RESUMEN

Previously, eEF-2 phosphorylation has been identified as a reversible mechanism involved in the inhibition of the elongation phase of translation. In this study, an increased level of phosphorylation of eukaryotic elongation factor-2 (eEF-2) was observed in the brains and livers of hibernating ground squirrels. In brain and liver from hibernators, eEF-2 kinase activity was increased relative to that of active animals. The activity of protein phosphatase 2A (PP2A), a phosphatase that dephosphorylates eEF-2, was also decreased in brain and liver from hibernators. This was associated with an increase in the level of inhibitor 2 of PP2A (I(2)(PP2A)), although there was an increase in the level of the catalytic subunit of PP2A (PP2A/C) in hibernating brains and livers. These results indicate that eEF-2 phosphorylation represents a specific and previously uncharacterized mechanism for inhibition of the elongation phase of protein synthesis during hibernation. Increased levels of eEF-2 phosphorylation in hibernators appear to be a component of the regulated shutdown of cellular functions that permits hibernating animals to tolerate severe reductions in cerebral blood flow and oxygen delivery capacity.

Asunto(s)

Encéfalo/metabolismo , Proteínas Quinasas Dependientes de Calcio-Calmodulina/metabolismo , Hibernación/fisiología , Hígado/metabolismo , Factor 2 de Elongación Peptídica/metabolismo , Sciuridae/fisiología , Animales , Catálisis , Citosol/enzimología , Quinasa del Factor 2 de Elongación , Fosfoproteínas Fosfatasas/metabolismo , Fosforilación , Proteína Fosfatasa 2 , Subunidades de Proteína

RESUMEN

Recombinant I(1)(PP2A) and I(2)(PP2A) did not affect the activity of the catalytic subunit of protein phosphatase 1 (PP1(C)) with (32)P-labeled myelin basic protein, histone H1, and phosphorylase when assayed in the absence of divalent cations. However, in the presence of Mn(2+), I(1)(PP2A) and I(2)(PP2A) stimulated PP1(C) activity by 15-20-fold with myelin basic protein and histone H1 but not phosphorylase. Half-maximal stimulation occurred at 2 and 4 nM I(1)(PP2A) and I(2)(PP2A), respectively. Moreover, I(1)(PP2A) and I(2)(PP2A) reduced the Mn(2+) requirement by about 30-fold to 10 microM. In contrast, PP1(C) activity was unaffected by I(1)(PP2A) and I(2)(PP2A) in the presence of Co(3+) (0.1 mM), Mg(2+) (2 mM), Ca(2+) (0.5 mM), and Zn(2+) (0.1 mM). Following gel filtration chromatography on Sephacryl S-200 in the presence of Mn(2+), PP1(C) coeluted with I(1)(PP2A) and I(2)(PP2A) in the void volume. However, when I(1)(PP2A) and I(2)(PP2A) or Mn(2+) were omitted, PP1(C) emerged with a V(e)/V(0) of approximately 1.6. The results demonstrate that I(1)(PP2A) and I(2)(PP2A) associate with and modify the substrate specificity of PP1(C) in the presence of physiological concentrations of Mn(2+). A novel role is suggested for I(1)(PP2A) and I(2)(PP2A) in the reciprocal regulation of two major mammalian serine/threonine phosphatases, PP1 and PP2A.

Asunto(s)

Inhibidores Enzimáticos/metabolismo , Isoenzimas/antagonistas & inhibidores , Isoenzimas/metabolismo , Fosfoproteínas Fosfatasas/antagonistas & inhibidores , Fosfoproteínas Fosfatasas/metabolismo , Animales , Cationes Bivalentes , Bovinos , Riñón/enzimología , Manganeso/metabolismo , Proteína Fosfatasa 1 , Proteína Fosfatasa 2 , Especificidad por Sustrato

RESUMEN

Transient expression of I2PP2A, a potent inhibitor of protein phosphatase 2A (PP2A), in HEK-293 cells increased the concentration and DNA binding of the proto-oncogene c-Jun. In contrast, expression of the catalytic subunit of PP2A (PP2AC) markedly decreased the concentration and DNA binding of c-Jun. Expression of I2PP2A also increased the transcriptional activity of activator protein-1, and this effect was diminished in a dose-dependent manner by expression of PP2AC. Densitometric analysis following Western blotting of extracts with antibodies specific for phospho-Ser63 and Ser73 suggests that the effects of I2PP2A and PP2AC expression might be mediated, in part, by changes in the phosphorylation of c-Jun at Ser63. The results indicate that I2PP2A elicits effects that are consistent with it acting as an inhibitor of PP2A in intact cells, and suggest that PP2A might exhibit site selectivity with respect to c-Jun phosphorylation.

