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1.
Int J Mol Sci ; 21(1)2019 Dec 25.
Artigo em Inglês | MEDLINE | ID: mdl-31881809

RESUMO

The spleen tyrosine kinase (Syk) is essential for immunoreceptor tyrosine-based activation motif (ITAM)-dependent platelet activation, and it is stimulated by Src-family kinase (SFK)-/Syk-mediated phosphorylation of Y352 (interdomain-B) and Y525/526 (kinase domain). Additional sites for Syk phosphorylation and protein interactions are known but remain elusive. Since Syk S297 phosphorylation (interdomain-B) was detected in platelets, we hypothesized that this phosphorylation site regulates Syk activity via protein kinase C (PKC)-and cyclic adenosine monophosphate (cAMP)-dependent pathways. ADP, the GPVI-agonist convulxin, and the GPIbα-agonist echicetin beads (EB) were used to stimulate human platelets with/without effectors. Platelet aggregation and intracellular messengers were analyzed, along with phosphoproteins, by immunoblotting using phosphosite-specific antibodies or phos-tags. ADP, convulxin, and EB upregulated Syk S297 phosphorylation, which was inhibited by iloprost (cAMP pathway). Convulxin-stimulated Syk S297 phosphorylation was stoichiometric, transient, abolished by the PKC inhibitor GF109203X, and mimicked by the PKC activator PDBu. Convulxin/EB stimulated Syk S297, Y352, and Y525/526 phosphorylation, which was inhibited by SFK and Syk inhibitors. GFX and iloprost inhibited convulxin/EB-induced Syk S297 phosphorylation but enhanced Syk tyrosine (Y352/Y525/526) and substrate (linker adaptor for T cells (LAT), phospholipase γ2 (PLC γ2)) phosphorylation. GFX enhanced convulxin/EB-increases of inositol monophosphate/Ca2+. ITAM-activated Syk stimulates PKC-dependent Syk S297 phosphorylation, which is reduced by SFK/Syk/PKC inhibition and cAMP. Inhibition of Syk S297 phosphorylation coincides with enhanced Syk activation, suggesting that S297 phosphorylation represents a mechanism for feedback inhibition in human platelets.


Assuntos
Plaquetas/metabolismo , Proteína Quinase C/metabolismo , Quinase Syk/metabolismo , Difosfato de Adenosina/farmacologia , Plaquetas/citologia , Cálcio/metabolismo , Venenos de Crotalídeos/farmacologia , Retroalimentação Fisiológica/efeitos dos fármacos , Humanos , Indóis/farmacologia , Lectinas Tipo C , Maleimidas/farmacologia , Fosfolipase C gama/metabolismo , Fosforilação/efeitos dos fármacos , Agregação Plaquetária/efeitos dos fármacos , Proteína Quinase C/antagonistas & inibidores , Proteína Quinase C/química , Quinase Syk/antagonistas & inibidores , Venenos de Víboras/farmacologia
2.
Phytomedicine ; 65: 153100, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31648127

RESUMO

BACKGROUND: The incidence of non-small cell lung cancer (NSCLC) accounts for approximately 85-90% of lung cancer, which has been shown to be challenging for treatment owing to poorly understanding of pathological mechanisms. Natural products serve as a source of almost all pharmaceutical preparations or offer guidance for those chemicals that have entered clinical trials, especially in NSCLC. PURPOSE: We investigated the effect of B10G5, a natural products isolated from the Croton tiglium, in human non-small cell lung canceras as a protein kinase C (PKC) activator. METHODS: The cell viability assay was evaluated by the MTT assay. The apoptosis and cell cycle distribution were assessed by flow cytometry. Reactive oxygen species (ROS) production was determined by using the fluorescent probe DCFDA. Cell migration ability of H1975 cells was analyzed by using the wound healing assay. The inhibiting effect of B10G5 against the phosphorylation level of the substrate by PKCs was assessed by using homogeneous time-resolved fluorescence (HTRF) technology. The correlation between PKCs and overall survival (OS) of Lung Adenocarcinoma (LUAD) patients was analysis by TCGA portal. The binding mode between B10G5 and the PKC isoforms was explored by molecular docking. Protein expression was detected by western blotting analysis. RESULTS: B10G5 suppressed cell proliferation and colony formation, as well as migration ability of NSCLC cells, without significant toxic effect on normal lung cells. B10G5 induced the cell apoptosis through the development of PARP cleavage, which is evidenced by means of the production of mitochondrial ROS. In addition, the B10G5 inhibitory effect was also related to the cell cycle arrest at G2/M phase. Mechanistically, molecular modelling technology suggested that the potential target of B10G5 was associated with PKC family. In vitro PKC kinase assay indicated that B10G5 effectively activated the PKC activity. Western blotting data revealed that B10G5 upregulated PKC to activate PKC-mediated RAF/MEK/ERK pathway. CONCLUSION: Our results showed that B10G5, a naturally occurring phorbol ester, considered to be a potential and a valuable therapeutic chemical in the treatment of NSCLC.


