RESUMEN
AIMS: Granulovacuolar degeneration involves the accumulation of large, double membrane-bound bodies within certain neurones during the course of Alzheimer's disease (AD) and other adult-onset dementias. Because of the two-layer membrane morphology, it has been proposed that the bodies are related to autophagic organelles. The aim of this study was to test this hypothesis, and determine the approximate stage at which the pathway stalls in AD. METHODS: Spatial colocalization of autophagic and endocytic markers with casein kinase 1 delta, a marker for granulovacuolar degeneration (GVD) bodies, was evaluated in hippocampal sections prepared from post mortem Braak stage IV and V AD cases using double-label confocal fluorescence microscopy. RESULTS: GVD bodies colocalized weakly with early-stage autophagy markers LC3 and p62, but strongly with late-stage marker lysosome-associated membrane protein 1 (LAMP1), which decorated their surrounding membranes. GVD bodies also colocalized strongly with charged multivesicular body protein 2B (CHMP2B), which colocalized with the core granule, but less strongly with lysosomal marker cathepsin D. CONCLUSIONS: The resultant immunohistochemical signature suggests that granulovacuolar degeneration bodies (GVBs) do contain late-stage autophagic markers, and accumulate at the nexus of autophagic and endocytic pathways. The data further suggest that failure to complete autolysosome formation may be an important correlate of GVB accumulation.
Asunto(s)
Enfermedad de Alzheimer/patología , Autofagia , Encéfalo/patología , Lisosomas/patología , Neuronas/patología , Anciano , Anciano de 80 o más Años , Femenino , Humanos , Inmunohistoquímica , Masculino , Microscopía Confocal , Persona de Mediana Edad , Orgánulos/patologíaRESUMEN
A mutant catalytic subunit of adenosine 3',5'-monophosphate (cAMP)-dependent protein kinase has been isolated from Saccharomyces cerevisiae that is no longer subject to regulation yet retains its catalytic activity. Biochemical analysis of the mutant subunit indicates a 100-fold decreased affinity for the regulatory subunit. The mutant catalytic subunit exhibits approximately a threefold increase in Michaelis constant for adenosine triphosphate and peptide cosubstrates, and is essentially unchanged in its catalytic rate. The nucleotide sequence of the mutant gene contains a single nucleotide change resulting in a threonine-to-alanine substitution at amino acid 241. This residue is conserved in other serine-threonine protein kinases. These results identify this threonine as an important contact between catalytic and regulatory subunits but only a minor contact in substrate recognition.
Asunto(s)
AMP Cíclico/farmacología , Proteínas Quinasas/genética , Saccharomyces cerevisiae/genética , Secuencia de Aminoácidos , Catálisis , Genes Fúngicos , Cinética , Sustancias Macromoleculares , Datos de Secuencia Molecular , Mutación , Fosforilación , Proteínas Quinasas/metabolismo , Saccharomyces cerevisiae/enzimología , Relación Estructura-Actividad , TreoninaRESUMEN
The GCS1 gene of the budding yeast Saccharomyces cerevisiae mediate the resumption of cell proliferation from the starved, stationary-phase state. Here we identify yeast genes that, in increased dosages, overcome the growth defect of gcs1 delta mutant cells. Among these are YCK1 (CK12) and YCK2 (CKI1), encoding membrane-associated casein kinase I, and YCK3, encoding a novel casein kinase I isoform. Some Yck3p gene product was found associated with the plasma membrane, like Yck1p and Yck2p, but most confractionated with the nucleus, like another yeast casein kinase I isoform, Hrr25p. Genetic studies showed that YCK3 and HRR25 constitute an essential gene family and that Yck3p can weakly substitute for Yck1p-Yck2p. For gcs1 delta suppression, both a protein kinase domain and a C-terminal prenylation motif were shown to be necessary. An impairment in endocytosis was found for gcs1 delta mutant cells, which was alleviated by an increased YCK2 gene dosage. The ability of an increased casein kinase I gene dosage to suppress the effects caused by the absence of Gcs1p suggests that Gcs1p and Yck1p-Yck2p affect parallel pathways.