Asunto(s)

Genes jun , Fosfoproteínas Fosfatasas/genética , Proteínas/genética , Transducción de Señal/genética , Factor de Transcripción AP-1/genética , Línea Celular , Proteínas Cromosómicas no Histona , Proteínas de Unión al ADN , Regulación de la Expresión Génica , Chaperonas de Histonas , Humanos , Fosfoproteínas Fosfatasas/antagonistas & inhibidores , Proteína Fosfatasa 2 , Proteínas/metabolismo , Proto-Oncogenes Mas , Factor de Transcripción AP-1/metabolismo , Factores de Transcripción , Transfección

RESUMEN

Phosphorylation modulates the activity of many proteins that interact with nucleic acids including DNA and RNA polymerases. The HIV-1 reverse transcriptase (RT) is essential during the replicative cycle of the HIV-1 virus. HIV-1 RT has several potential sites for phosphorylation that could regulate its activities. In this work, the phosphorylation of HIV-1 RT is examined in vitro and in vivo, to evaluate any role for this modification in regulating RT metabolism. Recombinant unphosphorylated HIV-1 RT heterodimer expressed in bacteria can be phosphorylated in vitro by several purified mammalian protein kinases. Seven kinases were tested, and five of these enzymes phosphorylated HIV-1 RT. Using an insect baculovirus expression system, the 66 kDa HIV-1 RT was also phosphorylated in vivo. However, HIV-1 RT immunoprecipitated from H9-lymphoma cells infected with HIV-1 showed negligible phosphorylation. Our results indicate that purified HIV-1 RT can be phosphorylated by several mammalian protein kinases in vitro and during expression in baculovirus infected insect cells.

Asunto(s)

Transcriptasa Inversa del VIH/química , Transcriptasa Inversa del VIH/metabolismo , Proteínas Quinasas/metabolismo , Secuencia de Aminoácidos , Animales , Anticuerpos Monoclonales , Línea Celular , Cinética , Datos de Secuencia Molecular , Fosfatos/metabolismo , Radioisótopos de Fósforo , Fosforilación , Proteínas Recombinantes/metabolismo , Spodoptera , Especificidad por Sustrato

Asunto(s)

Inhibidores Enzimáticos/aislamiento & purificación , Fosfoproteínas Fosfatasas/antagonistas & inhibidores , Proteínas/aislamiento & purificación , Animales , Bovinos , Proteínas Cromosómicas no Histona , Proteínas de Unión al ADN , Inhibidores Enzimáticos/metabolismo , Chaperonas de Histonas , Humanos , Péptidos y Proteínas de Señalización Intracelular , Riñón/metabolismo , Proteínas Nucleares , Fosfoproteínas Fosfatasas/biosíntesis , Proteína Fosfatasa 2 , Proteínas/metabolismo , Proteínas de Unión al ARN , Proteínas Recombinantes/aislamiento & purificación , Factores de Transcripción

RESUMEN

The amino acid sequences of two tryptic peptides derived from purified preparations of I1PP2A indicated that this potent heat-stable protein inhibitor of protein phosphatase 2A (PP2A) may be equivalent to putative histocompatibility leukocyte antigens class II-associated protein I (PHAP-I). Experiments using purified preparations of recombinant human PHAP-I confirmed that this protein inhibited PP2A. Half-maximal inhibition of the phosphatase occurred at about 4 nM PHAP-I, similar to the half-maximal inhibition obtained with purified preparations of bovine kidney I1PP2A. In addition, PHAP-I did not affect the activities of protein phosphatase 1, 2B, and 2C in a manner analogous to that of I1PP2A. Together, the results establish the identity of I1PP2A on a firm basis.

Asunto(s)

Proteínas Portadoras , Endorribonucleasas , Péptidos y Proteínas de Señalización Intracelular , Fosfoproteínas Fosfatasas/antagonistas & inhibidores , Proteínas/química , Proteínas de Unión al ARN , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Bovinos , Cartilla de ADN/química , Electroforesis en Gel de Poliacrilamida , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/farmacología , Regulación de la Expresión Génica , Humanos , Isopropil Tiogalactósido/farmacología , Datos de Secuencia Molecular , Peso Molecular , Proteínas Nucleares , Péptidos/química , Péptidos/metabolismo , Proteína Fosfatasa 1 , Proteína Fosfatasa 2 , Proteínas/farmacología , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/farmacología , Análisis de Secuencia , Tripsina/metabolismo

