESUMO

Cardiolipin is a glycerophospholipid found predominantly in the mitochondrial membranes of eukaryotes and in bacterial membranes. Cardiolipin interacts with protein complexes and plays pivotal roles in cellular energy metabolism, membrane dynamics, and stress responses. We recently identified the mitochondrial phosphatase, PTPMT1, as the enzyme that converts phosphatidylglycerolphosphate (PGP) to phosphatidylglycerol, a critical step in the de novo biosynthesis of cardiolipin. Upon examination of PTPMT1 evolutionary distribution, we found a PTPMT1-like phosphatase in the bacterium Rhodopirellula baltica. The purified recombinant enzyme dephosphorylated PGP in vitro. Moreover, its expression restored cardiolipin deficiency and reversed growth impairment in a Saccharomyces cerevisiae mutant lacking the yeast PGP phosphatase, suggesting that it is a bona fide PTPMT1 ortholog. When ectopically expressed, this bacterial PGP phosphatase was localized in the mitochondria of yeast and mammalian cells. Together, our results demonstrate the conservation of function between bacterial and mammalian PTPMT1 orthologs.


Assuntos
Bactérias/enzimologia , Regulação Bacteriana da Expressão Gênica , Regulação Enzimológica da Expressão Gênica , Fosfatidilgliceróis/química , Monoéster Fosfórico Hidrolases/química , Sequência de Aminoácidos , Animais , Cardiolipinas/química , Sequência Conservada , Drosophila melanogaster , Teste de Complementação Genética , Lipídeos/química , Camundongos , Mitocôndrias/metabolismo , Modelos Biológicos , Modelos Genéticos , Dados de Sequência Molecular , Saccharomyces cerevisiae/metabolismo , Homologia de Sequência de Aminoácidos
10.
Hum Mol Genet ; 21(7): 1604-10, 2012 Apr 01.
Artigo em Inglês | MEDLINE | ID: mdl-22186021

RESUMO

Lafora disease is a fatal, progressive myoclonus epilepsy caused in ~90% of cases by mutations in the EPM2A or EPM2B genes. Characteristic of the disease is the formation of Lafora bodies, insoluble deposits containing abnormal glycogen-like material in many tissues, including neurons, muscle, heart and liver. Because glycogen is important for glucose homeostasis, the aberrant glycogen metabolism in Lafora disease might disturb whole-body glucose handling. Indeed, Vernia et al. [Vernia, S., Heredia, M., Criado, O., Rodriguez de Cordoba, S., Garcia-Roves, P.M., Cansell, C., Denis, R., Luquet, S., Foufelle, F., Ferre, P. et al. (2011) Laforin, a dual-specificity phosphatase involved in Lafora disease, regulates insulin response and whole-body energy balance in mice. Hum. Mol. Genet., 20, 2571-2584] reported that Epm2a-/- mice had enhanced glucose disposal and insulin sensitivity, leading them to suggest that laforin, the Epm2a gene product, is involved in insulin signaling. We analyzed 3-month- and 6-7-month-old Epm2a-/- mice and observed no differences in glucose tolerance tests (GTTs) or insulin tolerance tests (ITTs) compared with wild-type mice of matched genetic background. At 3 months, Epm2b-/- mice also showed no differences in GTTs and ITTs. In the 6-7-month-old Epm2a-/- mice, there was no evidence for increased insulin stimulation of the phosphorylation of Akt, GSK-3 or S6 in skeletal muscle, liver and heart. From metabolic analyses, these animals were normal with regard to food intake, oxygen consumption, energy expenditure and respiratory exchange ratio. By dual-energy X-ray absorptiometry scan, body composition was unaltered at 3 or 6-7 months of age. Echocardiography showed no defects of cardiac function in Epm2a-/- or Epm2b-/- mice. We conclude that laforin and malin have no effect on whole-body glucose metabolism and insulin sensitivity, and that laforin is not involved in insulin signaling.