Asunto(s)
Quinasa de la Caseína I , Ciclo Celular , Isoenzimas/metabolismo , Proteínas Quinasas/química , Proteínas Quinasas/metabolismo , Estructura Secundaria de Proteína , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/fisiología , Secuencia de Aminoácidos , Animales , Caseína Quinasas , Bovinos , División Celular , Genes Fúngicos , Genotipo , Isoenzimas/biosíntesis , Isoenzimas/química , Modelos Estructurales , Datos de Secuencia Molecular , Mutagénesis , Proteína Quinasa C/metabolismo , Proteínas Quinasas/biosíntesis , Prenilación de Proteína , Mapeo Restrictivo , Saccharomyces cerevisiae/citología , Saccharomyces cerevisiae/crecimiento & desarrollo , Homología de Secuencia de Aminoácido , Supresión GenéticaRESUMEN
Two cDNAs encoding casein kinase-1 have been isolated from a yeast cDNA library and termed CKI1 and CKI2. Each clone encodes a protein of approximately 62,000 Da containing a highly conserved protein kinase domain surrounded by variable amino- and carboxy-terminal domains. The proteins also contain two conserved carboxy-terminal cysteine residues that comprise a consensus sequence for prenylation. Consistent with this posttranslational modification, cell fractionation experiments demonstrate that intact CKI1 is found exclusively in yeast cell membranes. Gene disruption experiments reveal that, although neither of the two CKI genes is essential by itself, at least one CKI gene is required for yeast cell viability. Spores deficient in both CKI1 and CKI2 fail to grow and, therefore, either fail to germinate or arrest as small cells before bud emergence. These results suggest that casein kinase-1, which is distributed widely in nature, plays a pivotal role in eukaryotic cell regulation.
Asunto(s)
Genes Fúngicos/genética , Proteínas Quinasas/genética , Saccharomyces cerevisiae/genética , Secuencia de Aminoácidos , Secuencia de Bases , Caseína Quinasas , Membrana Celular/enzimología , Mapeo Cromosómico , Clonación Molecular , ADN de Hongos/química , ADN de Hongos/genética , ADN de Hongos/aislamiento & purificación , Datos de Secuencia Molecular , Fenotipo , Proteínas Quinasas/química , Proteínas Quinasas/aislamiento & purificación , Saccharomyces cerevisiae/enzimologíaRESUMEN
We have examined the activity and substrate specificity of the Saccharomyces cerevisiae Hrr25p and the Schizosaccharomyces pombe Hhp1, Hhp2, and Cki1 protein kinase isoforms. These four gene products are isotypes of casein kinase I (CKI), and the sequence of these protein kinases predicts that they are protein serine/threonine kinases. However, each of these four protein kinases, when expressed in Escherichia coli in an active form, was recognized by anti-phosphotyrosine antibodies. Phosphoamino acid analysis of 32P-labeled proteins showed phosphorylation on serine, threonine, and tyrosine residues. The E. coli produced forms of Hhp1, Hhp2, and Cki1 were autophosphorylated on tyrosine, and both Hhp1 and Hhp2 were capable of phosphorylating the tyrosine-protein kinase synthetic peptide substrate polymer poly-E4Y1. Immune complex protein kinases assays from S. pombe cells showed that Hhp1-containing precipitates were associated with a protein-tyrosine kinase activity, and the Hhp1 present in these immunoprecipitates was phosphorylated on tyrosine residues. Although dephosphorylation of Hhp1 and Hhp2 by Ser/Thr phosphatase had little effect on the specific activity, tyrosine dephosphorylation of Hhp1 and Hhp2 caused a 1.8-to 3.1-fold increase in the Km for poly-E4Y1 and casein. These data demonstrate that four different CKI isoforms from two different yeasts are capable of protein-tyrosine kinase activity and encode dual-specificity protein kinases.
