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1.
Proc Natl Acad Sci U S A ; 118(33)2021 08 17.
Artigo em Inglês | MEDLINE | ID: mdl-34385319

RESUMO

The protein kinase Akt is one of the primary effectors of growth factor signaling in the cell. Akt responds specifically to the lipid second messengers phosphatidylinositol-3,4,5-trisphosphate [PI(3,4,5)P3] and phosphatidylinositol-3,4-bisphosphate [PI(3,4)P2] via its PH domain, leading to phosphorylation of its activation loop and the hydrophobic motif of its kinase domain, which are critical for activity. We have now determined the crystal structure of Akt1, revealing an autoinhibitory interface between the PH and kinase domains that is often mutated in cancer and overgrowth disorders. This interface persists even after stoichiometric phosphorylation, thereby restricting maximum Akt activity to PI(3,4,5)P3- or PI(3,4)P2-containing membranes. Our work helps to resolve the roles of lipids and phosphorylation in the activation of Akt and has wide implications for the spatiotemporal control of Akt and potentially lipid-activated kinase signaling in general.

2.
J Biol Chem ; 297(2): 100919, 2021 08.
Artigo em Inglês | MEDLINE | ID: mdl-34181950

RESUMO

Serum- and glucocorticoid-regulated kinase 3 (Sgk3) is a serine/threonine protein kinase activated by the phospholipid phosphatidylinositol 3-phosphate (PI3P) downstream of growth factor signaling via class I phosphatidylinositol 3-kinase (PI3K) signaling and by class III PI3K/Vps34-mediated PI3P production on endosomes. Upregulation of Sgk3 activity has recently been linked to a number of human cancers; however, the precise mechanism of activation of Sgk3 is unknown. Here, we use a wide range of cell biological, biochemical, and biophysical techniques, including hydrogen-deuterium exchange mass spectrometry, to investigate the mechanism of activation of Sgk3 by PI3P. We show that Sgk3 is regulated by a combination of phosphorylation and allosteric activation. We demonstrate that binding of Sgk3 to PI3P via its regulatory phox homology (PX) domain induces large conformational changes in Sgk3 associated with its activation and that the PI3P-binding pocket of the PX domain of Sgk3 is sequestered in its inactive conformation. Finally, we reconstitute Sgk3 activation via Vps34-mediated PI3P synthesis on phosphatidylinositol liposomes in vitro. In addition to identifying the mechanism of Sgk3 activation by PI3P, our findings open up potential therapeutic avenues in allosteric inhibitor development to target Sgk3 in cancer.

3.
PLoS One ; 15(12): e0242677, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-33296386

RESUMO

MORN (Membrane Occupation and Recognition Nexus) repeat proteins have a wide taxonomic distribution, being found in both prokaryotes and eukaryotes. Despite this ubiquity, they remain poorly characterised at both a structural and a functional level compared to other common repeats. In functional terms, they are often assumed to be lipid-binding modules that mediate membrane targeting. We addressed this putative activity by focusing on a protein composed solely of MORN repeats-Trypanosoma brucei MORN1. Surprisingly, no evidence for binding to membranes or lipid vesicles by TbMORN1 could be obtained either in vivo or in vitro. Conversely, TbMORN1 did interact with individual phospholipids. High- and low-resolution structures of the MORN1 protein from Trypanosoma brucei and homologous proteins from the parasites Toxoplasma gondii and Plasmodium falciparum were obtained using a combination of macromolecular crystallography, small-angle X-ray scattering, and electron microscopy. This enabled a first structure-based definition of the MORN repeat itself. Furthermore, all three structures dimerised via their C-termini in an antiparallel configuration. The dimers could form extended or V-shaped quaternary structures depending on the presence of specific interface residues. This work provides a new perspective on MORN repeats, showing that they are protein-protein interaction modules capable of mediating both dimerisation and oligomerisation.


