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1.
Int Microbiol ; 5(2): 91-4, 2002 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-12180786

RESUMO

Six receptor proteins, with molecular masses ranging from 94 to 27 kDa, that bind to Escherichia coli K88ab fimbriae were recovered from brush border membranes and were detected after solubilization with Triton X-114. The recovery of these receptor proteins in the aqueous phase suggests their peripheral localization. The 63-, 60- and 33-kDa K88ab binding proteins were recovered using gel-filtration chromatography of the aqueous phase.


Assuntos
Antígenos de Bactérias/isolamento & purificação , Aderência Bacteriana , Proteínas da Membrana Bacteriana Externa/metabolismo , Proteínas de Escherichia coli/isolamento & purificação , Escherichia coli/crescimento & desenvolvimento , Proteínas de Fímbrias/isolamento & purificação , Microvilosidades/microbiologia , Animais , Antígenos de Bactérias/metabolismo , Proteínas da Membrana Bacteriana Externa/isolamento & purificação , Proteínas de Escherichia coli/metabolismo , Proteínas de Fímbrias/metabolismo , Suínos
2.
Cell Growth Differ ; 12(12): 603-11, 2001 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-11751455

RESUMO

Rho family GTPases Rac and Cdc42 are pivotal regulators of apoptosis in multiple cell types. However, little is known about the mechanism by which these GTPases are regulated in response to apoptotic stimuli. Here, we demonstrate that TIAM1, a Rac-specific guanine nucleotide exchange factor, is cleaved by caspases during apoptosis. TIAM1 cleavage occurs in multiple cell lines in response to diverse apoptotic stimuli such as ceramide, Fas, and serum deprivation. Processing occurs at residue 993 of TIAM1 and removes the NH(2)-terminal of TIAM's two pleckstrin homology domains, leaving a stable fragment containing the Dbl homology and COOH-terminal pleckstrin homology domains. This leads to functional inactivation of TIAM1, as determined by failure of the cleavage product to stimulate GTP loading of Rac in vivo. Furthermore, this product is defective in signaling to two independent Rac effectors, c-Jun NH(2)-terminal kinase and serum response factor. Finally, we demonstrate that in cells treated with ceramide, cleavage of TIAM1 coincided with the inactivation of endogenous Rac. These results reveal a novel mechanism for regulating guanine nucleotide exchange factor activity and GTPase-mediated signaling pathways.


Assuntos
Apoptose , Caspases/metabolismo , Proteínas/metabolismo , Células 3T3 , Animais , Sítios de Ligação , Proteínas Sanguíneas/química , Células COS , Linhagem Celular , Membrana Celular/metabolismo , Ceramidas/metabolismo , Ceramidas/farmacologia , Ativação Enzimática , GTP Fosfo-Hidrolases/metabolismo , Fatores de Troca do Nucleotídeo Guanina , Humanos , Immunoblotting , Proteínas Quinases JNK Ativadas por Mitógeno , Células Jurkat , Luciferases/metabolismo , Camundongos , Microscopia de Fluorescência , Proteína Quinase 8 Ativada por Mitógeno , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Proteínas de Neoplasias , Células PC12 , Fosfoproteínas/química , Testes de Precipitina , Estrutura Terciária de Proteína , Ratos , Fator de Resposta Sérica/metabolismo , Transdução de Sinais , Proteína 1 Indutora de Invasão e Metástase de Linfoma de Células T , Fatores de Tempo , Células Tumorais Cultivadas , Proteínas rac de Ligação ao GTP/metabolismo
3.
Arch Biochem Biophys ; 388(1): 74-80, 2001 Apr 01.
Artigo em Inglês | MEDLINE | ID: mdl-11361144