RESUMEN

Two potent heat-stable protein phosphatase 2A (PP2A) inhibitor proteins designated I1PP2A and I2PP2A have been purified to apparent homogeneity from extracts of bovine kidney (Li, M., Guo, H., and Damuni, Z. (1995) Biochemistry 34, 1988-1996). N-terminal and internal amino acid sequencing indicated that I2PP2A was a truncated form of SET, a largely nuclear protein that is fused to nucleoporin Nup214 in acute non-lymphocytic myeloid leukemia. Experiments using purified preparations of recombinant human SET confirmed that this protein inhibited PP2A. Half-maximal inhibition of the phosphatase occurred at about 2 nM SET. By contrast, SET (up to 20 nM) did not affect the activities of purified preparations of protein phosphatases 1, 2B, and 2C. The results indicate that SET is a potent and specific inhibitor of PP2A and suggest that impaired regulation of PP2A may contribute to acute myeloid leukemogenesis.

Asunto(s)

Inhibidores Enzimáticos/farmacología , Fosfoproteínas Fosfatasas/antagonistas & inhibidores , Biosíntesis de Proteínas , Proteínas/farmacología , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Bovinos , Proteínas Cromosómicas no Histona , Proteínas de Unión al ADN , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/aislamiento & purificación , Chaperonas de Histonas , Humanos , Riñón/metabolismo , Leucemia Mieloide Aguda , Datos de Secuencia Molecular , Fragmentos de Péptidos/química , Mapeo Peptídico , Proteína Fosfatasa 2 , Proteínas/química , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/farmacología , Factores de Transcripción , Tripsina

RESUMEN

Insulin-stimulated protamine kinase (cPK) and protein kinase C (PKC) phosphorylated eukaryotic protein synthesis initiation factor 4E (eIF-4E) on serine and threonine residues located on an identical tryptic fragment as judged by two-dimensional phosphopeptide mapping. With cPK and PKC, the apparent Km for eIF-4E was about 1.2 and 50 microM, respectively. Relative to recombinant human eIF-4E, cPK exhibited about 100% and < or = 5% activity with eIF-4ES209A and eIF-4ET210A, respectively, and eIF-4ES209A was phosphorylated exclusively on threonines. Bovine kidney eIF-4E enhanced up to 1.8-fold globin synthesis in m7GTP-Sepharose-treated reticulocyte lysates. In contrast, following incubation with cPK, these eIF-4E preparations stimulated globin synthesis up to 6-fold. Compared to the dephosphorylation of the cPK-modified serine on eIF-4E, reticulocyte lysates and highly purified protein phosphatase 2A exhibited marked preference for the cPK-modified threonine. The results indicate that cPK phosphorylates eIF-4E on Ser209 and Thr210, that the hydroxyl group or phosphorylation of Thr210 is necessary for cPK to act on Ser209, and that Ser209 phosphorylation activates reticulocyte globin synthesis. The results suggest that cPK could contribute to the insulin-stimulated phosphorylation of eIF-4E, but that protein phosphatase 2A may confer the site specificity of this response.

Asunto(s)

Insulina/farmacología , Factores de Iniciación de Péptidos/metabolismo , Protamina Quinasa/metabolismo , Animales , Secuencia de Bases , Bovinos , ADN Complementario , Activación Enzimática , Factor 4E Eucariótico de Iniciación , Humanos , Datos de Secuencia Molecular , Mapeo Peptídico , Fosfopéptidos/química , Fosfopéptidos/metabolismo , Fosfoproteínas Fosfatasas/metabolismo , Fosforilación , Proteína Fosfatasa 2

RESUMEN

Tau is a neuron-specific, microtubule-associated protein that forms paired helical filaments (PHFs) of Alzheimer's disease when aberrantly phosphorylated. We have attempted to elucidate the protein kinases and phosphatases that regulate tau phosphorylation. Incubation of rat, human, and rhesus monkey temporal neocortex slices with the phosphatase inhibitor okadaic acid induced epitopes of tau similar to those found in PHFs. Okadaic acid (1-20 microM) induced variant forms of tau at 60-68 kDa, which were recognized by the monoclonal antibodies Alz-50 (in humans only) and 5E2 and two polyclonal antipeptide antisera, OK-1 and OK-2. The phosphorylation-sensitive monoclonal antibody Tau-1 failed to recognize the slowest mobility forms of tau after okadaic acid treatment. FK-520 (1-10 microM), a potent inhibitor of calcineurin activity, was tested in brain slices and found not to alter tau mobility. However, combinations of FK-520 (5 microM) and okadaic acid (100 nM) caused tau mobility shifts similar to those seen after 10 microM okadaic acid treatment; similar results were seen using the calcineurin-selective inhibitor cypermethrin. Treatment of human slices with 10 microM okadaic acid decreased both protein phosphatase 2A and calcineurin activity; FK-520 inhibited only protein phosphatase 2B activity. A proposed tau-directed kinase, 42-kDa mitogen-activated protein kinase (p42mapk), was activated by okadaic acid (> 100 nM) but not FK-520 (5 microM). Nerve growth factor (100 ng/ml) activated p42mapk, particularly when used in combination with 100 nM okadaic acid; changes in tau mobility were seen when this kinase was activated. Forskolin (2 microM) antagonized the effects of nerve growth factor on both p42mapk activity and tau phosphorylation; forskolin alone had little effect on PHF-like tau formation induced by phosphatase inhibitors. These results outline complex interactions between tau-directed protein kinases and protein phosphatases and suggest potential sites for therapeutic intervention.