Assuntos
Lipídeos/química , Proteínas de Protozoários/química , Sequências Repetitivas de Aminoácidos , Sequência de Aminoácidos , Membrana Celular/metabolismo , Cristalografia por Raios X , Citosol/metabolismo , Lipossomos , Fenótipo , Fosfolipídeos/metabolismo , Ligação Proteica , Multimerização Proteica , Proteínas de Protozoários/ultraestrutura , Proteínas Recombinantes/metabolismo , Trypanosoma brucei brucei/metabolismo
4.
Bioessays ; 42(4): e1900222, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-31997382

RESUMO

The recent discovery and structure determination of a novel ubiquitin-like dimerization domain in protein kinase D (PKD) has significant implications for its activation. PKD is a serine/threonine kinase activated by the lipid second messenger diacylglycerol (DAG). It is an essential and highly conserved protein that is implicated in plasma membrane directed trafficking processes from the trans-Golgi network. However, many open questions surround its mechanism of activation, its localization, and its role in the biogenesis of cargo transport carriers. In reviewing this field, the focus is primarily on the mechanisms that control the activation of PKD at precise locations in the cell. In light of the new structural findings, the understanding of the mechanisms underlying PKD activation is critically evaluated, with particular emphasis on the role of dimerization in PKD autophosphorylation, and the provenance and recognition of the DAG that activates PKD.


Assuntos
Diglicerídeos/metabolismo , Dimerização , Proteína Quinase C/química , Proteína Quinase C/metabolismo , Transdução de Sinais , Animais , Domínio Catalítico , Membrana Celular/metabolismo , Ativação Enzimática , Humanos , Fosforilação , Filogenia , Ligação Proteica , Domínios Proteicos , Proteína Quinase C/genética , Processamento de Proteína Pós-Traducional , Rede trans-Golgi/metabolismo
5.
J Biol Chem ; 294(39): 14422-14441, 2019 09 27.
Artigo em Inglês | MEDLINE | ID: mdl-31406020

RESUMO

Protein kinase D (PKD) is an essential Ser/Thr kinase in animals and controls a variety of diverse cellular functions, including vesicle trafficking and mitogenesis. PKD is activated by recruitment to membranes containing the lipid second messenger diacylglycerol (DAG) and subsequent phosphorylation of its activation loop. Here, we report the crystal structure of the PKD N terminus at 2.2 Å resolution containing a previously unannotated ubiquitin-like domain (ULD), which serves as a dimerization domain. A single point mutation in the dimerization interface of the ULD not only abrogated dimerization in cells but also prevented PKD activation loop phosphorylation upon DAG production. We further show that the kinase domain of PKD dimerizes in a concentration-dependent manner and autophosphorylates on a single residue in its activation loop. We also provide evidence that PKD is expressed at concentrations 2 orders of magnitude below the ULD dissociation constant in mammalian cells. We therefore propose a new model for PKD activation in which the production of DAG leads to the local accumulation of PKD at the membrane, which drives ULD-mediated dimerization and subsequent trans-autophosphorylation of the kinase domain.


Assuntos
Proteínas de Caenorhabditis elegans/química , Proteína Quinase C/química , Multimerização Proteica , Células 3T3 , Animais , Células COS , Caenorhabditis elegans , Proteínas de Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/metabolismo , Chlorocebus aethiops , Diglicerídeos/metabolismo , Células HEK293 , Humanos , Camundongos , Simulação de Acoplamento Molecular , Fosforilação , Mutação Puntual , Domínios Proteicos , Proteína Quinase C/genética , Proteína Quinase C/metabolismo , Transdução de Sinais
6.
Biochem Soc Trans ; 47(3): 897-908, 2019 06 28.
Artigo em Inglês | MEDLINE | ID: mdl-31147387

RESUMO

Akt is an essential protein kinase activated downstream of phosphoinositide 3-kinase and frequently hyperactivated in cancer. Canonically, Akt is activated by phosphoinositide-dependent kinase 1 and mechanistic target of rapamycin complex 2, which phosphorylate it on two regulatory residues in its kinase domain upon targeting of Akt to the plasma membrane by PI(3,4,5)P3 Recent evidence, however, has shown that, in addition to phosphorylation, Akt activity is allosterically coupled to the engagement of PI(3,4,5)P3 or PI(3,4)P2 in cellular membranes. Furthermore, the active membrane-bound conformation of Akt is protected from dephosphorylation, and Akt inactivation by phosphatases is rate-limited by its dissociation. Thus, Akt activity is restricted to membranes containing either PI(3,4,5)P3 or PI(3,4)P2 While PI(3,4,5)P3 has long been associated with signaling at the plasma membrane, PI(3,4)P2 is gaining increasing traction as a signaling lipid and has been implicated in controlling Akt activity throughout the endomembrane system. This has clear implications for the phosphorylation of both freely diffusible substrates and those localized to discrete subcellular compartments.