RESUMO

In this study we have evaluated the specificity of different PKC isozymes for the phosphorylation of the catalytic alpha1 subunit of rat renal Na+,K+-ATPase (alpha1 Na+,K+-ATPase). Using in vitro phosphotransferase assays we found that classical PKCs (cPKCs) alpha, betaI, and gamma efficiently phosphorylate alpha1 Na+,K+-ATPase. However, alpha1 Na+,K+-ATPase was a poor substrate for the novel PKCs (nPKCs) delta and epsilon. Two-dimensional phosphopeptide mapping revealed a similar pattern of phosphorylation by all cPKCs. The functional significance of this finding was evaluated by measuring Na+,K+-ATPase activity (assessed by 86Rb+ uptake) in COS-7 cells expressing the rat alpha1 Na+,K+-ATPase. 1-oleoyl-2-acetoyl-sn-glycerol (OAG), a nonselective PKC activator, inhibited Na+,K+-ATPase activity in this system. On the other hand, 12-deoxyphorbol-13-phenylacetate (DPP), which preferentially activates nPKCepsilon, did not affect 86Rb+ uptake. These results indicate a differential pattern of phosphorylation and regulation of rat renal Na+,K+-ATPase activity by PKC isoforms and suggest an important role for cPKCs in the physiological regulation of the pump.


Assuntos
Proteína Quinase C/química , ATPase Trocadora de Sódio-Potássio/metabolismo , Animais , Células COS , DNA Complementar/metabolismo , Relação Dose-Resposta a Droga , Ativação Enzimática , Irritantes/farmacologia , Isoenzimas , Ouabaína/farmacologia , Mapeamento de Peptídeos , Ésteres de Forbol/farmacologia , Fosforilação , Isoformas de Proteínas , Proteína Quinase C/metabolismo , Transporte Proteico , Ratos , Rubídio/farmacologia , Sódio/farmacologia , Fatores de Tempo , Transfecção
4.
J Biol Chem ; 276(21): 18303-12, 2001 May 25.
Artigo em Inglês | MEDLINE | ID: mdl-11278894

RESUMO

The novel phorbol ester receptor beta2-chimaerin is a Rac-GAP protein possessing a single copy of the C1 domain, a 50-amino acid motif initially identified in protein kinase C (PKC) isozymes that is involved in phorbol ester and diacylglycerol binding. We have previously shown that, like PKCs, beta2-chimaerin binds phorbol esters with high affinity in a phospholipid-dependent manner (Caloca, M. J., Fernandez, M. N., Lewin, N. E., Ching, D., Modali, R., Blumberg, P. M., and Kazanietz, M. G. (1997) J. Biol. Chem. 272, 26488-26496). In this paper we report that like PKC isozymes, beta2-chimaerin is translocated by phorbol esters from the cytosolic to particulate fraction. Phorbol esters also induce translocation of alpha1 (n)- and beta1-chimaerins, suggesting common regulatory mechanisms for all chimaerin isoforms. The subcellular redistribution of beta2-chimaerin by phorbol esters is entirely dependent on the C1 domain, as revealed by deletional analysis and site-directed mutagenesis. Interestingly, beta2-chimaerin translocates to the Golgi apparatus after phorbol ester treatment, as revealed by co-staining with the Golgi marker BODIPY-TR-ceramide. Structure relationship analysis of translocation using a series of PKC ligands revealed substantial differences between translocation of beta2-chimaerin and PKCalpha. Strikingly, the mezerein analog thymeleatoxin is not able to translocate beta2-chimaerin, although it very efficiently translocates PKCalpha. Phorbol esters also promote the association of beta2-chimaerin with Rac in cells. These data suggest that chimaerins can be positionally regulated by phorbol esters and that each phorbol ester receptor class has distinct pharmacological properties and targeting mechanisms. The identification of selective ligands for each phorbol ester receptor class represents an important step in dissecting their specific cellular functions.


Assuntos
Proteínas de Caenorhabditis elegans , Proteínas de Neoplasias/metabolismo , Ésteres de Forbol/metabolismo , Transdução de Sinais , Animais , Células COS , Proteínas de Transporte , Isoenzimas/metabolismo , Proteína Quinase C/metabolismo , Receptores de Droga/metabolismo
5.
Biochem Pharmacol ; 60(10): 1417-24, 2000 Nov 15.
Artigo em Inglês | MEDLINE | ID: mdl-11020443