Assuntos
Antineoplásicos Fitogênicos/farmacologia , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Croton/química , Ativadores de Enzimas/farmacologia , Neoplasias Pulmonares/tratamento farmacológico , Proteína Quinase C/metabolismo , Antineoplásicos Fitogênicos/química , Apoptose/efeitos dos fármacos , Carcinoma Pulmonar de Células não Pequenas/patologia , Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Ensaios de Seleção de Medicamentos Antitumorais , Ativadores de Enzimas/química , Humanos , Neoplasias Pulmonares/patologia , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Simulação de Acoplamento Molecular , Fosforilação/efeitos dos fármacos , Proteína Quinase C/química , Espécies Reativas de Oxigênio/metabolismo , Transdução de Sinais/efeitos dos fármacos
3.
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
4.
Int J Mol Sci ; 20(13)2019 Jul 05.
Artigo em Inglês | MEDLINE | ID: mdl-31284391

RESUMO

Self-incompatibility (SI) is a widespread mechanism in angiosperms that prevents inbreeding by rejecting self-pollen. However, the regulation of the SI response in Brassica napus is not well understood. Here, we report that the M-locus protein kinase (MLPK) BnaMLPKs, the functional homolog of BrMLPKs in Brassica rapa, controls SI in B. napus. We identified four paralogue MLPK genes in B. napus, including BnaA3.MLPK, BnaC3.MLPK, BnaA4.MLPK, and BnaC4.MLPK. Two transcripts of BnaA3.MLPK, BnaA3.MLPKf1 and BnaA3.MLPKf2, were generated by alternative splicing. Tissue expression pattern analysis demonstrated that BnaA3.MLPK, especially BnaA3.MLPKf2, is highly expressed in reproductive organs, particularly in stigmas. We subsequently created RNA-silencing lines and CRISPR/Cas9-induced quadruple mutants of BnaMLPKs in B. napus SI line S-70. Phenotypic analysis revealed that SI response is partially suppressed in RNA-silencing lines and is completely blocked in quadruple mutants. These results indicate the importance of BnaMLPKs in regulating the SI response of B. napus. We found that the expression of SI positive regulators S-locus receptor kinase (SRK) and Arm-Repeat Containing 1 (ARC1) are suppressed in bnmlpk mutant, whereas the self-compatibility (SC) element Glyoxalase I (GLO1) maintained a high expression level. Overall, our findings reveal a new regulatory mechanism of MLPK in the SI of B. napus.


Assuntos
Brassica napus/enzimologia , Brassica napus/fisiologia , Proteína Quinase C/metabolismo , Autoincompatibilidade em Angiospermas/fisiologia , Sequência de Aminoácidos , Sequência de Bases , Brassica napus/genética , Sistemas CRISPR-Cas/genética , Edição de Genes , Regulação da Expressão Gênica de Plantas , Mutação/genética , Especificidade de Órgãos/genética , Filogenia , Plantas Geneticamente Modificadas , Polinização , Isoformas de Proteínas/química , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Proteína Quinase C/química , Proteína Quinase C/genética , Interferência de RNA , RNA Mensageiro/genética , RNA Mensageiro/metabolismo
5.
Int J Mol Sci ; 20(14)2019 Jul 19.
Artigo em Inglês | MEDLINE | ID: mdl-31331067

RESUMO

Neurodegenerative diseases are among the leading causes of mortality and disability worldwide. However, current therapeutic approaches have failed to reach significant results in their prevention and cure. Protein Kinase Cs (PKCs) are kinases involved in the pathophysiology of neurodegenerative diseases, such as Alzheimer's Disease (AD) and cerebral ischemia. Specifically ε, δ, and γPKC are associated with the endogenous mechanism of protection referred to as ischemic preconditioning (IPC). Existing modulators of PKCs, in particular of εPKC, such as ψεReceptor for Activated C-Kinase (ψεRACK) and Resveratrol, have been proposed as a potential therapeutic strategy for cerebrovascular and cognitive diseases. PKCs change in expression during aging, which likely suggests their association with IPC-induced reduction against ischemia and increase of neuronal loss occurring in senescent brain. This review describes the link between PKCs and cerebrovascular and cognitive disorders, and proposes PKCs modulators as innovative candidates for their treatment. We report original data showing εPKC reduction in levels and activity in the hippocampus of old compared to young rats and a reduction in the levels of δPKC and γPKC in old hippocampus, without a change in their activity. These data, integrated with other findings discussed in this review, demonstrate that PKCs modulators may have potential to restore age-related reduction of endogenous mechanisms of protection against neurodegeneration.