Asunto(s)

Encéfalo/metabolismo , Fosfoproteínas Fosfatasas/fisiología , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Tirosina Quinasas/metabolismo , Proteínas tau/metabolismo , Secuencia de Aminoácidos , Calcineurina , Proteínas de Unión a Calmodulina/análisis , Colforsina/farmacología , Humanos , Proteína Quinasa 1 Activada por Mitógenos , Datos de Secuencia Molecular , Factores de Crecimiento Nervioso/farmacología , Fosfoproteínas Fosfatasas/análisis , Fosforilación , Proteína Fosfatasa 2

RESUMEN

Two heat-stable protein inhibitors of protein phosphatase 2A (PP2A), tentatively designated I1PP2A and I2PP2A, have been purified to apparent homogeneity from extracts of bovine kidney. The purified preparations of I1PP2A exhibited an apparent M(r) approximately 30,000 and 250,000 as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and gel permeation chromatography on Sephacryl S-300, respectively. In contrast, the purified preparations of I2PP2A exhibited an apparent M(r) approximately 20,000 and 80,000 as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and gel permeation chromatography on Sephacryl S-200, respectively. The purified preparations of I1PP2A and I2PP2A inhibited PP2A with 32P-labeled myelin basic protein, 32P-labeled histone H1, 32P-labeled pyruvate dehydrogenase complex, 32P-labeled phosphorylase, and protamine kinase as substrates. By contrast, I1PP2A and I2PP2A exhibited little effect, if any, on the activity of PP2A with 32P-labeled casein, and did not prevent the autodephosphorylation of PP2A in incubations with the autophosphorylation-activated protein kinase [Guo, H., & Damuni, Z. (1993) Proc. Natl. Acad. Sci. U.S.A. 90, 2500-2504]. The purified preparations of I1PP2A and I2PP2A had little effect, if any, on the activities of protein phosphatase 1, protein phosphatase 2B, protein phosphatase 2C, and pyruvate dehydrogenase phosphatase. With 32P-labeled MBP as a substrate, kinetic analysis according to Henderson showed that I1PP2A and I2PP2A were noncompetitive and displayed a Ki of about 30 and 25 nM, respectively. Following cleavage with Staphylococcus aureus V8 protease, I1PP2A and I2PP2A displayed distinct peptide patterns, indicating that these inhibitor proteins are the products of distinct genes. The N-terminal amino acid sequences of the purified preparations indicate that I1PP2A and I2PP2A are novel proteins.

Asunto(s)

Calor , Riñón/enzimología , Fosfoproteínas Fosfatasas/antagonistas & inhibidores , Proteínas/aislamiento & purificación , Secuencia de Aminoácidos , Animales , Caseínas/metabolismo , Bovinos , Cromatografía en Gel , Proteínas Cromosómicas no Histona , Proteínas de Unión al ADN , Estabilidad de Medicamentos , Electroforesis en Gel de Poliacrilamida , Chaperonas de Histonas , Histonas/metabolismo , Péptidos y Proteínas de Señalización Intracelular , Datos de Secuencia Molecular , Peso Molecular , Proteína Básica de Mielina/metabolismo , Proteínas Nucleares , Mapeo Peptídico , Fosfoproteínas Fosfatasas/metabolismo , Radioisótopos de Fósforo , Fosforilación , Protamina Quinasa/metabolismo , Proteína Fosfatasa 1 , Proteína Fosfatasa 2 , Proteínas/química , Proteínas/farmacología , Complejo Piruvato Deshidrogenasa/metabolismo , Proteínas de Unión al ARN , Factores de Transcripción