RESUMO

The phorbol ester tumor promoters and related analogs are widely used as potent activators of protein kinase C (PKC). The phorbol esters mimic the action of the lipid second messenger diacylglycerol (DAG). The aim of this commentary is to highlight a series of important and controversial concepts in the pharmacology and regulation of phorbol ester receptors. First, phorbol ester analogs have marked differences in their biological properties. This may be related to a differential regulation of PKC isozymes by distinct analogs. Moreover, it seems that marked differences exist in the ligand recognition properties of the C1 domains, the phorbol ester/DAG binding sites in PKC isozymes. Second, an emerging theme that we discuss here is that phorbol esters also target receptors unrelated to PKC isozymes, a concept that has been largely ignored. These novel receptors lacking kinase activity include chimaerins (a family of Rac-GTPase-activating proteins), RasGRP (a Ras exchange factor), and Unc-13/Munc-13 (a family of proteins involved in exocytosis). Unlike the classical and novel PKCs, these "non-kinase" phorbol ester receptors possess a single copy of the C1 domain. Interestingly, each receptor class has unique pharmacological properties and biochemical regulation. Lastly, it is well established that phorbol esters and related analogs can translocate each receptor to different intracellular compartments. The differential pharmacological properties of the phorbol ester receptors can be exploited to generate specific agonists and antagonists that will be helpful tools to dissect their cellular function.


Assuntos
Proteínas de Caenorhabditis elegans , Carcinógenos/farmacologia , Diglicerídeos/metabolismo , Ésteres de Forbol/farmacologia , Proteína Quinase C/metabolismo , Receptores de Droga/metabolismo , Animais , Transporte Biológico , Carcinógenos/toxicidade , Proteínas de Transporte , Sequência Conservada , Diglicerídeos/química , Ativação Enzimática , Humanos , Isoenzimas/química , Isoenzimas/metabolismo , Mimetismo Molecular , Ésteres de Forbol/toxicidade , Conformação Proteica , Proteína Quinase C/química , Proteína Quinase C/efeitos dos fármacos , Receptores de Droga/química , Receptores de Droga/efeitos dos fármacos , Sistemas do Segundo Mensageiro
6.
Endocrinology ; 141(1): 146-52, 2000 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-10614633

RESUMO

Several reports have indicated that protein kinase C (PKC) is an important regulator of proliferation in thyroid cells. Unlike TSH, the mitogenic effects of phorbol esters are accompanied by de-differentiation. The role of individual PKC isoforms in thyroid cell proliferation and differentiation has not been examined. Recent studies have implicated the atypical PKCzeta, a phorbol ester-unresponsive isozyme, in cell proliferation, death, and survival. We overexpressed PKCzeta in Wistar rat thyroid (WRT) cells and determined that PKCzeta conferred TSH-independent DNA synthesis and cell proliferation. Cells overexpressing PKCzeta show higher levels of phosphorylated p42/p44 MAPK compared with vector-transfected cells. Experiments using a luciferase reporter for Elk-1 revealed that PKCzeta overexpressing cells exhibit higher basal Elk-1 transcriptional activity than vector-transfected control cells. Interestingly, stimulation of Elk-1 transcriptional activity by MEK1, a p42/p44 MAPK kinase, was significantly enhanced in cells overexpressing PKCzeta. Strikingly, TSH retained the ability to stimulate Tg expression in cells expressing PKCzeta. These results suggest that PKCzeta stimulates TSH-independent mitogenesis through a p42/p44 MAPK-dependent pathway. Unlike overexpression of Ras or phorbol ester treatment, PKC overexpression does not impair thyroglobulin (Tg) expression.


Assuntos
Proteínas de Ligação a DNA , Proteína Quinase C/fisiologia , Glândula Tireoide/citologia , Tireotropina/fisiologia , Animais , Western Blotting , Divisão Celular , Células Cultivadas , DNA/biossíntese , Regulação Enzimológica da Expressão Gênica/genética , Regulação Enzimológica da Expressão Gênica/fisiologia , Vetores Genéticos/genética , Proteína Quinase 1 Ativada por Mitógeno/fisiologia , Proteína Quinase C/biossíntese , Proteína Quinase C/genética , Proteínas Proto-Oncogênicas/genética , Ratos , Ratos Wistar , Fatores de Transcrição/genética , Proteínas Elk-1 do Domínio ets
7.
Proc Natl Acad Sci U S A ; 96(21): 11854-9, 1999 Oct 12.
Artigo em Inglês | MEDLINE | ID: mdl-10518540