Assuntos
Glicemia/análise , Fosfatases de Especificidade Dupla/genética , Resistência à Insulina , Ubiquitina-Proteína Ligases/genética , Animais , Coração/fisiologia , Insulina/farmacologia , Camundongos , Camundongos Knockout , Proteínas Tirosina Fosfatases não Receptoras , Transdução de Sinais
11.
Trends Biochem Sci ; 34(12): 628-39, 2009 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-19818631

RESUMO

Reversible phosphorylation modulates nearly every step of glycogenesis and glycogenolysis. Multiple metabolic disorders are the result of defective enzymes that control these phosphorylation events, enzymes that were identified biochemically before the advent of the molecular biology era. Lafora disease is a metabolic disorder resulting in accumulation of water-insoluble glucan in the cytoplasm, and manifests as a debilitating neurodegeneration that ends with the death of the patient. Unlike most metabolic disorders, the link between Lafora disease and metabolism has not been defined in almost 100 years. The results of recent studies with mammalian cells, mouse models, eukaryotic algae, and plants have begun to define the molecular mechanisms that cause Lafora disease. The emerging theme identifies a new phosphorylation substrate in glycogen metabolism, the glucan itself.


Assuntos
Doença de Lafora/genética , Doença de Lafora/metabolismo , Doenças Neurodegenerativas/metabolismo , Animais , Proteínas de Transporte/genética , Proteínas de Transporte/fisiologia , Humanos , Modelos Biológicos , Doenças Neurodegenerativas/etiologia , Proteínas Tirosina Fosfatases não Receptoras/genética , Proteínas Tirosina Fosfatases não Receptoras/fisiologia , Ubiquitina-Proteína Ligases
12.
Anal Biochem ; 435(1): 54-6, 2013 Apr 01.
Artigo em Inglês | MEDLINE | ID: mdl-23201267

RESUMO

With the recent discovery of a unique class of dual-specificity phosphatases that dephosphorylate glucans, we report an in vitro assay tailored for the detection of phosphatase activity against phosphorylated glucans. We demonstrate that, in contrast to a general phosphatase assay using a synthetic substrate, only phosphatases that possess glucan phosphatase activity liberate phosphate from the phosphorylated glucan amylopectin using the described assay. This assay is simple and cost-effective, providing reproducible results that clearly establish the presence or absence of glucan phosphatase activity. The assay described will be a useful tool in characterizing emerging members of the glucan phosphatase family.


Assuntos
Amilopectina/metabolismo , Fosfatases de Especificidade Dupla/metabolismo , Ensaios Enzimáticos/métodos , Corantes de Rosanilina/análise , Animais , Arabidopsis/enzimologia , Ensaios Enzimáticos/economia , Glicogênio/metabolismo , Humanos , Fosforilação , Coelhos , Corantes de Rosanilina/metabolismo , Amido/metabolismo , Especificidade por Substrato
13.
J Biol Chem ; 286(37): 32834-42, 2011 Sep 16.
Artigo em Inglês | MEDLINE | ID: mdl-21795713

RESUMO

A new family of adenylyltransferases, defined by the presence of a Fic domain, was recently discovered to catalyze the addition of adenosine monophosphate (AMP) to Rho GTPases (Yarbrough, M. L., Li, Y., Kinch, L. N., Grishin, N. V., Ball, H. L., and Orth, K. (2009) Science 323, 269-272; Worby, C. A., Mattoo, S., Kruger, R. P., Corbeil, L. B., Koller, A., Mendez, J. C., Zekarias, B., Lazar, C., and Dixon, J. E. (2009) Mol. Cell 34, 93-103). This adenylylation event inactivates Rho GTPases by preventing them from binding to their downstream effectors. We reported that the Fic domain(s) of the immunoglobulin-binding protein A (IbpA) from the pathogenic bacterium Histophilus somni adenylylates mammalian Rho GTPases, RhoA, Rac1, and Cdc42, thereby inducing host cytoskeletal collapse, which allows H. somni to breach alveolar barriers and cause septicemia. The IbpA-mediated adenylylation occurs on a functionally critical tyrosine in the switch 1 region of these GTPases. Here, we conduct a detailed characterization of the IbpA Fic2 domain and compare its activity with other known Fic adenylyltransferases, VopS (Vibrio outer protein S) from the bacterial pathogen Vibrio parahaemolyticus and the human protein HYPE (huntingtin yeast interacting protein E; also called FicD). We also included the Fic domains of the secreted protein, PfhB2, from the opportunistic pathogen Pasteurella multocida, in our analysis. PfhB2 shares a common domain architecture with IbpA and contains two Fic domains. We demonstrate that the PfhB2 Fic domains also possess adenylyltransferase activity that targets the switch 1 tyrosine of Rho GTPases. Comparative kinetic and phylogenetic analyses of IbpA-Fic2 with the Fic domains of PfhB2, VopS, and HYPE reveal important aspects of their specificities for Rho GTPases and nucleotide usage and offer mechanistic insights for determining nucleotide and substrate specificities for these enzymes. Finally, we compare the evolutionary lineages of Fic proteins with those of other known adenylyltransferases.