Asunto(s)
Isoenzimas/metabolismo , Proteínas Quinasas/metabolismo , Saccharomyces cerevisiae/enzimología , Schizosaccharomyces/enzimología , Secuencia de Aminoácidos , Caseína Quinasas , Genes Fúngicos , Isoenzimas/genética , Datos de Secuencia Molecular , Péptidos/química , Fosforilación , Proteínas Quinasas/genética , Saccharomyces cerevisiae/genética , Schizosaccharomyces/genética , Especificidad por Sustrato , TirosinaRESUMEN
Inhibitor-2 was phosphorylated by casein kinase-II in vitro at a rate similar to that of glycogen synthase, a physiological substrate of this protein kinase. The major phosphorylation sites were identified as serines-86, -120 and -121, the peptide containing serines-120 and -121 being labelled about 2.5-fold more rapidly than that containing serine-86. The 13 residues C-terminal to serine-121 (SGEEDSDLSPEERE) contain seven acidic amino acids, while the six residues following serine-86 (SDTETTE) contain three. These results are consistent with the known specificity requirements of casein kinase-II. The three serines are C-terminal to the threonine (residue 72) whose phosphorylation by glycogen synthase kinase-3 is potentiated by prior phosphorylation with casein kinase-II. This reinforces the view that a C-terminal phosphoserine residue is important for the specificity of glycogen synthase kinase-3. Identification of the residues phosphorylated by casein kinase-II will facilitate further studies on the in vivo phosphorylation state of inhibitor-2.
Asunto(s)
Músculos/enzimología , Proteínas Quinasas/metabolismo , Proteínas/metabolismo , Secuencia de Aminoácidos , Animales , Sitios de Unión , Proteínas Quinasas Dependientes de Calcio-Calmodulina , Caseína Quinasas , Cromatografía Líquida de Alta Presión , Glucógeno Sintasa Quinasas , Fosforilación , Conejos , Relación Estructura-Actividad , Tripsina/metabolismoRESUMEN
The distribution of casein kinase 1 delta (Cki delta) was studied by immunohistochemistry and correlated with other pathological hallmarks in Alzheimer's disease (AD), Down syndrome (DS), progressive supranuclear palsy (PSP), parkinsonism dementia complex of Guam (PDC), Pick's disease (PiD), pallido-ponto-nigral degeneration (PPND), Parkinson's disease (PD), dementia with Lewy bodies (DLB), amyotrophic lateral sclerosis (ALS), and elderly controls. Cki delta was found to be associated generally with granulovacuolar bodies and tau-containing neurofibrillary tangles in AD, DS, PSP, PDC, PPND, and controls, and Pick bodies and ballooned neurons in PiD. It was not associated with tau-containing inclusions in astroglia and oligodendroglia in PPND, PSP, and PDC. It was also not associated with tau-negative Lewy bodies in PD and DLB, Hirano bodies in PDC, Marinesco bodies in PD, AD, and controls and "skein"-like inclusions in anterior motor neurons in ALS. The colocalization of the kinase Cki delta and its apparent substrate tau suggests a function for Cki delta in the abnormal processing of tau.
Asunto(s)
Enfermedades Neurodegenerativas/metabolismo , Enfermedades Neurodegenerativas/patología , Proteínas Quinasas/metabolismo , Proteínas tau/metabolismo , Anciano , Anciano de 80 o más Años , Enfermedad de Alzheimer/metabolismo , Enfermedad de Alzheimer/patología , Esclerosis Amiotrófica Lateral/metabolismo , Esclerosis Amiotrófica Lateral/patología , Especificidad de Anticuerpos , Encéfalo/enzimología , Encéfalo/patología , Caseína Quinasas , Femenino , Humanos , Inmunohistoquímica , Cuerpos de Inclusión/química , Cuerpos de Inclusión/enzimología , Cuerpos de Lewy/química , Cuerpos de Lewy/enzimología , Masculino , Persona de Mediana Edad , Ovillos Neurofibrilares/química , Ovillos Neurofibrilares/enzimología , Neuroglía/química , Neuroglía/enzimología , Enfermedad de Parkinson/metabolismo , Enfermedad de Parkinson/patología , Proteínas Quinasas/análisis , Proteínas Quinasas/inmunología , Proteínas tau/análisis , Proteínas tau/inmunologíaRESUMEN
Rat liver nuclei contain a protein phosphatase that is indistinguishable from the catalytic subunit of protein phosphatase-1 in its molecular mass, sensitivity to inhibitor-1 and inhibitor-2 and specificity for the beta-subunit of phosphorylase kinase. This activity is not bound to the outer nuclear membrane, but located within the nucleus. The average level of protein phosphatase-1 activity in nuclei is at least 5-fold higher than its average extranuclear concentration.