RESUMEN

Phosphorylation of the catalytic subunit of protein phosphatase 2A (PP2A) on threonines with a distinct autophosphorylation-activated protein kinase [Guo and Damuni (1993) Proc. Natl. Acad. Sci. USA 90, 2500-2504] inactivated the phosphatase with 32P-labelled myelin basic protein prepared by incubation with the kinase domain of the epidermal growth factor receptor, the src-family protein kinases p56lck and p60c-src, myelin basic protein kinase-1, or protamine kinase. Phosphoamino acid analysis demonstrated that the kinase domain of the epidermal growth factor receptor, p56lck and p60c-src phosphorylated myelin basic protein on tyrosines, that the protamine kinase phosphorylated myelin basic protein on serines, and that myelin basic protein kinase-1 phosphorylated myelin basic protein on threonines. The results demonstrate that the autophosphorylation-activated protein kinase not only inactivates the protein serine/threonine phosphatase, but also the protein tyrosine phosphatase activity of PP2A. This autophosphorylation-activated protein kinase-mediated inactivation of PP2A may, in response to extracellular stimuli, not only contribute to the enhanced phosphorylation of cellular proteins on serines and threonines but also on tyrosines.

Asunto(s)

Fosfoproteínas Fosfatasas/antagonistas & inhibidores , Proteínas Quinasas/metabolismo , Proteínas Tirosina Fosfatasas/antagonistas & inhibidores , Adenosina Trifosfato/metabolismo , Proteínas Quinasas Dependientes de Calcio-Calmodulina/metabolismo , Activación Enzimática , Receptores ErbB/metabolismo , Glucógeno Sintasa Quinasa 3 , Cinética , Proteína Tirosina Quinasa p56(lck) Específica de Linfocito , Radioisótopos de Fósforo , Fosforilación , Fosfoserina/análisis , Fosfotreonina/análisis , Fosfotirosina , Protamina Quinasa/metabolismo , Proteína Fosfatasa 2 , Proteínas Tirosina Quinasas/metabolismo , Proteínas Proto-Oncogénicas pp60(c-src)/metabolismo , Proteínas Recombinantes/metabolismo , Especificidad por Sustrato , Tirosina/análogos & derivados , Tirosina/análisis

RESUMEN

Phosphorylation in vivo of several proteins in the mammalian heterogeneous nuclear ribonucleoprotein complex (hnRNP), including A1, has been observed and proposed as a regulatory step in pre-mRNA splicing [Maryland, S. H., Dwen, P., & Pederson, T. (1993) Proc. Natl. Acad. Sci. U.S.A. 90, 7764-7768]. We examined the ability of recombinant hnRNP protein A1 to act as a substrate for a number of purified Ser/Thr protein kinases in vitro. A survey of seven protein kinases showed that A1 was heavily phosphorylated by protein kinase C (PKC) and also was phosphorylated by casein kinase II, protamine kinase, and protein kinase A. In contrast, autophosphorylation-activated protein kinase and two forms of myelin basic protein kinase failed to phosphorylate A1. Proteolysis with trypsin and V8 protease revealed that PKC phosphorylates A1 at three main sites, two in the N-terminal domain (spanning residues 2-196) and one in the C-terminal domain (spanning residues 197-320). Amino acid sequencing revealed that these sites were Ser95, Ser192, and Ser199; phosphorylation at Ser192 was more abundant than at Ser95 and Ser199. Phosphorylation by PKC inhibited the strand annealing activity of A1. Protein phosphatase 2A, but not protein phosphatase 1, dephosphorylated A1 and reversed the inhibitory effect of PKC phosphorylation on the strand annealing activity. A conformational change in the C-terminal domain of A1 was observed upon PKC phosphorylation, and this was associated with a decrease in A1's affinity for single-stranded polynucleotides. The results are consistent with a role of phosphorylation of A1 in regulating its strand annealing activity in vivo.

Asunto(s)

Ribonucleoproteína Heterogénea-Nuclear Grupo A-B , Proteínas Quinasas/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Ribonucleoproteínas/metabolismo , Secuencia de Aminoácidos , Animales , Proteínas Quinasas Dependientes de Calcio-Calmodulina/metabolismo , Quinasa de la Caseína II , Dicroismo Circular , Secuencia de Consenso , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Electroforesis en Gel de Poliacrilamida , Glucógeno Sintasa Quinasa 3 , Ribonucleoproteína Nuclear Heterogénea A1 , Ribonucleoproteínas Nucleares Heterogéneas , Cinética , Mamíferos , Datos de Secuencia Molecular , Peso Molecular , Fosfoproteínas Fosfatasas/metabolismo , Fosforilación , Protamina Quinasa/metabolismo , Conformación Proteica , Proteína Quinasa C/metabolismo , Proteína Fosfatasa 1 , Proteína Fosfatasa 2 , Precursores del ARN/metabolismo , Empalme del ARN , ARN Nuclear Heterogéneo/metabolismo , Ratas , Proteínas Recombinantes/química , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismo , Ribonucleoproteínas/química , Ribonucleoproteínas/aislamiento & purificación