RESUMO

The members of the chimaerin family of Rac-GTPase-activating proteins possess a single C1 domain with high homology to those present in protein kinase C (PKC) isozymes. This domain in PKCs is involved in phorbol ester and diacylglycerol (DAG) binding. We previously have demonstrated that one of the chimaerin isoforms, beta2-chimaerin, binds phorbol esters with high affinity. In this study we analyzed the properties of beta2-chimaerin as a DAG receptor by using a series of conformationally constrained cyclic DAG analogues (DAG lactones) as probes. We identified analogs that bind to beta2-chimaerin with more than 100-fold higher affinity than 1-oleoyl-2-acetylglycerol. The potencies of these analogs approach those of the potent phorbol ester tumor promoters. The different DAG lactones show some selectivity for this novel receptor compared with PKCalpha. Cellular studies revealed that these DAG analogs induce translocation of beta2-chimaerin from cytosolic (soluble) to particulate fractions. Using green fluorescent protein-fusion proteins for beta2-chimaerin we determined that this novel receptor translocates to the perinuclear region after treatment with DAG lactones. Binding and translocation were prevented by mutation of the conserved Cys-246 in the C1 domain. The structural homology between the C1 domain of beta2-chimaerin and the C1b domain of PKCdelta also was confirmed by modeling analysis. Our results demonstrate that beta2-chimaerin is a high affinity receptor for DAG through binding to its C1 domain and supports the emerging concept that multiple pathways transduce signaling through DAG and the phorbol esters.


Assuntos
Diglicerídeos/metabolismo , Lactonas/metabolismo , Proteínas de Neoplasias/metabolismo , Sequência de Aminoácidos , Animais , Transporte Biológico , Células COS , Proteínas de Fluorescência Verde , Cinética , Ligantes , Proteínas Luminescentes/metabolismo , Modelos Moleculares , Dados de Sequência Molecular , Mutagênese , Ligação Proteica , Proteínas Recombinantes/metabolismo , Homologia de Sequência de Aminoácidos , Fatores de Tempo
8.
Vet Microbiol ; 61(1-2): 51-8, 1998 Mar 15.
Artigo em Inglês | MEDLINE | ID: mdl-9646465

RESUMO

The characteristics of the binding of biotinylated E. coli K88ab fimbriae to guinea pig erythrocyte membranes, as a possible model of the target host cell were studied. Binding showed sigmoidal dependence, with an apparent saturation at about 0.8 ng of fimbriae. Hill coefficient values (h) were about 2-2.4, which indicated that the receptor population showed positive cooperativity with at least three binding sites. Apparent binding constants to the first and third binding sites (1K3 and 3K3) were determined. Three K88ab binding proteins, of 67, 63 and 48 kDa, were identified on solubilized erythrocyte membranes and were recovered mainly in a detergent phase, suggesting a possible integral localization of the receptors.


Assuntos
Aderência Bacteriana/fisiologia , Membrana Eritrocítica/fisiologia , Escherichia coli/fisiologia , Fímbrias Bacterianas/fisiologia , Adesinas Bacterianas/análise , Animais , Biotina , Western Blotting , Proteínas de Transporte/sangue , Proteínas de Transporte/isolamento & purificação , Cobaias , Cinética
9.
J Biol Chem ; 272(42): 26488-96, 1997 Oct 17.
Artigo em Inglês | MEDLINE | ID: mdl-9334226