Assuntos
Encéfalo/metabolismo , Neuroproteção , Proteína Quinase C/metabolismo , Fatores Etários , Envelhecimento/metabolismo , Animais , Biomarcadores , Suscetibilidade a Doenças , Desenvolvimento de Medicamentos , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Terapia de Alvo Molecular , Doenças do Sistema Nervoso/diagnóstico , Doenças do Sistema Nervoso/tratamento farmacológico , Doenças do Sistema Nervoso/etiologia , Doenças do Sistema Nervoso/metabolismo , Proteína Quinase C/química , Proteína Quinase C/genética , Transdução de Sinais/efeitos dos fármacos
6.
J Biosci ; 44(2)2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-31180068

RESUMO

Laminins are a major constituent of the extracellular matrix (ECM). Laminin-111, the most extensively studied laminin isoform, consists of the α1, the ß1 and the γ1 chain, and is involved in many cellular processes, like adhesion, migration and differentiation. Given the regulatory role of phosphorylation in protein function, it is important to identify the phosphorylation sites of human laminin ß1-chain sequence (LAMB1). Therefore, we computationally predicted all possible phosphorylation sites in LAMB1. For the first time, we identified the possibly responsible kinases for already in vitro experimentally observed phosphorylated residues in LAMB1. All known functional (active) sites of LAMB1, were recorded after an extensive literature search and combined with the experimentally observed and our predicted phosphorylated residues. This generated a detailed phosphorylation map of LAMB1. Five kinases (PKA, PKC, CKII, CKI and GPCR1) were indicated important, while the role of PKA, PKC and CKII, kinases known for ectophosphorylation activity, was highlighted. The activity of PKA and PKC was associated with the active site RIQNLLKITNLRIKFVKLHTLGDNLLDS. Also, predicted phosphorylations inside two amyloidogenic (DSITKYFQMSLE, VILQHSAADIAR) and two anti-cancerous (YIGSR and PDSGR) sites suggested a possible role in the development of the corresponding diseases.


Assuntos
Biologia Computacional/métodos , Laminina/química , Mapeamento de Peptídeos/métodos , Processamento de Proteína Pós-Traducional , Sequência de Aminoácidos , Caseína Quinase I/química , Caseína Quinase I/metabolismo , Caseína Quinase II/química , Caseína Quinase II/metabolismo , Domínio Catalítico , Proteínas Quinases Dependentes de AMP Cíclico/química , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Receptor Quinase 1 Acoplada a Proteína G/química , Receptor Quinase 1 Acoplada a Proteína G/metabolismo , Expressão Gênica , Humanos , Laminina/genética , Laminina/metabolismo , Fosforilação , Proteína Quinase C/química , Proteína Quinase C/metabolismo
7.
Proc Natl Acad Sci U S A ; 116(22): 10804-10812, 2019 05 28.
Artigo em Inglês | MEDLINE | ID: mdl-31088962

RESUMO

Metazoan cell polarity is controlled by a set of highly conserved proteins. Lethal giant larvae (Lgl) functions in apical-basal polarity through phosphorylation-dependent interactions with several other proteins as well as the plasma membrane. Phosphorylation of Lgl by atypical protein kinase C (aPKC), a component of the partitioning-defective (Par) complex in epithelial cells, excludes Lgl from the apical membrane, a crucial step in the establishment of epithelial cell polarity. We present the crystal structures of human Lgl2 in both its unphosphorylated and aPKC-phosphorylated states. Lgl2 adopts a double ß-propeller structure that is unchanged by aPKC phosphorylation of an unstructured loop in its second ß-propeller, ruling out models of phosphorylation-dependent conformational change. We demonstrate that phosphorylation controls the direct binding of purified Lgl2 to negative phospholipids in vitro. We also show that a coil-helix transition of this region that is promoted by phosphatidylinositol 4,5-bisphosphate (PIP2) is also phosphorylation-dependent, implying a highly effective phosphorylative switch for membrane association.