Asunto(s)
Hígado/enzimología , Fosfoproteínas Fosfatasas/análisis , Animales , Núcleo Celular/enzimología , AMP Cíclico/fisiología , Femenino , Fosfoproteínas Fosfatasas/antagonistas & inhibidores , Fosfoproteínas Fosfatasas/metabolismo , Fosforilasa Fosfatasa/análisis , Proteínas Quinasas/análisis , Proteína Fosfatasa 1 , Ratas , Ratas EndogámicasRESUMEN
The casein kinase-1 (Ck1) family are serine/threonine specific protein kinases. They are highly associated with Alzheimer disease (AD) brain-derived tau filaments and granulovacuolar bodies. Recently we have demonstrated that one family member, Ckidelta, colocalizes with tau containing neurofibrillary tangles (NFTs) and other tau deposits in a number of neurodegenerative diseases. Here we show that the association in AD is accompanied by a sharp upregulation of Ckidelta mRNA in brain but not in peripheral organs. The degree of upregulation in AD brain is correlated with the degree of regional pathology. There was a 24.4-fold increase of Ckidelta mRNA in AD hippocampus compared with control, 8.04-fold in the amygdala, 7.45 in the entorhinal cortex and 7.30-fold in the midtemporal gyrus. These are areas with a high burden of NFTs, neuropil threads and dystrophic neurites. In areas almost devoid of this tau pathology, such as the caudate nucleus, occipital cortex and cerebellum, the increases in AD compared to control brain were only 2.21-, 1.89- and 1.87-fold, respectively. Western blot analysis showed that the upregulation of Ckidelta mRNA was paralleled by an upregulation of Ckidelta protein. These data establish that the association of Ckidelta with the tau pathology of AD is reflective of an increase in gene transcription. Since Alzheimer-like phosphoepitopes of tau can be generated by Ck1, the Ckidelta isoform may play an important role in this fundamental aspect of AD pathology.
Asunto(s)
Enfermedad de Alzheimer/metabolismo , Enfermedad de Alzheimer/fisiopatología , Encéfalo/metabolismo , Encéfalo/fisiopatología , Proteínas Quinasas/genética , Proteínas Quinasas/metabolismo , ARN Mensajero/metabolismo , Regulación hacia Arriba/fisiología , Anciano , Enfermedad de Alzheimer/patología , Western Blotting , Encéfalo/patología , Caseína Quinasas , Humanos , Proteínas Quinasas/análisis , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transcripción Genética/fisiologíaAsunto(s)
Diabetes Mellitus Experimental/terapia , Trasplante de Islotes Pancreáticos , Trasplante de Páncreas , Envejecimiento , Animales , Femenino , Feto , Prueba de Tolerancia a la Glucosa , Insulina/metabolismo , Secreción de Insulina , Islotes Pancreáticos/crecimiento & desarrollo , Masculino , Embarazo , Ratas , Ratas Endogámicas Lew , Trasplante HomólogoRESUMEN
Tau aggregation is an appealing target for therapeutic intervention. However, conformational change or aggregation needs to be targeted without inhibiting the normal biology of tau and its role in microtubule stabilization. The number of compound classes being tested at this time are very limited and include Congo red derivatives [2], anthraquinones (Pickhardt et al. 2005 [3], disputed in Crowe et al. 2007 [4]), 2,3-di(furan-2-yl)-quinoxalines , phenylthiazolyl-hydrazide (PTH) [5], polyphenols and porphyrins [6] and cyanine dyes [1, 7, 8]. Herein we have utilized a member of the cyanine dye family (C11) in an organotypic slice culture model of tangle formation. Our results demonstrate that C11 is capable of affecting tau polymerization in a biphasic, dose dependent manner. At submicromolar concentrations (0.001 microM) C11 reduced levels of aggregated tau. However, higher doses resulted in an increase in tau polymerization. These effects can also be seen at the level of individual filaments with changes in filament length and number mirroring the pattern seen via immunoblotting. In addition, this effect is achieved without altering levels of phosphorylation at disease and microtubule binding relevant epitopes.
