RESUMO
The peptidyl-prolyl isomerase Pin1 has been implicated in regulating cell cycle progression. Pin1 was found to be required for the DNA replication checkpoint in Xenopus laevis. Egg extracts depleted of Pin1 inappropriately transited from the G2 to the M phase of the cell cycle in the presence of the DNA replication inhibitor aphidicolin. This defect in replication checkpoint function was reversed after the addition of recombinant wild-type Pin1, but not an isomerase-inactive mutant, to the depleted extract. Premature mitotic entry in the absence of Pin1 was accompanied by hyperphosphorylation of Cdc25, activation of Cdc2/cyclin B, and generation of epitopes recognized by the mitotic phosphoprotein antibody, MPM-2. Therefore, Pin1 appears to be required for the checkpoint delaying the onset of mitosis in response to incomplete replication.
Assuntos
Proteínas de Ciclo Celular , Replicação do DNA , Mitose , Proteínas Nucleares , Peptidilprolil Isomerase/metabolismo , Proteínas de Xenopus , Animais , Afidicolina/farmacologia , Ciclo Celular , Ciclina B/metabolismo , Inibidores Enzimáticos/farmacologia , Fase G2 , Peptidilprolil Isomerase de Interação com NIMA , Inibidores da Síntese de Ácido Nucleico , Oócitos , Peptidilprolil Isomerase/genética , Peptidilprolil Isomerase/farmacologia , Mutação Puntual , Proteínas Quinases/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Tirosina Quinases/metabolismo , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/farmacologia , Xenopus laevis , Fosfatases cdc25/metabolismoRESUMO
G protein-coupled receptor (GPCR) sequestration to endosomes is proposed to be the mechanism by which G protein-coupled receptor kinase (GRK)-phosphorylated receptors are dephosphorylated and resensitized. The identification of beta-arrestins as GPCR trafficking molecules suggested that beta-arrestins might represent critical determinants for GPCR resensitization. Therefore, we tested whether beta2-adrenergic receptor (beta2AR) resensitization was dependent upon beta-arrestins and an intact clathrin-coated vesicle endocytic pathway. The overexpression of either the beta-arrestin 1-V53D dominant negative inhibitor of beta2AR sequestration or dynamin I-K44A to block clathrin-coated vesicle-mediated endocytosis impaired both beta2AR dephosphorylation and resensitization. In contrast, resensitization of a sequestration-impaired beta2AR mutant (Y326A) was reestablished following the overexpression of either GRK2 or beta-arrestin 1. Moreover, beta2ARs did not resensitize in COS-7 cells as the consequence of impaired sequestration and dephosphorylation. However, beta2AR resensitization was restored in these cells following the overexpression of beta-arrestin 2. These findings demonstrate, using both loss and gain of function paradigms, that beta2AR dephosphorylation and resensitization are dependent upon an intact sequestration pathway. These studies also indicate that beta-arrestins play an integral role in regulating not only the desensitization and intracellular trafficking of GPCRs but their ability to resensitize. beta-Arrestin expression levels appear to underlie cell type-specific differences in the regulation of GPCR resensitization.
Assuntos
Adenilil Ciclases/metabolismo , Arrestinas/farmacologia , Clatrina/metabolismo , Invaginações Revestidas da Membrana Celular/fisiologia , Isoproterenol/farmacologia , Receptores Adrenérgicos beta 2/fisiologia , Substituição de Aminoácidos , Animais , Arrestinas/biossíntese , Células COS , Linhagem Celular , Dinamina I , Dinaminas , Endocitose , GTP Fosfo-Hidrolases/biossíntese , GTP Fosfo-Hidrolases/farmacologia , Proteínas de Ligação ao GTP/metabolismo , Humanos , Rim , Cinética , Modelos Biológicos , Mutagênese Sítio-Dirigida , Receptores Adrenérgicos beta 2/efeitos dos fármacos , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/farmacologia , Transfecção , beta-Arrestina 1 , beta-Arrestina 2 , beta-ArrestinasRESUMO
Calcium/calmodulin (CaM) directly activates CaM-dependent protein kinase I (CaMKI) by binding to the enzyme and indirectly promotes the phosphorylation and synergistic activation of CaMKI by an exogenous kinase. We have evaluated the initial CaM-dependent activation of the unphosphorylated form of CaMKI. The kinetics of bacterially expressed human CaMKI show that the peptide syntide-2 is a relatively poor substrate, whereas the synapsin site-1 peptide is 17-fold more specific. The peptide ADR1G is 400-fold more specific than syntide-2, and its catalytic rate is among the highest reported for a kinase peptide substrate. To understand how CaM activates CaMKI, we have characterized the activation of the enzyme by CaM mutants with substitutions at hydrophobic residues. The point mutant M124Q located in the C-terminal domain of CaM produced a 57-fold increase in the CaM activation constant for CaMKI and suggests the involvement of methionine 124 in an important hydrophobic interaction with tryptophan 303 of CaMKI. Substituting two, three, and five hydrophobic residues in the N-terminal domain of CaM increased the CaM activation constant for CaMKI by 10-190-fold and lowered the maximal enzyme activity by more than 80%. Two of these N-terminal mutants of CaM do not affect the Km for peptide substrate but instead produce a 5-10-fold higher Km for ATP. This result demonstrates the critical role of the N-terminal domain of CaM in regulating the access of ATP to CaMKI.