Assuntos
Polaridade Celular/fisiologia , Proteínas do Citoesqueleto , Proteína Quinase C , Proteínas do Citoesqueleto/química , Proteínas do Citoesqueleto/metabolismo , Humanos , Modelos Moleculares , Fosfatidilinositol 4,5-Difosfato , Fosforilação , Proteína Quinase C/química , Proteína Quinase C/metabolismo
8.
Int J Mol Sci ; 20(9)2019 Apr 29.
Artigo em Inglês | MEDLINE | ID: mdl-31035721

RESUMO

Protein kinase Mζ is considered important for memory formation and maintenance in different species, including invertebrates. PKMζ participates in multiple molecular pathways in neurons, regulating translation initiation rate, AMPA receptors turnover, synaptic scaffolding assembly, and other processes. Here, for the first time, we established the sequence of mRNA encoding PKMζ homolog in land snail Helix lucorum. We annotated important features of this mRNA: domains, putative capping sites, translation starts, and splicing sites. We discovered that this mRNA has at least two isoforms, and one of them lacks sequence encoding C1 domain. C1 deletion may be unique for snail because it has not been previously found in other species. We performed behavioral experiments with snails, measured expression levels of identified isoforms, and confirmed that their expression correlates with one type of learning.


Assuntos
Aprendizagem , Proteína Quinase C/metabolismo , Fatores de Transcrição da Família Snail/metabolismo , Processamento Alternativo , Sequência de Aminoácidos , Isoenzimas , Modelos Biológicos , Família Multigênica , Domínios e Motivos de Interação entre Proteínas , Proteína Quinase C/química , Proteína Quinase C/genética , Sítios de Splice de RNA , Relação Estrutura-Atividade , Transcrição Genética
9.
Sci Adv ; 5(3): eaav0482, 2019 03.
Artigo em Inglês | MEDLINE | ID: mdl-30906862

RESUMO

The question vis-à-vis the chemistry of phosphoryl group transfer catalyzed by protein kinases remains a major challenge. The neutron diffraction structure of the catalytic subunit of cAMP-dependent protein kinase (PKA-C) provides a more complete chemical portrait of key proton interactions at the active site. By using a high-affinity protein kinase substrate (PKS) peptide, we captured the reaction products, dephosphorylated nucleotide [adenosine diphosphate (ADP)] and phosphorylated PKS (pPKS), bound at the active site. In the complex, the phosphoryl group of the peptide is protonated, whereas the carboxyl group of the catalytic Asp166 is not. Our structure, including conserved waters, shows how the peptide links the distal parts of the cleft together, creating a network that engages the entire molecule. By comparing slow-exchanging backbone amides to those determined by the NMR analysis of PKA-C with ADP and inhibitor peptide (PKI), we identified exchangeable amides that likely distinguish catalytic and inhibited states.


Assuntos
Proteínas Quinases Dependentes de AMP Cíclico/química , Nêutrons , Peptídeos/química , Proteína Quinase C/química , Difosfato de Adenosina/química , Amidas/química , Sequência de Aminoácidos , Animais , Sítios de Ligação , Catálise , Domínio Catalítico/genética , Cristalografia por Raios X , Espectroscopia de Ressonância Magnética , Camundongos , Modelos Moleculares , Nucleotídeos/química , Fosforilação , Conformação Proteica , Prótons , Especificidade por Substrato , Água/química
10.
Fish Shellfish Immunol ; 87: 590-599, 2019 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-30738864

RESUMO

Complementary (c)DNA encoding novel protein kinase C (PKC) messenger (m)RNA of the white shrimp Litopenaeus vannamei, consisted of 2454-bp cDNA containing an open reading frame (ORF) of 2232 bp, belonging to the novel (n)PKC family of proteins characterized by their containing two phorbol ester/diacylglycerol-binding domains (C1 domain), a C2 domain, and a catalytic domain of the serine/threonine kinase, designated LvnPKC. A comparison of amino acid sequences showed that LvnPKC was closely related to arthropod nPKC. LvnPKC cDNA was detected in all tested tissues with a real-time PCR including the hepatopancreas, gills, muscles, subcuticular epithelium, abdominal nerve, thoracic nerve, brain, the stomach, heart, and especially in hemocytes and the intestines. Moreover, significantly upregulated LvnPKC expression was only observed in the eyestalk, brain, and hepatopancreas of shrimp transferred from 28 °C to 18 °C for 30 min. Induction of LvnPKC expression in hemocytes of L. vannamei injected with Vibrio alginolyticus at 105 cfu shrimp-1 was detected earlier than in those injected with 103 cfu shrimp-1. Shrimp received LvnPKC-dsRNA for 1 days specifically depleted the expression of LvnPKC mRNA in hemocytes compared those of diethylpyrocarbonate water treatment. After that, significantly decreased expressions of lipopolysaccharide - and ß-1,3-glucan-binding protein, prophenoloxidase-activating enzyme, peroxinectin, prophenoloxidase I, and prophenoloxidase II in the prophenoloxidase-activating system; lysozyme and cytosolic manganese superoxide dismutase and mitochondrial manganese superoxide dismutase in the antioxidant system were observed. We therefore concluded that LvnPKC is involved in immune defense of L. vannamei exposed to hypothermal stress or infected with V. alginolyticus.