Assuntos
Proteínas de Bactérias/química , Evolução Molecular , Proteínas de Membrana/química , Nucleotidiltransferases/química , Pasteurellaceae/enzimologia , Proteínas de Bactérias/genética , Humanos , Proteínas de Membrana/genética , Nucleotidiltransferases/genética , Pasteurellaceae/genética , Estrutura Terciária de Proteína , Vibrio parahaemolyticus/enzimologia , Vibrio parahaemolyticus/genética
14.
J Cell Biol ; 178(3): 477-88, 2007 Jul 30.
Artigo em Inglês | MEDLINE | ID: mdl-17646401

RESUMO

Lafora disease (LD) is a progressive myoclonic epilepsy resulting in severe neurodegeneration followed by death. A hallmark of LD is the accumulation of insoluble polyglucosans called Lafora bodies (LBs). LD is caused by mutations in the gene encoding the phosphatase laforin, which reportedly exists solely in vertebrates. We utilized a bioinformatics screen to identify laforin orthologues in five protists. These protists evolved from a progenitor red alga and synthesize an insoluble carbohydrate whose composition closely resembles LBs. Furthermore, we show that the kingdom Plantae, which lacks laforin, possesses a protein with laforin-like properties called starch excess 4 (SEX4). Mutations in the Arabidopsis thaliana SEX4 gene results in a starch excess phenotype reminiscent of LD. We demonstrate that Homo sapiens laforin complements the sex4 phenotype and propose that laforin and SEX4 are functional equivalents. Finally, we show that laforins and SEX4 dephosphorylate a complex carbohydrate and form the only family of phosphatases with this activity. These results provide a molecular explanation for the etiology of LD.


Assuntos
Evolução Biológica , Metabolismo dos Carboidratos , Doença de Lafora , Isoformas de Proteínas , Proteínas Tirosina Fosfatases não Receptoras , Sequência de Aminoácidos , Animais , Proteínas de Arabidopsis/classificação , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Biologia Computacional , Evolução Molecular , Glicogênio/metabolismo , Humanos , Doença de Lafora/genética , Doença de Lafora/metabolismo , Doença de Lafora/patologia , Doença de Lafora/fisiopatologia , Dados de Sequência Molecular , Filogenia , Isoformas de Proteínas/classificação , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Proteínas Tirosina Fosfatases não Receptoras/classificação , Proteínas Tirosina Fosfatases não Receptoras/genética , Proteínas Tirosina Fosfatases não Receptoras/metabolismo , Alinhamento de Sequência , Amido/metabolismo
15.
Biochem J ; 439(2): 265-75, 2011 Oct 15.
Artigo em Inglês | MEDLINE | ID: mdl-21728993

RESUMO

Lafora progressive myoclonus epilepsy [LD (Lafora disease)] is a fatal autosomal recessive neurodegenerative disorder caused by loss-of-function mutations in either the EPM2A gene, encoding the dual-specificity phosphatase laforin, or the EPM2B gene, encoding the E3-ubiquitin ligase malin. Previously, we and others showed that laforin and malin form a functional complex that regulates multiple aspects of glycogen metabolism, and that the interaction between laforin and malin is enhanced by conditions activating AMPK (AMP-activated protein kinase). In the present study, we demonstrate that laforin is a phosphoprotein, as indicated by two-dimensional electrophoresis, and we identify Ser(25) as the residue involved in this modification. We also show that Ser(25) is phosphorylated both in vitro and in vivo by AMPK. Lastly, we demonstrate that this residue plays a critical role for both the phosphatase activity and the ability of laforin to interact with itself and with previously established binding partners. The results of the present study suggest that phosphorylation of laforin-Ser(25) by AMPK provides a mechanism to modulate the interaction between laforin and malin. Regulation of this complex is necessary to maintain normal glycogen metabolism. Importantly, Ser(25) is mutated in some LD patients (S25P), and our results begin to elucidate the mechanism of disease in these patients.