Asunto(s)
Carbocianinas/farmacología , Ovillos Neurofibrilares/efectos de los fármacos , Ovillos Neurofibrilares/metabolismo , Tauopatías/tratamiento farmacológico , Tauopatías/metabolismo , Proteínas tau/efectos de los fármacos , Proteínas tau/metabolismo , Animales , Sitios de Unión/efectos de los fármacos , Sitios de Unión/fisiología , Carbocianinas/uso terapéutico , Relación Dosis-Respuesta a Droga , Evaluación Preclínica de Medicamentos/métodos , Ratones , Microtúbulos/efectos de los fármacos , Microtúbulos/metabolismo , Técnicas de Cultivo de Órganos , Polímeros/química , Relación Estructura-Actividad , Tauopatías/fisiopatologíaRESUMEN
Alzheimer's disease is diagnosed by postmortem detection of pathological lesions that accumulate in specific brain regions. Although the presence of both beta-amyloid plaques and tau-bearing neurofibrillary lesions defines Alzheimer's disease, the distribution of neurofibrillary lesions alone correlates strongly with neurodegeneration and cognitive decline. A whole-brain imaging test capable of detecting these lesions in premortem cases could have great potential for staging and differentially diagnosing Alzheimer's disease. Here we discuss the challenges in developing a whole-brain imaging approach for detection of this intracellular target.
Asunto(s)
Enfermedad de Alzheimer/diagnóstico , Encéfalo/patología , Diagnóstico por Imagen/métodos , Ovillos Neurofibrilares/patología , Tauopatías/diagnóstico , Péptidos beta-Amiloides/análisis , Péptidos beta-Amiloides/química , Péptidos beta-Amiloides/metabolismo , Unión Competitiva/fisiología , Biomarcadores/análisis , Encéfalo/metabolismo , Encéfalo/fisiopatología , Colorantes/química , Colorantes/metabolismo , Diagnóstico por Imagen/tendencias , Humanos , Ovillos Neurofibrilares/metabolismo , Unión Proteica/fisiología , Proteínas tau/análisis , Proteínas tau/química , Proteínas tau/metabolismoRESUMEN
The multifunctional Ca2+/calmodulin-dependent protein kinase purified from rat brain cytosol undergoes a self-phosphorylation or autophosphorylation reaction. Our conclusion that this reaction is autocatalytic is based on the following lines of evidence: The autophosphorylation reaction and the protein kinase activity toward other substrates are absolutely dependent on the presence of both Ca2+ and calmodulin; autophosphorylation and phosvitin kinase activity show a similar time course and indistinguishable heat lability; the reaction is a consistent property of every preparation of rat brain kinase; the reaction is present in both crude and highly purified preparations of similar kinases or isozymes from rat lung, spleen, heart, bovine brain, and a neuronal tissue from Aplysia californica, a marine mollusk; phosphorylation of the kinase subunits is not mimicked by addition of cAMP, cGMP, Ca2+ plus diglyceride, or addition of the cAMP-dependent protein kinase, and is not blocked by the heat-stable inhibitor protein of the cAMP-dependent protein kinase; and the reaction is intramolecular. Autophosphorylation results in the stoichiometric incorporation of phosphate into both the 51,000- and 60,000-dalton subunits.