Assuntos
Regulação da Expressão Gênica/imunologia , Imunidade Inata/genética , Penaeidae/genética , Penaeidae/imunologia , Proteína Quinase C/genética , Proteína Quinase C/imunologia , Sequência de Aminoácidos , Animais , Proteínas de Artrópodes/química , Proteínas de Artrópodes/genética , Proteínas de Artrópodes/imunologia , Sequência de Bases , Perfilação da Expressão Gênica , Imunocompetência , Filogenia , Proteína Quinase C/química , Vibrio alginolyticus/fisiologia
11.
IUBMB Life ; 71(6): 697-705, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-30393952

RESUMO

Protein kinase C (PKC) is activated by 1,2-diacylglycerol as a second messenger in the signaling mechanism coupled with the hydrolysis of membrane inositol phospholipids, although it was not found by screening for a 1,2-diacylglycerol-dependent enzyme. PKC is also a receptor for the tumor-promoting phorbol esters, but it was not identified by its property of binding phorbol esters, either. Instead, the discovery and characterization of PKC, now known to comprise a family with multiple isoforms, was through a circuitous voyage filled with unexpected twists and turns. This review summarizes the discovery and the initial experiments of PKC as a historical perspective of the enzyme family in the context of the progress in the studies on protein phosphorylation. © 2018 IUBMB Life, 71(6):697-705, 2019.


Assuntos
Fosfatidilinositóis/metabolismo , Proteína Quinase C/genética , Proteínas/química , Diglicerídeos/química , Diglicerídeos/metabolismo , Humanos , Hidrólise , Ésteres de Forbol/metabolismo , Fosforilação/genética , Ligação Proteica/genética , Proteína Quinase C/química , Proteínas/genética
12.
Fungal Genet Biol ; 122: 21-30, 2019 01.
Artigo em Inglês | MEDLINE | ID: mdl-30391723

RESUMO

The Aspergillus nidulans orthologue of Protein kinase C (PkcA) and the A. nidulans formin SepA participate in polarized growth. PkcA localizes to growing hyphal apices and septation sites, and amino acid sequences within PkcA that are required for PkcA to localize to these sites of cell wall synthesis have been identified. SepA is associated with the contractile actomyosin ring (CAR), and it localizes at hyphal tips in association with the Spitzenkörper (SPK) and as an apical dome. A mutation in the sepA gene (sepA1) renders A. nidulans aseptate at elevated temperature. Progress towards understanding the spatiotemporal relationship between PkcA and SepA during polarized growth is presented here. Fluorescent chimeras of PkcA and SepA strongly overlapped in some hyphal tips in a dome pattern, while other tips displayed SepA SPK and PkcA dome localization within the same tip. At septation sites PkcA and SepA consistently colocalized through late stages of CAR constriction. Bimolecular fluorescence complementation experimental results provide evidence that SepA and PkcA are both present in complexes at both hyphal tip domes and at cortical rings. A Gal4-based yeast two-hybrid analysis confirmed the physical interaction between SepA and PkcA, and indicted that the FH2 domain of SepA is involved in its physical interaction with PkcA. A functional interaction between PkcA and SepA was shown through complementation of the pkcA calC2 mutant's hypersensitivity to cell wall perturbing agents by overexpressed sepA and by the ability of the sepA1 mutation to block PkcA's ability to form cortical rings. Taken together these results suggest that a PkcA/SepA complex is involved in polarized growth. Through experiments using the actin disrupter latrunculin B, evidence is presented suggesting that actin plays a role in the PkcA/SepA complex.