RESUMEN

The catalytic C subunit of protein phosphatase 2A2 was methylated with an apparent km of about 0.1 microM by purified preparations of a methyltransferase from bovine brain. This methylation was inhibited by okadaic acid and microcystin-LR half-maximally at 40 nM and 60 nM, respectively. The extent of inhibition depended on the protein phosphatase concentration in the incubations, but was independent of the methyltransferase concentration. The results demonstrate that okadaic acid and microcystin-LR directly inhibit the methylation of protein phosphatase 2A. The results are consistent with the idea that okadaic acid and microcystin-LR act, at least in part, by binding to the carboxyl terminus of the C subunit of protein phosphatase 2A thereby preventing access of the methyltransferase to its target site, the C subunit carboxyl terminal Leu309.

Asunto(s)

Éteres Cíclicos/farmacología , Metiltransferasas/metabolismo , Péptidos Cíclicos/farmacología , Fosfoproteínas Fosfatasas/metabolismo , Animales , Sitios de Unión , Encéfalo/enzimología , Bovinos , Electroforesis en Gel de Poliacrilamida , Cinética , Toxinas Marinas , Metilación , Metiltransferasas/antagonistas & inhibidores , Microcistinas , Ácido Ocadaico , Proteína Fosfatasa 2

RESUMEN

Microtubule-associated protein tau is abnormally hyperphosphorylated and forms the major protein subunit of paired helical filaments (PHF) in Alzheimer disease brains. The abnormally phosphorylated sites Ser-199, Ser-202, Ser-396 and Ser-404 but not Ser-46 and Ser-235 of Alzheimer tau were found to be dephosphorylated by protein phosphatase-1 and this dephosphorylation was activated by Mn2+. In contrast, protein phosphatase-2C did not dephosphorylate any of these sites. Both protein phosphatase-1 and -2C had high activities towards [32P]tau phosphorylated by cAMP-dependent protein kinase. These results suggest that both protein phosphatase-1 and -2C might be associated with normal phosphorylation state of tau, but only the former and not the latter phosphatase is involved in its abnormal phosphorylation in Alzheimer disease.

Asunto(s)

Enfermedad de Alzheimer/metabolismo , Fosfoproteínas Fosfatasas/metabolismo , Proteínas tau/metabolismo , Anciano , Enfermedad de Alzheimer/enzimología , Animales , Bovinos , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Humanos , Masculino , Persona de Mediana Edad , Fosforilación , Proteína Fosfatasa 1 , Conejos , Serina/metabolismo

RESUMEN

Two myelin basic protein kinases designated MBPK-1 and MBPK-2 were purified to apparent homogeneity from extracts of bovine kidney cortex. The purified preparations exhibited an apparent M(r) approximately 40,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and approximately 42,000 (MBPK-1) and 45,000 (MBPK-2) by gel permeation chromatography. Up to 0.4 and 1.8 mol of phosphoryl groups were incorporated per mol of MBPK-1 and MBPK-2, respectively, on threonines following incubation with ATP. Autophosphorylation, incubation with protein phosphatase 2A2 (PP2A2), CD45, or T-cell protein tyrosine phosphatase did not affect MBPK-1 activity. Autophosphorylation increased by about 3-fold MBPK-2 activity. This autophosphorylation and activation was reversed by PP2A2 but not by CD45 or T-cell protein tyrosine phosphatase. MBPK-1 and MBPK-2 displayed a positive reaction with an antibody to mitogen-activated protein kinase. Purified preparations of protamine kinase were activated by about 1.5-6-fold and, after inactivation with PP2A2, were reactivated by about 30% by MBPK-1 and MBPK-2. Activation and reactivation correlated with the incorporation, respectively, of 0.1-0.5 and 0.5 mol of phosphoryl groups/mol of the protamine kinase on serines. The results show that MBPK-1 and MBPK-2 are protamine kinase-activating kinases and suggest that MBPK-1 and MBPK-2 may be related to mitogen-activated protein kinase.