Asunto(s)
Calcio/farmacología , Calmodulina/farmacología , Proteínas Quinasas/metabolismo , Animales , Encéfalo/enzimología , Citosol/enzimología , Técnicas In Vitro , Cinética , Peso Molecular , Fosforilación , Conformación Proteica , RatasRESUMEN
Cassette mutagenesis was used to synthesize an Escherichia coli expression library of unique phosphorylation sites. The cassette encodes a central serine residue surrounded by every combination of Ala, Arg, Gln, Glu, Gly, and Pro residues over a 7-residue segment (a total of 6(7) approximately 2.8 x 10(5) sequences). The cassette was inserted into the gene of a suitable carrier protein and expressed in E. coli with the T7 expression system, and the resultant library was subjected to solid-phase protein phosphorylation assays on nitrocellulose filters. When the library was screened with TPK1 delta, the modified catalytic subunit of the Saccharomyces cerevisiae cAMP-dependent protein kinase, individual colonies that expressed substrates for this kinase were identified. By DNA sequencing through the cassette region of positive clones, the consensus recognition sequence for TPK1 delta was deduced and found to conform with the well-established substrate selectivity of its mammalian homolog (Arg-Arg-Xaa-Ser). Because a large number of clones can be sequenced rapidly, and the positions of invariant residues composing a recognition site identified, this approach may be useful as a general screen of protein kinase substrate selectivity.
Asunto(s)
Proteínas Quinasas/metabolismo , Proteínas/metabolismo , Secuencia de Aminoácidos , Secuencia de Bases , ADN/química , ADN/genética , Electroforesis en Gel de Poliacrilamida , Escherichia coli/genética , Expresión Génica , Biblioteca de Genes , Datos de Secuencia Molecular , Mutagénesis Insercional , Fosforilación , Proteínas/química , Proteínas/genética , Especificidad por SustratoRESUMEN
A soluble Ca2+/calmodulin-dependent protein kinase has been purified from rat brain to near homogeneity by using casein as substrate. The enzyme was purified by using hydroxylapatite adsorption chromatography, phosphocellulose ion-exchange chromatography, Sepharose 6B gel filtration, affinity chromatography using calmodulin-Sepharose 4B, and ammonium sulfate precipitation. On sodium dodecyl sulfate (NaDodSO4)-polyacrylamide gels, the purified enzyme consists of three protein bands: a single polypeptide of 51 000 daltons and a doublet of 60 000 daltons. Measurements of the Stokes radius by gel filtration (81.3 +/- 3.7 A) and the sedimentation coefficient by sucrose density sedimentation (13.7 +/- 0.7 S) were used to calculate a native molecular mass of 460 000 +/- 29 000 daltons. The kinase autophosphorylated both the 51 000-dalton polypeptide and the 60 000-dalton doublet, resulting in a decreased mobility in NaDodSO4 gels. Comparison of the phosphopeptides produced by partial proteolysis of autophosphorylated enzyme reveals substantial similarities between subunits. These patterns, however, suggest that the 51 000-dalton subunit is not a proteolytic fragment of the 60 000-dalton doublet. Purified Ca2+/calmodulin-dependent casein kinase activity was dependent upon Ca2+, calmodulin, and ATP X Mg2+ or ATP X Mn2+ when measured under saturating casein concentrations. Co2+, Mn2+, and La3+ could substitute for Ca2+ in the presence of Mg2+ and saturating calmodulin concentrations. In addition to casein, the purified enzyme displayed a broad substrate specificity which suggests that it may be a "general" protein kinase with the potential for mediating numerous processes in brain and possibly other tissues.
Asunto(s)
Encéfalo/enzimología , Calcio/farmacología , Calmodulina/farmacología , Proteínas Quinasas/metabolismo , Animales , Calmodulina/metabolismo , Fenómenos Químicos , Química Física , Cromatografía , Estabilidad de Medicamentos , Activación Enzimática , Cinética , Sustancias Macromoleculares , Masculino , Peso Molecular , Fragmentos de Péptidos , Fosforilación , Proteínas Quinasas/aislamiento & purificación , Ratas , Ratas Endogámicas , Especificidad por SustratoRESUMEN
A truncated variant of TPK1, the yeast cAMP-dependent protein kinase catalytic subunit, was overexpressed in an engineered strain of Saccharomyces cerevisiae, purified by liquid chromatography, and crystallized from solutions of 2-propanol and magnesium at alkaline pH. The crystals are hexagonal dipyramids, space group P6(1)22 (P6(5)22), with unit-cell parameters a = b = 61 A, c = 320 A. Large single crystals suitable for diffraction analysis are obtainable by microseeding, and diffract beyond 2.8-A resolution. Crystal density measurements reveal 12 kinase monomers per unit cell with a single kinase monomer per asymmetric unit.