Assuntos
Proteínas Quinases Dependentes de Cálcio-Calmodulina/metabolismo , Calmodulina/metabolismo , Proteínas de Saccharomyces cerevisiae , Trifosfato de Adenosina/metabolismo , Sequência de Aminoácidos , Animais , Sítios de Ligação/genética , Proteína Quinase Tipo 1 Dependente de Cálcio-Calmodulina , Proteínas Quinases Dependentes de Cálcio-Calmodulina/química , Calmodulina/genética , Cristalografia por Raios X , Proteínas de Ligação a DNA/metabolismo , Ativação Enzimática , Humanos , Peptídeos e Proteínas de Sinalização Intercelular , Cinética , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Peptídeos/metabolismo , Fosforilação , Ratos , Alinhamento de Sequência , Relação Estrutura-Atividade , Sinapsinas/metabolismo , Fatores de Transcrição/metabolismoRESUMO
HepG2 cells and medium were assayed for cholesteryl ester hydrolase (CEH) activity in the presence and absence of sodium cholate. Although bile salt-dependent CEH activity was measured in the medium at 6 to 96 h (up to 4500 pmol/h per mg cell protein), there was very little activity detected in the corresponding cell homogenates (less than 70 pmol/h per mg cell protein). Activity in the medium was expressed only in the presence of trihydroxy bile salts and was maximal at 40 mM cholate and pH 7.5. Incubation of HepG2 cells with brefeldin A resulted in an 80 to 90% inhibition of secretion of the bile salt-dependent CEH activity, while only inhibiting total protein secretion by 42%. Bile salt-dependent CEH activity could also be detected in rat liver perfusates. Although there was measurable activity in all of 14 livers analyzed (47 +/- 10 and 53 +/- 17 nmol/h per g liver per h perfusion during two 5-min collections after 15 and 30 min of perfusion, respectively), it did not correlate with the activity found in corresponding liver homogenates, as only four livers had detectable bile salt-dependent CEH activity. These results provide evidence for the secretion of a bile salt-dependent CEH activity, from both a hepatic cell line and the intact liver, that has similar properties to the enzyme previously isolated from rat liver homogenates and rat pancreas.
Assuntos
Ácidos e Sais Biliares/fisiologia , Fígado/enzimologia , Esterol Esterase/metabolismo , Animais , Brefeldina A , Ciclopentanos/farmacologia , Humanos , Fígado/metabolismo , Masculino , Monensin/farmacologia , Perfusão , Ratos , Ratos Endogâmicos , Células Tumorais CultivadasRESUMO
Retinol esterification was examined in microsomes from rat liver and lactating mammary gland as a function of the form of retinol substrate, dependence on fatty acyl CoA, and sensitivity to phenylmethylsulfonyl fluoride (PMSF). Retinol bound to cellular retinol-binding protein (CRBP) or dispersed in solvent was esterified in a fatty acyl CoA-independent, PMSF-sensitive reaction, consistent with lecithin:retinol acyltransferase (LRAT) activity. LRAT activity exhibited the same Km (2 microM retinol) between tissues but a higher Vmax in liver as compared to that in mammary gland (47 vs 8 pmol/min/mg microsome protein, respectively). Solvent-dispersed retinol was also esterified in a fatty acyl CoA-dependent, PMSF-resistant reaction, consistent with acyl CoA:retinol acyltransferase (ARAT) activity. Retinol bound to CRBP was not a good substrate for this reaction. ARAT activity displayed a similar Vmax (300 pmol/min/mg microsome protein) between tissues but Km values of 15 and 5 microM for retinol and fatty acyl CoA in mammary gland as compared to 30 and 25 microM, respectively, in the liver. Thus, when substrate was near or below Km, retinol esterification occurred predominantly by LRAT in the liver and ARAT in the mammary gland, respectively. The concentration of CRBP in the cytosol, determined by Western blotting, was approximately 2 microM in the liver but was almost nondetectable in the mammary gland. These data suggest that retinol esterification is regulated via different mechanisms in liver and mammary gland and support a specific role for CRBP in the liver.