Assuntos
Metabolismo dos Lipídeos , Proteínas Proto-Oncogênicas c-akt/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Fosforilação , Transdução de Sinais
7.
Neuron ; 100(6): 1354-1368.e5, 2018 12 19.
Artigo em Inglês | MEDLINE | ID: mdl-30449657

RESUMO

Corpus callosum malformations are associated with a broad range of neurodevelopmental diseases. We report that de novo mutations in MAST1 cause mega-corpus-callosum syndrome with cerebellar hypoplasia and cortical malformations (MCC-CH-CM) in the absence of megalencephaly. We show that MAST1 is a microtubule-associated protein that is predominantly expressed in post-mitotic neurons and is present in both dendritic and axonal compartments. We further show that Mast1 null animals are phenotypically normal, whereas the deletion of a single amino acid (L278del) recapitulates the distinct neurological phenotype observed in patients. In animals harboring Mast1 microdeletions, we find that the PI3K/AKT3/mTOR pathway is unperturbed, whereas Mast2 and Mast3 levels are diminished, indicative of a dominant-negative mode of action. Finally, we report that de novo MAST1 substitutions are present in patients with autism and microcephaly, raising the prospect that mutations in this gene give rise to a spectrum of neurodevelopmental diseases.


Assuntos
Agenesia do Corpo Caloso/genética , Cerebelo/anormalidades , Regulação da Expressão Gênica no Desenvolvimento/genética , Malformações do Desenvolvimento Cortical/genética , Proteínas Associadas aos Microtúbulos/genética , Mutação/genética , Malformações do Sistema Nervoso/genética , Agenesia do Corpo Caloso/complicações , Agenesia do Corpo Caloso/diagnóstico por imagem , Agenesia do Corpo Caloso/patologia , Animais , Animais Recém-Nascidos , Apoptose/genética , Encéfalo/metabolismo , Encéfalo/patologia , Células Cultivadas , Cerebelo/diagnóstico por imagem , Criança , Deficiências do Desenvolvimento/complicações , Deficiências do Desenvolvimento/diagnóstico por imagem , Deficiências do Desenvolvimento/genética , Modelos Animais de Doenças , Embrião de Mamíferos , Feminino , Humanos , Masculino , Malformações do Desenvolvimento Cortical/complicações , Malformações do Desenvolvimento Cortical/diagnóstico por imagem , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteínas Associadas aos Microtúbulos/deficiência , Proteínas do Tecido Nervoso/metabolismo , Malformações do Sistema Nervoso/complicações , Malformações do Sistema Nervoso/diagnóstico por imagem , Fator de Transcrição PAX6/metabolismo
9.
Proc Natl Acad Sci U S A ; 115(17): E3940-E3949, 2018 04 24.
Artigo em Inglês | MEDLINE | ID: mdl-29632185

RESUMO

The protein kinase Akt controls myriad signaling processes in cells, ranging from growth and proliferation to differentiation and metabolism. Akt is activated by a combination of binding to the lipid second messenger PI(3,4,5)P3 and its subsequent phosphorylation by phosphoinositide-dependent kinase 1 and mechanistic target of rapamycin complex 2. The relative contributions of these mechanisms to Akt activity and signaling have hitherto not been understood. Here, we show that phosphorylation and activation by membrane binding are mutually interdependent. Moreover, the converse is also true: Akt is more rapidly dephosphorylated in the absence of PIP3, an autoinhibitory process driven by the interaction of its PH and kinase domains. We present biophysical evidence for the conformational changes in Akt that accompany its activation on membranes, show that Akt is robustly autoinhibited in the absence of PIP3 irrespective of its phosphorylation, and map the autoinhibitory PH-kinase interface. Finally, we present a model for the activation and inactivation of Akt by an ordered series of membrane binding, phosphorylation, dissociation, and dephosphorylation events.