RESUMO

Beta2-chimaerin, a member of the GTPase-activating proteins for the small GTP-binding protein p21Rac, possesses a single cysteine-rich domain with high homology to those implicated in phorbol ester and diacylglycerol binding in protein kinase C (PKC) isozymes. We have expressed beta2-chimaerin in Sf9 insect cells using the baculovirus expression system and determined that, like PKCs, beta2-chimaerin binds phorbol esters with high affinity in the presence of phosphatidylserine as a cofactor. Scatchard plot analysis using the radioligand [3H]phorbol 12,13-dibutyrate revealed a dissociation constant of 1.9 +/- 0.2 nM for beta2-chimaerin. Likewise, beta2-chimaerin is a high affinity receptor for the bryostatins, a class of atypical PKC activators. A detailed comparison of structure-activity relations using several phorbol ester analogs revealed striking differences in binding recognition between beta2-chimaerin and PKCalpha. Although the diacylglycerol 1-oleoyl-2-acetylglycerol binds with similar potency to both beta2-chimaerin and PKCalpha, the mezerein analog thymeleatoxin has 56-fold less affinity for binding to beta2-chimaerin. To establish whether beta2-chimaerin responds to phorbol esters in cellular systems, we overexpressed beta2-chimaerin in COS-7 cells and monitored its subcellular distribution after phorbol ester treatment. Interestingly, as described previously for PKC isozymes, beta2-chimaerin translocates from cytosolic to particulate fractions as a consequence of phorbol ester treatment. Our results demonstrate that beta2-chimaerin is a novel target for the phorbol ester tumor promoters. The expansion of the family of phorbol ester receptors strongly suggests a potential for the "non-kinase" receptors as cellular mediators of the phorbol ester responses.


Assuntos
Carcinógenos/metabolismo , Proteínas de Neoplasias/metabolismo , Dibutirato de 12,13-Forbol/metabolismo , Animais , Transporte Biológico , Células COS , Compartimento Celular , Linhagem Celular , Ativação Enzimática , Ligação Proteica , Proteína Quinase C/metabolismo , Proteínas Recombinantes/metabolismo , Spodoptera
10.
Vet Microbiol ; 58(1): 45-52, 1997 Oct 31.
Artigo em Inglês | MEDLINE | ID: mdl-9451460

RESUMO

Red cells glycolytic enzymes attached and nonattached to K88+ Escherichia coli were assayed. Hexokinase, glucose-6-phosphate dehydrogenase, 6-phosphogluconate dehydrogenase, pyruvate kinase, glyceraldehyde-3-phosphate dehydrogenase and glutathione reductase activities, were measured. E. coli with K88ab fimbriae, E. coli with K88ac fimbriae, and isolated K88ab fimbriae were investigated for their effect on the above enzymes. Different changes were obtained with K88ab + bacteria compared with K88ac + bacteria. Purified fimbriae gave a third set of responses.


Assuntos
Antígenos de Bactérias , Aderência Bacteriana , Eritrócitos/efeitos dos fármacos , Eritrócitos/enzimologia , Proteínas de Escherichia coli , Escherichia coli/imunologia , Escherichia coli/patogenicidade , Proteínas de Fímbrias , Animais , Antígenos de Superfície/análise , Galinhas , Enzimas/sangue , Fímbrias Bacterianas/imunologia , Glucosefosfato Desidrogenase/sangue , Glutationa Redutase/sangue , Gliceraldeído-3-Fosfato Desidrogenases/sangue , Cobaias , Hexoquinase/sangue , Testes de Fixação do Látex/veterinária , Fosfogluconato Desidrogenase/sangue , Piruvato Quinase/sangue , Suínos
11.
Infect Immun ; 64(8): 3416-8, 1996 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-8757886

RESUMO

Nontoxigenic Escherichia coli strains bearing K88 fimbriae have been associated with diarrhea in piglets. We have used guinea pig erythrocytes as a model of the host cell to study the cellular alterations after adherence of purified K88ab fimbriae. Although Mg2+-dependent ATPase was inhibited (up to 61%), Na/K ATPase was not. Metabolic enzymes were not significantly affected.


Assuntos
Adenosina Trifosfatases/metabolismo , Antígenos de Bactérias , Antígenos de Superfície/metabolismo , Membrana Eritrocítica/metabolismo , Proteínas de Escherichia coli , Escherichia coli/fisiologia , Proteínas de Fímbrias , Fímbrias Bacterianas/metabolismo , Animais , Aderência Bacteriana , ATPase de Ca(2+) e Mg(2+)/metabolismo , Membrana Eritrocítica/enzimologia , Escherichia coli/patogenicidade , Cobaias , Plasmídeos , Ligação Proteica , ATPase Trocadora de Sódio-Potássio/metabolismo
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