Assuntos
Aspergillus nidulans/genética , Complexos Multiproteicos/genética , Peptídeo Hidrolases/genética , Proteína Quinase C/genética , Citoesqueleto de Actina/química , Citoesqueleto de Actina/genética , Aspergillus nidulans/química , Polaridade Celular/genética , Parede Celular/genética , Citocinese/genética , Proteínas Fúngicas/química , Proteínas Fúngicas/genética , Hifas/genética , Hifas/crescimento & desenvolvimento , Complexos Multiproteicos/química , Mutação , Peptídeo Hidrolases/química , Proteína Quinase C/química
13.
Biochemistry ; 58(6): 509-513, 2019 02 12.
Artigo em Inglês | MEDLINE | ID: mdl-30584764

RESUMO

Cd2+ and Pb2+ are xenobiotic heavy metal ions that use ionic mimicry to interfere with the cellular function of biomacromolecules. Using a combination of SAXS, electron microscopy, FRET, and solution NMR spectroscopy, we demonstrate that treatment with Cd2+ and Pb2+ causes self-assembly of protein kinase C regulatory domains that peripherally associate with membranes. The self-assembly process successfully competes with ionic mimicry and is mediated by conserved protein regions that are distinct from the canonical Ca2+-binding motifs of protein kinase C. The ability of protein oligomers to interact with anionic membranes is enhanced compared to the monomeric species. Our findings suggest that metal-ion-dependent peripheral membrane domains can be utilized for generating protein-metal-ion nanoclusters and serve as biotemplates for the design of sequestration agents.


Assuntos
Cádmio/farmacologia , Membrana Celular/metabolismo , Chumbo/farmacologia , Nanocompostos/química , Proteína Quinase C/química , Proteína Quinase C/metabolismo , Membrana Celular/efeitos dos fármacos , Humanos , Conformação Proteica , Domínios Proteicos
14.
J Mol Biol ; 430(21): 4275-4292, 2018 10 19.
Artigo em Inglês | MEDLINE | ID: mdl-30189200

RESUMO

Protein tyrosine phosphatase PTPN13, also known as PTP-BL in mice, represents a large multi-domain non-transmembrane scaffolding protein that contains five consecutive PDZ domains. Here, we report the solution structures of the extended murine PTPN13 PDZ3 domain in its apo form and in complex with its physiological ligand, the carboxy-terminus of protein kinase C-related kinase-2 (PRK2), determined by multidimensional NMR spectroscopy. Both in its ligand-free state and when complexed to PRK2, PDZ3 of PTPN13 adopts the classical compact, globular D/E fold. PDZ3 of PTPN13 binds five carboxy-terminal amino acids of PRK2 via a groove located between the EB-strand and the DB-helix. The PRK2 peptide resides in the canonical PDZ3 binding cleft in an elongated manner and the amino acid side chains in position P0 and P-2, cysteine and aspartate, of the ligand face the groove between EB-strand and DB-helix, whereas the PRK2 side chains of tryptophan and alanine located in position P-1 and P-3 point away from the binding cleft. These structures are rare examples of selective class III ligand recognition by a PDZ domain and now provide a basis for the detailed structural investigation of the promiscuous interaction between the PDZ domains of PTPN13 and their ligands. They will also lead to a better understanding of the proposed scaffolding function of these domains in multi-protein complexes assembled by PTPN13 and could ultimately contribute to low molecular weight antagonists that might even act on the PRK2 signaling pathway to modulate rearrangements of the actin cytoskeleton.


Assuntos
Proteína Quinase C/química , Proteína Quinase C/metabolismo , Proteína Tirosina Fosfatase não Receptora Tipo 13/química , Proteína Tirosina Fosfatase não Receptora Tipo 13/metabolismo , Sítios de Ligação , Humanos , Ligantes , Modelos Moleculares , Domínios PDZ , Ligação Proteica , Conformação Proteica
15.
J Cancer Res Clin Oncol ; 144(12): 2303-2311, 2018 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-30116883

RESUMO

PURPOSE: Autophagy, as a highly conserved cellular degradation and recycling process, plays an important part in maintaining cellular homeostasis. PKC signaling is involved in multiple pathways including cell cycle progression, tumorigenesis, migration and autophagy. METHODS: Literatures about PKC and autophagy from PubMed databases were reviewed in this study. RESULTS: Studies regarding the association of PKC and autophagy remain debatable. Different duration of the stimulation of autophagy and distinct cell contexts result in different function of PKC in regulating autophagy. The subcellular localization of PKCs and their downstream regulators may influence the autophagy regulation as well. As important intracellular components, the mitochondria play an important role in regulating autophagy, by metabolic modulation and structural derangement. CONCLUSION: Phase II studies regarding PKC-ß inhibitor, enzastaurin, showed promising results in MCL, DLBCL and recurrent high-grade gliomas. However, the detailed mechanism is still in need. The mechanism of PKC-ß in mediating autophagy in lymphoma and high-grade gliomas remains elusive as well. Moreover, several studies were in agreement that rottlerin enhanced autophagy in breast cancer cells, which warrants further clinical studies to verify PKC-δ as a therapeutic target. Thus, identifying the function of PKC in modulating autophagy and conducting related clinical studies help find novel target for chemotherapy.