Asunto(s)

Isoenzimas/metabolismo , Corteza Renal/enzimología , Proteínas Quinasas/metabolismo , Secuencia de Aminoácidos , Animales , Western Blotting , Proteínas Quinasas Dependientes de Calcio-Calmodulina , Catálisis , Bovinos , Cromatografía en Gel , Electroforesis en Gel de Poliacrilamida , Activación Enzimática , Glucógeno Sintasa Quinasa 3 , Isoenzimas/aislamiento & purificación , Datos de Secuencia Molecular , Fosforilación , Protamina Quinasa , Proteínas Quinasas/inmunología , Proteínas Quinasas/aislamiento & purificación

RESUMEN

Protein phosphatase 2A2 is inactivated by phosphorylation following incubation with purified preparations of an autophosphorylation-activated protein kinase (Hong Guo and Zahi Damuni (1992) Proc. Natl. Acad. Sci. U.S.A. 90, 2500-2504). This protein kinase was purified about 250,000-fold from extracts of bovine kidney to apparent homogeneity. The purified preparations exhibited a single polypeptide of apparent M(r) approximately 36,000. Up to 1 mol of phosphoryl groups was incorporated per mol of the purified kinase following incubation with ATP. This autophosphorylation reaction (t1/2 approximately 0.5-1 min) was accompanied by a approximately 10-fold activation of the kinase. Autophosphorylation and activation were reversed by protein phosphatase 2A2 or the catalytic subunit of protein phosphatase 1. Phosphoamino acid analysis indicated that the kinase underwent autophosphorylation on threonines. The rate of autophosphorylation was independent of the concentration of the enzyme and a slope of 0.97 (gamma = 0.998) was obtained by van't Hoff's plot indicating that autoposphorylation was intramolecular. Relative to myelin basic protein, the enzyme exhibited about 8, 62, 130, 33, 5, and < 0.1% activity with histones H1, H2A, H2B, H3, and H4 and with glycogen synthase alpha, respectively. Heparin inhibited the activity of the enzyme half-maximally at about 20 micrograms/ml. The results indicate that this autophosphorylation-activated kinase is a new protein kinase.

Asunto(s)

Riñón/enzimología , Fosfoproteínas Fosfatasas/metabolismo , Proteínas Serina-Treonina Quinasas/aislamiento & purificación , Proteínas Serina-Treonina Quinasas/metabolismo , Adenosina Trifosfato/metabolismo , Animales , Bovinos , Cromatografía de Afinidad , Cromatografía DEAE-Celulosa , Cromatografía en Gel , Cromatografía por Intercambio Iónico , Electroforesis en Gel de Poliacrilamida , Cinética , Peso Molecular , Fosfoproteínas Fosfatasas/antagonistas & inhibidores , Fosforilación , Proteína Fosfatasa 1 , Proteína Fosfatasa 2 , Especificidad por Sustrato

RESUMEN

Purified preparations of a distinct autophosphorylation-activated protein kinase from bovine kidney phosphorylated and inactivated purified preparations of protein phosphatase 2A2 (PP2A2) by about 80% with the autophosphorylation-activated protein kinase, protamine kinase, and 32P-labeled myelin basic protein as substrates. Analysis of incubations performed in the presence of 0.2 mM [gamma-32P]ATP by autoradiography following SDS/PAGE and by FPLC gel permeation chromatography on Superose 12 demonstrated that the catalytic subunit of PP2A2 was phosphorylated in the incubation mixtures containing the kinase and phosphatase. Up to 0.3 mol of phosphate groups was incorporated per mol of the catalytic subunit of PP2A2 following incubation with the kinase. This phosphorylation was enhanced about 5-fold in the presence of 0.4 microM microcystin-LR. In addition, up to 1 mol of phosphate groups was incorporated per mol of the PP2A2 subunit of apparent M(r) approximately 60,000 when microcystin-LR was included. Analysis by thin-layer chromatography indicated that PP2A2 catalyzed an autodephosphorylation reaction which was inhibited by microcystin-LR. Phospho amino acid analysis showed that the catalytic subunit of PP2A2 was phosphorylated on threonine residues by the autophosphorylation-activated protein kinase. Together with previous observations, the results suggest that inactivation of PP2A by phosphorylation catalyzed by the autophosphorylation-activated protein kinase could contribute to the marked increase in the phosphorylation of cellular proteins in response to insulin and other mitogens.