Assuntos
Proteínas Quinases Ativadas por AMP/metabolismo , Doença de Lafora/enzimologia , Proteínas Tirosina Fosfatases não Receptoras/metabolismo , Serina/metabolismo , Linhagem Celular , Eletroforese em Gel Bidimensional , Humanos , Modelos Moleculares , Fosforilação , Conformação Proteica , Proteínas Tirosina Fosfatases não Receptoras/química , Técnicas do Sistema de Duplo-Híbrido
16.
Proc Natl Acad Sci U S A ; 105(23): 7970-5, 2008 Jun 10.
Artigo em Inglês | MEDLINE | ID: mdl-18524949

RESUMO

Phosphatidylinositol lipids play diverse physiological roles, and their concentrations are tightly regulated by various kinases and phosphatases. The enzymatic activity of Ciona intestinalis voltage sensor-containing phosphatase (Ci-VSP), recently identified as a member of the PTEN (phosphatase and tensin homolog deleted on chromosome 10) family of phosphatidylinositol phosphatases, is regulated by its own voltage-sensor domain in a voltage-dependent manner. However, a detailed mechanism of Ci-VSP regulation and its substrate specificity remain unknown. Here we determined the in vitro substrate specificity of Ci-VSP by measuring the phosphoinositide phosphatase activity of the Ci-VSP cytoplasmic phosphatase domain. Despite the high degree of identity shared between the active sites of PTEN and Ci-VSP, Ci-VSP dephosphorylates not only the PTEN substrate, phosphatidylinositol 3,4,5-trisphosphate [PI(3,4,5)P3], but also, unlike PTEN, phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2]. Enzymatic action on PI(4,5)P2 removes the phosphate at position 5 of the inositol ring, resulting in the production of phosphatidylinositol 4-phosphate [PI(4)P]. The active site Cys-X(5)-Arg (CX(5)R) sequence of Ci-VSP differs with that of PTEN only at amino acid 365 where a glycine residue in Ci-VSP is replaced by an alanine in PTEN. Ci-VSP with a G365A mutation no longer dephosphorylates PI(4,5)P2 and is not capable of inducing depolarization-dependent rundown of a PI(4,5)P2-dependent potassium channel. These results indicate that Ci-VSP is a PI(3,4,5)P3/PI(4,5)P2 phosphatase that uniquely functions in the voltage-dependent regulation of ion channels through regulation of PI(4,5)P2 levels.


Assuntos
Ciona intestinalis/enzimologia , PTEN Fosfo-Hidrolase/química , Fosfatidilinositol 4,5-Difosfato/metabolismo , Monoéster Fosfórico Hidrolases/química , Monoéster Fosfórico Hidrolases/metabolismo , Homologia de Sequência de Aminoácidos , Sequência de Aminoácidos , Substituição de Aminoácidos , Animais , Sítios de Ligação , Glicina/metabolismo , Ativação do Canal Iônico , Canais Iônicos/metabolismo , Dados de Sequência Molecular , Proteínas Mutantes/metabolismo , Fosforilação , Especificidade por Substrato , Xenopus
17.
J Cell Biol ; 218(11): 3795-3811, 2019 11 04.
Artigo em Inglês | MEDLINE | ID: mdl-31541016

RESUMO

Fam20C is a secreted protein kinase mutated in Raine syndrome, a human skeletal disorder. In vertebrates, bone and enamel proteins are major Fam20C substrates. However, Fam20 kinases are conserved in invertebrates lacking bone and enamel, suggesting other ancestral functions. We show that FAMK-1, the Caenorhabditis elegans Fam20C orthologue, contributes to fertility, embryogenesis, and development. These functions are not fulfilled when FAMK-1 is retained in the early secretory pathway. During embryogenesis, FAMK-1 maintains intercellular partitions and prevents multinucleation; notably, temperature elevation or lowering cortical stiffness reduces requirement for FAMK-1 in these contexts. FAMK-1 is expressed in multiple adult tissues that undergo repeated mechanical strain, and selective expression in the spermatheca restores fertility. Informatic, biochemical, and functional analysis implicate lectins as FAMK-1 substrates. These findings suggest that FAMK-1 phosphorylation of substrates, including lectins, in the late secretory pathway is important in embryonic and tissue contexts where cells are subjected to mechanical strain.