Assuntos
Autofagia , Proteína Quinase C/metabolismo , Animais , Autofagia/efeitos dos fármacos , Autofagia/genética , Regulação da Expressão Gênica , Humanos , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/genética , Mitocôndrias/metabolismo , Família Multigênica , Proteína Quinase C/química , Proteína Quinase C/genética , Transdução de Sinais
16.
J Membr Biol ; 251(5-6): 653-666, 2018 12.
Artigo em Inglês | MEDLINE | ID: mdl-30056551

RESUMO

The catalytic α-subunits of both the Na+,K+-ATPase and the gastric H+,K+-ATPase possess lysine-rich N-termini which project into the cytoplasm. Due to conflicting experimental results, it is currently unclear whether the N-termini play a role in ion pump function or regulation, and, if they do, by what mechanism. Comparison of the lysine frequencies of the N-termini of both proteins with those of all of their extramembrane domains showed that the N-terminal lysine frequencies are far higher than one would expect simply from exposure to the aqueous solvent. The lysine frequency was found to vary significantly between different vertebrate classes, but this is due predominantly to a change in N-terminal length. As evidenced by a comparison between fish and mammals, an evolutionary trend towards an increase of the length of the N-terminus of the H+,K+-ATPase on going from an ancestral fish to mammals could be identified. This evolutionary trend supports the hypothesis that the N-terminus is important in ion pump function or regulation. In placental mammals, one of the lysines is replaced by serine (Ser-27), which is a target for protein kinase C. In most other animal species, a lysine occupies this position and hence no protein kinase C target is present. Interaction with protein kinase C is thus not the primary role of the lysine-rich N-terminus. The disordered structure of the N-terminus may, via increased flexibility, facilitate interaction with another binding partner, e.g. the surrounding membrane, or help to stabilise particular enzyme conformations via the increased entropy it produces.


Assuntos
ATPase Trocadora de Hidrogênio-Potássio/química , ATPase Trocadora de Hidrogênio-Potássio/metabolismo , Lisina/química , ATPase Trocadora de Sódio-Potássio/química , ATPase Trocadora de Sódio-Potássio/metabolismo , Animais , ATPase Trocadora de Hidrogênio-Potássio/classificação , Modelos Moleculares , Conformação Proteica , Proteína Quinase C/química , Proteína Quinase C/classificação , Proteína Quinase C/metabolismo , Análise de Sequência de Proteína , ATPase Trocadora de Sódio-Potássio/classificação
17.
FEBS Lett ; 592(14): 2432-2443, 2018 07.
Artigo em Inglês | MEDLINE | ID: mdl-29933512

RESUMO

The protein kinase D (PKD) family is regulated through multi-site phosphorylation, including autophosphorylation. For example, PKD displays in vivo autophosphorylation on Ser-742 (and Ser-738 in vitro) in the activation loop and Ser-910 in the C-tail (hPKD1 numbering). In this paper, we describe the surprising observation that PKD also displays in vitro autocatalytic activity towards a Tyr residue in the P + 1 loop of the activation segment. We define the molecular determinants for this unusual activity and identify a Cys residue (C705 in PKD1) in the catalytic loop as of utmost importance. In cells, PKD Tyr autophosphorylation is suppressed through the association of an inhibitory factor. Our findings provide important novel insights into PKD (auto)regulation.


Assuntos
Proteína Quinase C/química , Proteína Quinase C/metabolismo , Tirosina/metabolismo , Sequência de Aminoácidos , Animais , Células COS , Células Cultivadas , Chlorocebus aethiops , Drosophila , Ativação Enzimática/genética , Células HEK293 , Homeostase/genética , Humanos , Mutagênese Sítio-Dirigida , Fosforilação/genética , Proteína Quinase C/genética , Tirosina/genética
18.
Curr Protoc Chem Biol ; 10(2): e42, 2018 06.
Artigo em Inglês | MEDLINE | ID: mdl-29927112