Assuntos
Acil Coenzima A/metabolismo , Lactação/fisiologia , Fígado/metabolismo , Glândulas Mamárias Animais/metabolismo , Microssomos Hepáticos/metabolismo , Microssomos/metabolismo , Vitamina A/metabolismo , Animais , Citosol/metabolismo , Ésteres , Feminino , Cinética , Masculino , Palmitoil Coenzima A/metabolismo , Ratos , Ratos Endogâmicos Lew , Ratos Endogâmicos , Proteínas de Ligação ao Retinol/metabolismo , Proteínas Celulares de Ligação ao RetinolRESUMO
Nascent high density lipoprotein (HDL) (1.063 less than d less than 1.21 g/ml) was isolated from recirculating rat liver perfusates and separated by heparin-Sepharose chromatography into a nonretained fraction (NR) and a fraction (R) that eluted with 0.5 M NaCl. Fractions NR and R contained 70% and 30% of the nascent HDL protein, respectively. ApoB-containing particles were removed from fraction R by chromatography on concanavalin A. The protein composition of fractions NR and R was 40% and 29%, respectively. Fraction NR contained 25% apoA-I, 11% apoA-IV, 24% apoE, and 38% apoC. Fraction R contained primarily apoE (81% of total protein). The lipid composition of NR and R, respectively, was: triglyceride 44% and 26%, phospholipid 41% and 57%, cholesterol 8% and 13%, and cholesteryl ester 7% and 4%. Fractions NR and R had molecular weights of 400,000 and 860,000, respectively, as calculated from the Stokes radius. Negative staining electron microscopy indicated that both fractions consisted mainly of spherical particles (260-280 A) but some stacked disks were seen in fraction R. Livers perfused by the single-pass technique produced fractions NR and R in the same ratio as livers perfused by recirculation. The apolipoprotein compositions were similar to those in the recirculating perfusion; however, both fractions NR and R had more triglyceride (greater than 50% of total lipid). An HDL fraction was also isolated from liver perfusates by a combination of molecular sieve and heparin-Sepharose affinity chromatography. This HDL contained triglyceride but no apoB, indicating that triglyceride-rich HDL particles are not an artifact of ultracentrifugation.(ABSTRACT TRUNCATED AT 250 WORDS)
Assuntos
Lipoproteínas HDL/isolamento & purificação , Fígado/análise , Animais , Apolipoproteínas/isolamento & purificação , Cromatografia de Afinidade , Cromatografia em Gel , Lipoproteínas HDL/classificação , Lipoproteínas HDL/ultraestrutura , Lipoproteínas VLDL/isolamento & purificação , Masculino , Perfusão , Fosfatidilcolina-Esterol O-Aciltransferase/metabolismo , Fosfolipídeos/análise , Ratos , Ratos Endogâmicos , Triglicerídeos/análiseRESUMO
Nascent high density lipoprotein (HDL) and nascent very low density lipoprotein (VLDL) were isolated from rat livers that had been perfused with [3H]glycerol to label the triglyceride. When injected into intact rats, the labeled HDL-triglyceride disappeared as rapidly as the VLDL-triglyceride, with only 10% of the injected label remaining in the plasma after 30 min. The protein moiety of nascent HDL was labeled with [35S]methionine in a similar fashion and the labeled nascent HDL was separated into nonretained (NR) and retained (R) fractions by heparin-Sepharose affinity chromatography. When injected into rats, 55% of the injected label in nascent fraction NR and 72% of that in nascent fraction R was recovered from plasma at 30 min, compared to only 10% of the triglyceride label from unfractionated nascent HDL, indicating dissociation of triglyceride and apolipoprotein clearance. The plasma decay curves for both triglyceride and protein were biexponential. By 5 min, 15% of the 35S label remaining in plasma represented apoE and apoC that had been transferred from nascent HDL fractions NR and R to the d less than 1.063 g/ml fraction of plasma. Plasma HDL was labeled in vivo with [35S]methionine, separated into fractions NR and R, and the clearance of the two plasma HDL fractions was compared with that of the corresponding nascent HDL fractions. Except for a faster rate of removal of the nascent HDL fractions during the first 5 min, the serum decay curves were very similar.(ABSTRACT TRUNCATED AT 250 WORDS)
Assuntos
Lipoproteínas HDL/metabolismo , Fígado/metabolismo , Triglicerídeos/metabolismo , Animais , Apolipoproteínas/metabolismo , Lipoproteínas VLDL/metabolismo , Masculino , Taxa de Depuração Metabólica , Metionina , Perfusão , Ratos , Ratos Endogâmicos , Radioisótopos de EnxofreRESUMO
Rat livers were perfused by the nonrecirculating technique with medium containing [15N]glycine and sodium benzoate. At various times, the isotopic enrichment of hepatic free glycine, hepatic glycyl-tRNA, and perfusate hippurate was measured by GLC-MS. After 60 min, these parameters had reached approximately maximal values. At 90 min, the perfusate hippurate had a 30% greater enrichment of 15N than the intracellular glycine or glycyl-tRNA. Hippurate enrichment was half that of the medium glycine. The rat livers secreted apolipoprotein B (B-100 plus B-48) at a rate of 22 micrograms/g per h. From the 15N enrichment and the secretion rate, an intrahepatic pool size of 86 micrograms/g of apoB was calculated. From the minimal intracellular transit time of 30 min, an apoB fractional synthetic rate (FSR) of 2 pools/h was indicated, whereas the FSR estimated from the 15N-enrichment was 0.26/h. A possible explanation for the discrepancy is that apoB may recycle within the hepatocyte. On the basis of the present experiments, when hippurate enrichment is used as a measure of the enrichment of intrahepatic glycine in in vivo studies with 15N-labeled glycine, a correction should be applied, under normal metabolic circumstances, of approximately 20-30%.