Assuntos
Modelos Biológicos , Fosfatidilinositol 3-Quinases/metabolismo , Fosfatos de Fosfatidilinositol/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Ativação Enzimática , Humanos , Fosfatidilinositol 3-Quinases/química , Fosfatidilinositol 3-Quinases/genética , Fosfatos de Fosfatidilinositol/química , Fosfatos de Fosfatidilinositol/genética , Fosforilação , Domínios Proteicos , Proteínas Proto-Oncogênicas c-akt/química , Proteínas Proto-Oncogênicas c-akt/genética
10.
Sci Rep ; 7(1): 17405, 2017 12 12.
Artigo em Inglês | MEDLINE | ID: mdl-29234112

RESUMO

The Tec kinases, closely related to Src family kinases, are essential for lymphocyte function in the adaptive immune system. Whilst the Src and Abl kinases are regulated by tail phosphorylation and N-terminal myristoylation respectively, the Tec kinases are notable for the absence of either regulatory element. We have found that the inactive conformations of the Tec kinase Itk and Src preferentially bind ADP over ATP, stabilising both proteins. We demonstrate that Itk adopts the same conformation as Src and that the autoinhibited conformation of Src is independent of its C-terminal tail. Allosteric activation of both Itk and Src depends critically on the disruption of a conserved hydrophobic stack that accompanies regulatory domain displacement. We show that a conformational switch permits the exchange of ADP for ATP, leading to efficient autophosphorylation and full activation. In summary, we propose a universal mechanism for the activation and autoinhibition of the Src and Tec kinases.


Assuntos
Difosfato de Adenosina/metabolismo , Trifosfato de Adenosina/metabolismo , Proteínas Tirosina Quinases/metabolismo , Quinases da Família src/metabolismo , Regulação Alostérica , Animais , Humanos , Camundongos , Simulação de Dinâmica Molecular , Fosforilação/fisiologia , Mutação Puntual , Ligação Proteica , Conformação Proteica , Proteínas Tirosina Quinases/genética , Quinases da Família src/genética
11.
Mol Cell ; 65(3): 416-431.e6, 2017 Feb 02.
Artigo em Inglês | MEDLINE | ID: mdl-28157504

RESUMO

Protein kinase B/Akt regulates cellular metabolism, survival, and proliferation in response to hormones and growth factors. Hyperactivation of Akt is frequently observed in cancer, while Akt inactivation is associated with severe diabetes. Here, we investigated the molecular and cellular mechanisms that maintain Akt activity proportional to the activating stimulus. We show that binding of phosphatidylinositol-3,4,5-trisphosphate (PIP3) or PI(3,4)P2 to the PH domain allosterically activates Akt by promoting high-affinity substrate binding. Conversely, dissociation from PIP3 was rate limiting for Akt dephosphorylation, dependent on the presence of the PH domain. In cells, active Akt associated primarily with cellular membranes. In contrast, a transforming mutation that uncouples kinase activation from PIP3 resulted in the accumulation of hyperphosphorylated, active Akt in the cytosol. Our results suggest that intramolecular allosteric and cellular mechanisms cooperate to restrict Akt activity to cellular membranes, thereby enhancing the fidelity of Akt signaling and the specificity of downstream substrate phosphorylation.


Assuntos
Membrana Celular/metabolismo , Fosfatidilinositóis/metabolismo , Proteínas Proto-Oncogênicas c-akt/química , Proteínas Proto-Oncogênicas c-akt/metabolismo , Regulação Alostérica , Sítios de Ligação , Regulação da Expressão Gênica , Células HeLa , Humanos , Células MCF-7 , Mutação , Fosforilação , Ligação Proteica , Proteínas Proto-Oncogênicas c-akt/genética , Especificidade por Substrato
12.
Bioessays ; 38(9): 903-16, 2016 09.
Artigo em Inglês | MEDLINE | ID: mdl-27492088

RESUMO

Coiled-coils are found in proteins throughout all three kingdoms of life. Coiled-coil domains of some proteins are almost invariant in sequence and length, betraying a structural and functional role for amino acids along the entire length of the coiled-coil. Other coiled-coils are divergent in sequence, but conserved in length, thereby functioning as molecular spacers. In this capacity, coiled-coil proteins influence the architecture of organelles such as centrioles and the Golgi, as well as permit the tethering of transport vesicles. Specialized coiled-coils, such as those found in motor proteins, are capable of propagating conformational changes along their length that regulate cargo binding and motor processivity. Coiled-coil domains have also been identified in enzymes, where they function as molecular rulers, positioning catalytic activities at fixed distances. Finally, while coiled-coils have been extensively discussed for their potential to nucleate and scaffold large macromolecular complexes, structural evidence to substantiate this claim is relatively scarce.