Assuntos
Caenorhabditis elegans/enzimologia , Caseína Quinase I/metabolismo , Animais , Caenorhabditis elegans/metabolismo , Caseína Quinase I/genética , Células HEK293 , Humanos
18.
Science ; 336(6085): 1150-3, 2012 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-22582013

RESUMO

Protein phosphorylation is a fundamental mechanism regulating nearly every aspect of cellular life. Several secreted proteins are phosphorylated, but the kinases responsible are unknown. We identified a family of atypical protein kinases that localize within the Golgi apparatus and are secreted. Fam20C appears to be the Golgi casein kinase that phosphorylates secretory pathway proteins within S-x-E motifs. Fam20C phosphorylates the caseins and several secreted proteins implicated in biomineralization, including the small integrin-binding ligand, N-linked glycoproteins (SIBLINGs). Consequently, mutations in Fam20C cause an osteosclerotic bone dysplasia in humans known as Raine syndrome. Fam20C is thus a protein kinase dedicated to the phosphorylation of extracellular proteins.


Assuntos
Caseínas/metabolismo , Proteínas da Matriz Extracelular/metabolismo , Complexo de Golgi/enzimologia , Via Secretória , Anormalidades Múltiplas/genética , Anormalidades Múltiplas/metabolismo , Motivos de Aminoácidos , Sequência de Aminoácidos , Animais , Calcificação Fisiológica , Caseína Quinase I , Caseína Quinases/metabolismo , Bovinos , Linhagem Celular Tumoral , Fissura Palatina/genética , Fissura Palatina/metabolismo , Exoftalmia/genética , Exoftalmia/metabolismo , Proteínas da Matriz Extracelular/química , Proteínas da Matriz Extracelular/genética , Glicoproteínas/metabolismo , Células HEK293 , Células HeLa , Humanos , Microcefalia/genética , Microcefalia/metabolismo , Leite/enzimologia , Dados de Sequência Molecular , Mutação , Osteopontina , Osteosclerose/genética , Osteosclerose/metabolismo , Fosforilação , Sinais Direcionadores de Proteínas , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/metabolismo , Especificidade por Substrato
19.
Cell Metab ; 13(3): 233-4, 2011 Mar 02.
Artigo em Inglês | MEDLINE | ID: mdl-21356510

RESUMO

Phosphorylation of glycogen has been known for decades; however, the basic metabolic pathways responsible for this modification are unknown. In this issue, Tagliabracci et al. (2011) report the enzyme responsible for incorporating phosphate and the chemical nature of the phosphate linkage, providing a framework for expanding our understanding of a devastating form of epilepsy.

20.
Cell Metab ; 13(6): 690-700, 2011 Jun 08.
Artigo em Inglês | MEDLINE | ID: mdl-21641550

RESUMO

PTPMT1 was the first protein tyrosine phosphatase found localized to the mitochondria, but its biological function was unknown. Herein, we demonstrate that whole body deletion of Ptpmt1 in mice leads to embryonic lethality, suggesting an indispensable role for PTPMT1 during development. Ptpmt1 deficiency in mouse embryonic fibroblasts compromises mitochondrial respiration and results in abnormal mitochondrial morphology. Lipid analysis of Ptpmt1-deficient fibroblasts reveals an accumulation of phosphatidylglycerophosphate (PGP) along with a concomitant decrease in phosphatidylglycerol. PGP is an essential intermediate in the biosynthetic pathway of cardiolipin, a mitochondrial-specific phospholipid regulating the membrane integrity and activities of the organelle. We further demonstrate that PTPMT1 specifically dephosphorylates PGP in vitro. Loss of PTPMT1 leads to dramatic diminution of cardiolipin, which can be partially reversed by the expression of catalytic active PTPMT1. Our study identifies PTPMT1 as the mammalian PGP phosphatase and points to its role as a regulator of cardiolipin biosynthesis.


Assuntos
Cardiolipinas/biossíntese , PTEN Fosfo-Hidrolase/metabolismo , Trifosfato de Adenosina/metabolismo , Animais , Linhagem Celular , Respiração Celular/genética , Clonagem Molecular , Embrião de Mamíferos/metabolismo , Engenharia Genética , Genótipo , Camundongos , Camundongos Knockout , Mitocôndrias/genética , Mitocôndrias/metabolismo , Membranas Mitocondriais/metabolismo , Mutagênese Sítio-Dirigida , Mutação , PTEN Fosfo-Hidrolase/genética , Fosfatidilgliceróis/metabolismo , Monoéster Fosfórico Hidrolases/genética , Monoéster Fosfórico Hidrolases/metabolismo , Proteínas Recombinantes/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo
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