RESUMO

The protein kinase C (PKC) family of serine/ threonine kinases has been shown to play active roles as either suppressors or promoters of carcinogenesis in different types of tumors. Using antibodies that preferentially recognize the active conformation of classical PKCs (cPKCs), we have previously shown that in breast cancer samples the expression levels of cPKCs were similar in estrogen receptor-positive (ER+ ) as compared to triple-negative tumors; however, the levels of active cPKCs were different. Determining the activation status of PKCs and other kinases in tumors may thus aid therapeutic decisions. Further, in basic science these tools may be used to understand the spatial and temporal dynamics of PKC signaling under different stimuli and for co-immunoprecipitation studies to detect binding partners and substrates of active cPKCs. In this article, we describe how monoclonal and polyclonal anti-active state PKC antibodies can be obtained using rational approaches to select bona fide epitopes through inspection of the crystal structure of classical PKCs coupled to molecular modeling studies. We believe that this methodology can be used for other kinases and multi-domain enzymes that undergo changes in their conformation upon activation. © 2018 by John Wiley & Sons, Inc.


Assuntos
Anticorpos/química , Anticorpos/imunologia , Proteína Quinase C/química , Proteína Quinase C/imunologia , Animais , Anticorpos Monoclonais/química , Anticorpos Monoclonais/imunologia , Domínio Catalítico , Humanos , Conformação Proteica , Proteína Quinase C/metabolismo
19.
J Pharmacol Sci ; 137(1): 20-29, 2018 May.
Artigo em Inglês | MEDLINE | ID: mdl-29699771

RESUMO

Propofol is the most commonly used anesthetic. Immunohistochemical studies have reported that propofol translocated protein kinase Cs (PKCs) in cardiomyocyte in a subtype-specific manner; however detailed features of the propofol-induced translocation of PKCs remain unknown. In this study, we performed real-time observation of propofol-induced PKC translocation in SH-SY5Y cells expressing PKCs fused with a fluorescent protein. Propofol unidirectionally translocated γPKC-GFP, a conventional PKC, and ζPKC-GFP, an atypical PKC, to the plasma membrane and nucleus, respectively, whereas the propofol-induced translocation of novel PKCs was diverse and subtype-specific among δPKC, εPKC and ηPKC. The propofol-induced translocation of εPKC-GFP was especially complicated and diverse, that is, 200 µM propofol first translocated εPKC-GFP to the perinuclear region. Thereafter, εPKC was translocated to the nucleus, followed by translocation to the plasma membrane. Analysis using a mutant εPKC in which the C1 domain was deleted demonstrated that the C1b domain of εPKC was indispensable for its translocation to the perinuclear region and plasma membrane, but not for its nuclear translocation. An in vitro kinase assay revealed that propofol increased the activities of the PKCs activities at the concentration that triggered the translocation. These results suggest that propofol could translocate PKCs to their appropriate target sites in a subtype-specific manner and concomitantly activated PKCs at these sites, contributing to its beneficial or adverse effects.


Assuntos
Anestésicos/farmacologia , Propofol/farmacologia , Proteína Quinase C/metabolismo , Animais , Células COS , Membrana Celular/metabolismo , Núcleo Celular/metabolismo , Chlorocebus aethiops , Proteína Quinase C/química , Proteína Quinase C/classificação , Transporte Proteico/efeitos dos fármacos
20.
PLoS One ; 13(4): e0195668, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29641588

RESUMO

Protein kinase C (PKC) isoforms play a pivotal role in the regulation of numerous cellular functions, making them extensively studied and highly attractive drug targets. Utilizing the crystal structure of the PKCδ C1B domain, we have developed hydrophobic isophthalic acid derivatives that modify PKC functions by binding to the C1 domain of the enzyme. In the present study, we aimed to improve the drug-like properties of the isophthalic acid derivatives by increasing their solubility and enhancing the binding affinity. Here we describe the design and synthesis of a series of multisubstituted pyrimidines as analogs of C1 domain-targeted isophthalates and characterize their binding affinities to the PKCα isoform. In contrast to our computational predictions, the scaffold hopping from phenyl to pyrimidine core diminished the binding affinity. Although the novel pyrimidines did not establish improved binding affinity for PKCα compared to our previous isophthalic acid derivatives, the present results provide useful structure-activity relationship data for further development of ligands targeted to the C1 domain of PKC.


Assuntos
Ácidos Ftálicos/química , Proteína Quinase C/química , Proteína Quinase C/metabolismo , Pirimidinas/química , Pirimidinas/metabolismo , Desenho de Fármacos , Modelos Moleculares , Ligação Proteica , Conformação Proteica , Domínios Proteicos , Relação Estrutura-Atividade
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