Assuntos
Conformação Proteica , Proteínas/metabolismo , Animais , Bactérias/metabolismo , Eucariotos/metabolismo , Humanos
13.
Small GTPases ; 7(2): 82-92, 2016 04 02.
Artigo em Inglês | MEDLINE | ID: mdl-27070834

RESUMO

The Rho-associated coiled-coil containing kinases (ROCK) were first identified as effectors of the small GTPase RhoA, hence their nomenclature. Since their discovery, two decades ago, scientists have sought to unravel the structure, regulation, and function of these essential kinases. During that time, a consensus model has formed, in which ROCK activity is regulated via both Rho-dependent and independent mechanisms. However, recent findings have raised significant questions regarding this model. In their recent publication in Nature Communications, Truebestein and colleagues present the structure of a full-length Rho kinase for the first time. In contrast to previous reports, the authors could find no evidence for autoinhibition, RhoA binding, or regulation of kinase activity by phosphorylation. Instead, they propose that ROCK functions as a molecular ruler, in which the central coiled-coil bridges the membrane-binding regulatory domains to the kinase domains at a fixed distance from the plasma membrane. Here, we explore the consequences of the new findings, re-examine old data in the context of this model, and emphasize outstanding questions in the field.


Assuntos
Quinases Associadas a rho/metabolismo , Animais , Apoptose , Humanos , Fosforilação , Transporte Proteico
14.
J Mol Biol ; 428(1): 121-141, 2016 Jan 16.
Artigo em Inglês | MEDLINE | ID: mdl-26582574

RESUMO

Diacylglycerol (DAG) activates the eight conventional and novel isozymes of protein kinase C (PKC) by binding to their C1 domains. The crystal structure of PKCßII in a partially activated conformation showed how the C1B domain regulates activity by clamping a helix in the C-terminal AGC extension of the kinase domain. Here we show that the global three-dimensional shape of the conventional and novel PKCs is conserved despite differences in the order of the domains in their primary sequences. The membrane translocation phenotypes of mutants in the C1B clamp are consistent across all DAG-activated PKCs, demonstrating conservation of this regulatory interface. We now identify a novel interface that sequesters the C1A domain in PKCßII in a membrane-inaccessible state and we generalize this to all DAG-activated PKCs. In the conventional PKCs, we identify a novel element of their C2 domains that additionally contributes to the stability of the inactive conformation. We demonstrate that the interdomain linkers play important roles in permitting and stabilizing this state. We propose a multi-step activation mechanism in which the sequential and cooperative binding of the regulatory domains to the membrane is coupled to allosteric activation of the kinase domain by DAG and that acquisition of full catalytic activity requires DAG binding to the C1B domain. In light of the conservation of shape and intramolecular architecture, we propose that this mechanism is common to all DAG-activated PKCs.


Assuntos
Diglicerídeos/metabolismo , Ativadores de Enzimas/metabolismo , Isoenzimas/química , Isoenzimas/metabolismo , Proteína Quinase C/química , Proteína Quinase C/metabolismo , Animais , Humanos , Camundongos , Modelos Moleculares , Conformação Proteica , Ratos , Espalhamento a Baixo Ângulo
15.
Nat Commun ; 6: 10029, 2015 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-26620183

RESUMO

The Rho-associated coiled-coil kinases (ROCK) are essential regulators of the actin cytoskeleton; however, the structure of a full-length ROCK is unknown and the mechanisms by which its kinase activity is controlled are not well understood. Here we determine the low-resolution structure of human ROCK2 using electron microscopy, revealing it to be a constitutive dimer, 120 nm in length, with a long coiled-coil tether linking the kinase and membrane-binding domains. We find, in contrast to previous reports, that ROCK2 activity does not appear to be directly regulated by binding to membranes, RhoA, or by phosphorylation. Instead, we show that changing the length of the tether modulates ROCK2 function in cells, suggesting that it acts as a molecular ruler. We present a model in which ROCK activity is restricted to a discrete region of the actin cytoskeleton, governed by the length of its coiled-coil. This represents a new type of spatial control, and hence a new paradigm for kinase regulation.


