Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 10 de 10
Filtrar
Mais filtros










Base de dados
Intervalo de ano de publicação
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.
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
4.
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
5.
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
6.
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
7.
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
8.
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
9.
J Biol Chem ; 287(25): 20931-41, 2012 Jun 15.
Artigo em Inglês | MEDLINE | ID: mdl-22535953

RESUMO

Protective proteases are key elements of protein quality control pathways that are up-regulated, for example, under various protein folding stresses. These proteases are employed to prevent the accumulation and aggregation of misfolded proteins that can impose severe damage to cells. The high temperature requirement A (HtrA) family of serine proteases has evolved to perform important aspects of ATP-independent protein quality control. So far, however, no HtrA protease is known that degrades protein aggregates. We show here that human HTRA1 degrades aggregated and fibrillar tau, a protein that is critically involved in various neurological disorders. Neuronal cells and patient brains accumulate less tau, neurofibrillary tangles, and neuritic plaques, respectively, when HTRA1 is expressed at elevated levels. Furthermore, HTRA1 mRNA and HTRA1 activity are up-regulated in response to elevated tau concentrations. These data suggest that HTRA1 is performing regulated proteolysis during protein quality control, the implications of which are discussed.


Assuntos
Proteínas do Tecido Nervoso/química , Dobramento de Proteína , Proteólise , Serina Endopeptidases/química , Proteínas tau/química , Encéfalo/metabolismo , Encéfalo/patologia , Regulação Enzimológica da Expressão Gênica , Serina Peptidase 1 de Requerimento de Alta Temperatura A , Humanos , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Neuritos/enzimologia , Neuritos/patologia , Serina Endopeptidases/genética , Serina Endopeptidases/metabolismo , Tauopatias/enzimologia , Tauopatias/patologia , Proteínas tau/genética , Proteínas tau/metabolismo
10.
Nat Struct Mol Biol ; 18(3): 386-8, 2011 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-21297635

RESUMO

Crystal structures of active and inactive conformations of the human serine protease HTRA1 reveal that substrate binding to the active site is sufficient to stimulate proteolytic activity. HTRA1 attaches to liposomes, digests misfolded proteins into defined fragments and undergoes substrate-mediated oligomer conversion. In contrast to those of other serine proteases, the PDZ domain of HTRA1 is dispensable for activation or lipid attachment, indicative of different underlying mechanistic features.


Assuntos
Serina Endopeptidases/metabolismo , Domínio Catalítico , Cristalografia por Raios X , Serina Peptidase 1 de Requerimento de Alta Temperatura A , Humanos , Hidrólise , Modelos Moleculares , Domínios PDZ , Ligação Proteica , Dobramento de Proteína , Serina Endopeptidases/química
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA
...