Asunto(s)

Fosfoproteínas Fosfatasas/metabolismo , Proteínas Quinasas/metabolismo , Adenosina Trifosfato/metabolismo , Aminoácidos/análisis , Animales , Autorradiografía , Bovinos , Cromatografía Líquida de Alta Presión , Electroforesis en Gel de Poliacrilamida , Activación Enzimática , Corteza Renal/enzimología , Cinética , Proteína Básica de Mielina/metabolismo , Fosfoproteínas Fosfatasas/antagonistas & inhibidores , Fosfoproteínas Fosfatasas/aislamiento & purificación , Radioisótopos de Fósforo , Fosforilación , Proteínas Quinasas/aislamiento & purificación , Proteína Fosfatasa 2

RESUMEN

Purified preparations of a protamine protein kinase from bovine kidney cytosol [Damuni, Amick & Sneed (1989) J. Biol. Chem. 264, 6412-6416] were inactivated after incubation with near-homogeneous preparations of protein phosphatase 2A1 and protein phosphatase 2A2. These protein phosphatase 2A-mediated inactivations of the protamine kinase were unaffected by highly purified preparations of inhibitor 2, but were prevented when the incubations were performed in the presence of 100 nM microcystin-LR, 100 nM okadaic acid or 0.2 mM-ATP. By contrast, highly purified preparations of protein phosphatase 2B, protein phosphatase 2C, the catalytic subunit of protein phosphatase 1, and two forms of a protein tyrosine phosphatase, designated PTPase 1B and T-cell PTPase, had little effect, if any, on protamine kinase activity. Purified preparations of the protamine kinase did not react with anti-phosphotyrosine antibodies, as determined by Western blotting and immunoprecipitation analysis. The results indicate that protein phosphatase 2A is a specific protamine-kinase-inactivating phosphatase.

Asunto(s)

Fosfoproteínas Fosfatasas/metabolismo , Inhibidores de Proteínas Quinasas , Animales , Bovinos , Escherichia coli , Cinética , Protamina Quinasa , Proteína Fosfatasa 1 , Proteína Fosfatasa 2

RESUMEN

About an eightfold increase in protamine kinase activity was detected following extraction of highly purified microsomes from bovine kidney with 1% Triton X-100. Relative to the soluble fraction, the microsomes contained about 30% protamine kinase activity. The microsomal protamine kinase was purified to apparent homogeneity. The purified enzyme exhibited an apparent M(r) approximately 45,000 as estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and by gel permeation chromatography on Sephacryl S-200. Relative to protamine, the purified kinase exhibited about 100% activity with the synthetic peptide RRLSSLRA and about 5, 8, and less than 0.1% activity with casein, histone H2B, and histone H1, respectively. The purified kinase phosphorylated several 40 S ribosome polypeptides. One of these polypeptides was identified as ribosomal protein S6 by N-terminal sequencing. About 2.5 mol of phosphoryl groups was incorporated per mole of ribosomal protein S6 following incubation of the 40 S ribosomes with the purified kinase. Following incubation with protein phosphatase 2A2, purified preparations of the protamine kinase were inactivated. These properties were identical to those of purified preparations of a protamine kinase from extracts of bovine kidney cytosol (Z. Damuni, G.D. Amick, and T.R. Sneed, 1989, J. Biol. Chem. 264, 6412-6418). Near identical peptide patterns were obtained following incubation of purified preparations of the microsomal and cytosolic protamine kinases with Staphylococcus aureus V8 proteinase. The results indicate that a form of the cytosolic protamine kinase is present in microsomes.

Asunto(s)

Riñón/enzimología , Microsomas/enzimología , Proteínas Quinasas/aislamiento & purificación , Proteínas Quinasas/metabolismo , Animales , Bovinos , Cromatografía de Afinidad , Cromatografía en Gel , Cromatografía por Intercambio Iónico , Cinética , Peso Molecular , Fosforilación , Protamina Quinasa , Ribosomas/metabolismo , Especificidad por Sustrato

RESUMEN

Up to 1 mol of phosphoryl groups was incorporated per mol of eukaryotic protein synthesis initiation factor (eIF) 4E following incubation of purified preparations of this factor with purified preparations of a protamine kinase from bovine kidney cytosol. By contrast, purified preparations of two forms of mitogen-activated protein kinase, casein kinase II and two forms of a distinct autophosphorylation-activated protein kinase exhibited little activity, if any, with eIF-4E. Together with previous observations, the results indicate that the protamine kinase could contribute to the insulin-stimulated phosphorylation of eIF-4E.

Asunto(s)

Factores de Iniciación de Péptidos/metabolismo , Proteínas Quinasas/metabolismo , Animales , Bovinos , Factor 4E Eucariótico de Iniciación , Riñón/enzimología , Riñón/metabolismo , Factores de Iniciación de Péptidos/aislamiento & purificación , Fosforilación , Protamina Quinasa , Proteínas Quinasas/aislamiento & purificación , Especificidad por Sustrato