Assuntos
Citoesqueleto/enzimologia , Quinases Associadas a rho/metabolismo , Actinas/metabolismo , Citoesqueleto/genética , Citoesqueleto/metabolismo , Dimerização , Humanos , Estrutura Terciária de Proteína , Quinases Associadas a rho/química , Quinases Associadas a rho/genética , Proteína rhoA de Ligação ao GTP/genética , Proteína rhoA de Ligação ao GTP/metabolismo
16.
Nat Commun ; 4: 1849, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-23673634

RESUMO

Chimaerins, a family of GTPase activating proteins for the small G-protein Rac, have been implicated in development, neuritogenesis and cancer. These Rac-GTPase activating proteins are regulated by the lipid second messenger diacylglycerol generated by tyrosine kinases such as the epidermal growth factor receptor. Here we identify an atypical proline-rich motif in chimaerins that binds to the adaptor protein Nck1. Unlike most Nck1 partners, chimaerins bind to the third SH3 domain of Nck1. This association is mediated by electrostatic interactions of basic residues within the Pro-rich motif with acidic clusters in the SH3 domain. Epidermal growth factor promotes the binding of ß2-chimaerin to Nck1 in the cell periphery in a diacylglycerol-dependent manner. Moreover, ß2-chimaerin translocation to the plasma membrane and its peripheral association with Rac1 requires Nck1. Our studies underscore a coordinated mechanism for ß2-chimaerin activation that involves lipid interactions via the C1 domain and protein-protein interactions via the N-terminal proline-rich region.


Assuntos
Diglicerídeos/metabolismo , Proteínas Ativadoras de GTPase/metabolismo , Proteínas de Neoplasias/química , Proteínas de Neoplasias/metabolismo , Domínios Proteicos Ricos em Prolina , Proteínas rac de Ligação ao GTP/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/química , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Sequência de Aminoácidos , Aminoácidos/metabolismo , Animais , Células COS , Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Chlorocebus aethiops , Fator de Crescimento Epidérmico/farmacologia , Proteínas Ativadoras de GTPase/química , Células HeLa , Humanos , Modelos Moleculares , Dados de Sequência Molecular , Proteínas Oncogênicas/química , Proteínas Oncogênicas/metabolismo , Ligação Proteica/efeitos dos fármacos , Transporte Proteico/efeitos dos fármacos , Relação Estrutura-Atividade , Vanadatos/farmacologia , Proteínas rac de Ligação ao GTP/química , Domínios de Homologia de src
17.
FEBS Lett ; 587(11): 1610-6, 2013 Jun 05.
Artigo em Inglês | MEDLINE | ID: mdl-23603387

RESUMO

Sec14 family homologs are the major yeast phosphatidylinositol/phosphatidylcholine transfer proteins regulating lipid metabolism and vesicle trafficking. The structure of Saccharomyces cerevisiae Sfh3 displays a conserved Sec14 scaffold and reveals determinants for the specific recognition of phosphatidylinositol ligand. Apo-Sfh3 forms a dimer through the hydrophobic interaction of gating helices. Binding of phosphatidylinositol leads to dissociation of the dimer into monomers in a reversible manner. This study suggests that the substrate induced dimer-monomer transformation is an essential part of lipid transfer cycles by Sfh3.


Assuntos
Fosfatidilinositóis/química , Proteínas de Transferência de Fosfolipídeos/química , Proteínas de Saccharomyces cerevisiae/química , Saccharomyces cerevisiae , Sequência de Aminoácidos , Cristalografia por Raios X , Ligação de Hidrogênio , Interações Hidrofóbicas e Hidrofílicas , Modelos Moleculares , Dados de Sequência Molecular , Ligação Proteica , Multimerização Proteica , Estrutura Quaternária de Proteína , Estrutura Secundária de Proteína , Especificidade por Substrato
18.
Curr Opin Struct Biol ; 21(6): 785-91, 2011 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-22142590

RESUMO

Membranes are sites of intense signaling activity within the cell, serving as dynamic scaffolds for the recruitment of signaling molecules and their substrates. The specific and reversible localization of these signaling molecules to membranes is critical for the appropriate activation of downstream signaling pathways. Phospholipid-binding domains, including C1, C2, PH, and PX domains, play critical roles in the membrane targeting of protein kinases. Recent structural studies have identified a new membrane association domain, the Kinase Associated 1 (KA1) domain, which targets a number of yeast and mammalian protein kinases to membranes containing acidic phospholipids. Despite an abundance of localization studies on lipid-binding proteins and structural studies of the isolated lipid-binding domains, the question of how membrane binding is coupled to the activation of the kinase catalytic domain has been virtually untouched. Recently, structural studies on protein kinase C (PKC) have provided some of the first structural insights into the allosteric regulation of protein kinases by lipid second messengers.


Assuntos
Fosfolipídeos/metabolismo , Proteínas Quinases/metabolismo , Regulação Alostérica , Animais , Sítios de Ligação , Humanos , Conformação Proteica , Proteína Quinase C/química , Proteína Quinase C/metabolismo , Proteínas Quinases/química , Transdução de Sinais
19.
Cell ; 144(1): 55-66, 2011 Jan 07.
Artigo em Inglês | MEDLINE | ID: mdl-21215369

RESUMO

Protein kinase C (PKC) isozymes are the paradigmatic effectors of lipid signaling. PKCs translocate to cell membranes and are allosterically activated upon binding of the lipid diacylglycerol to their C1A and C1B domains. The crystal structure of full-length protein kinase C ßII was determined at 4.0 Å, revealing the conformation of an unexpected intermediate in the activation pathway. Here, the kinase active site is accessible to substrate, yet the conformation of the active site corresponds to a low-activity state because the ATP-binding side chain of Phe629 of the conserved NFD motif is displaced. The C1B domain clamps the NFD helix in a low-activity conformation, which is reversed upon membrane binding. A low-resolution solution structure of the closed conformation of PKCßII was derived from small-angle X-ray scattering. Together, these results show how PKCßII is allosterically regulated in two steps, with the second step defining a novel protein kinase regulatory mechanism.


Assuntos
Proteína Quinase C/química , Regulação Alostérica , Sequência de Aminoácidos , Animais , Catálise , Ativação Enzimática , Humanos , Modelos Moleculares , Dados de Sequência Molecular , Mutação , Proteína Quinase C/genética , Proteína Quinase C/metabolismo , Proteína Quinase C beta , Ratos , Espalhamento a Baixo Ângulo , Alinhamento de Sequência , Difração de Raios X
20.
EMBO J ; 29(12): 1988-2001, 2010 Jun 16.
Artigo em Inglês | MEDLINE | ID: mdl-20502438

RESUMO

DivIVA is a conserved protein in Gram-positive bacteria that localizes at the poles and division sites, presumably through direct sensing of membrane curvature. DivIVA functions as a scaffold and is vital for septum site selection during vegetative growth and chromosome anchoring during sporulation. DivIVA deletion causes filamentous growth in Bacillus subtilis, whereas overexpression causes hyphal branching in Streptomyces coelicolor. We have determined the crystal structure of the N-terminal (Nt) domain of DivIVA, and show that it forms a parallel coiled-coil. It is capped with two unique crossed and intertwined loops, exposing hydrophobic and positively charged residues that we show here are essential for membrane binding. An intragenic suppressor introducing a positive charge restores membrane binding after mutating the hydrophobic residues. We propose that the hydrophobic residues insert into the membrane and that the positively charged residues bind to the membrane surface. A low-resolution crystal structure of the C-terminal (Ct) domain displays a curved tetramer made from two parallel coiled-coils. The Nt and Ct parts were then merged into a model of the full length, 30 nm long DivIVA protein.


Assuntos
Bacillus subtilis/química , Bacillus subtilis/fisiologia , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Proteínas de Ciclo Celular/química , Proteínas de Ciclo Celular/metabolismo , Membrana Celular/metabolismo , Sequência de Aminoácidos , Cristalografia por Raios X , Modelos Moleculares , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Ligação Proteica , Estrutura Terciária de Proteína , Alinhamento de Sequência , Supressão Genética
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