Asunto(s)
Proteínas Quinasas/química , Saccharomyces cerevisiae/enzimología , Secuencia de Bases , Catálisis , Cromatografía Liquida , Cristalización , Electroforesis en Gel de Poliacrilamida , Datos de Secuencia Molecular , Difracción de Rayos XRESUMEN
A casein kinase was highly purified from rabbit skeletal muscle whose substrate specificity and enzymatic properties were virtually identical to those of casein kinase-I from rabbit reticulocytes. Prolonged incubation of glycogen synthase with high concentrations of skeletal muscle casein kinase-I and Mg-ATP resulted in the incorporation of greater than 6 mol phosphate/mol subunit and decreased the activity ratio (+/- glucose-6P) from 0.8 to less than 0.02. The sites phosphorylated by casein kinase-I were all located in the N and C-terminal cyanogen bromide peptides, termed CB-1 and CB-2. At an incorporation of 6 mol phosphate/mol subunit, approximately equal to 2 mol/mol was present in CB-1 and approximately equal to 4 mol/mol in CB-2. Within CB-1, casein kinase-I phosphorylated the serines that were 3, 7 and 10 residues from the N-terminus of glycogen synthase, with minor phosphorylation at threonine-5. Within CB-2, approximately equal to 90% of the phosphate incorporated was located between residues 28 and 53, and at least five of the seven serine residues in this region were phosphorylated. The remaining 10% of phosphate incorporated into CB-2 was located between residues 98 and 123, mainly at a serine residue(s). Two of the major sites labelled by casein kinase-I (serine-3 and serine-10 of CB-1) are not phosphorylated by any other protein kinase. This will enable the role of casein kinase-I as a glycogen synthase kinase in vivo to be evaluated.
Asunto(s)
Glucógeno Sintasa/metabolismo , Músculos/enzimología , Proteínas Quinasas/metabolismo , Secuencia de Aminoácidos , Animales , Sitios de Unión , Caseína Quinasas , Cromatografía en Gel , Electroforesis en Gel de Poliacrilamida , Técnicas In Vitro , Fosforilación , Proteínas Quinasas/aislamiento & purificación , Conejos , Reticulocitos/enzimologíaRESUMEN
The subcellular distribution of three casein kinase I (CK1) homologs, encoded by the YCK1, YCK2, and HRR25 genes, has been determined in budding yeast through a combination of subcellular fractionation and immunofluorescence methods. Both Yck proteins are tightly associated with the plasma membrane or underlying cytoskeleton and require both high-salt and nonionic detergent for extraction. Association is mediated primarily by the prenylation motif found at the C terminus of both Yck proteins. In contrast, the third CK1 homolog, Hrr25p, is found predominantly in the nucleus and only partially in the plasma membrane. Despite partial colocalization with the Yck proteins, Hrr25p is unable to rescue the yck1 delta yck2 delta phenotype. However, a chimeric kinase containing the N-terminal kinase domain of Hrr25p and the C-terminal region of Yck2p contains full Yck activity in vivo. These data suggest that members of the casein kinase I family have distinct but partially overlapping distributions in the cell that are mediated by their unique C-terminal regions.
Asunto(s)
Quinasa de la Caseína I , Compartimento Celular , Membrana Celular/fisiología , Proteínas Quinasas/metabolismo , Prenilación de Proteína , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/fisiología , Secuencia de Bases , Biomarcadores , Caseína Quinasas , División Celular/fisiología , Fraccionamiento Celular , Análisis Mutacional de ADN , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Proteínas Quinasas/genética , Proteínas Quinasas/aislamiento & purificaciónRESUMEN
The cDNAs for two casein kinase-1 homologs, hhp1 and hhp2, have been isolated from Schizosaccharomyces pombe and characterized. Their corresponding genes reside on chromosomes II and I, respectively, and encode approximately 42-46 kDa proteins that are related structurally to the HRR25 gene product of budding yeast. On the basis of multiple sequence alignment, the CK1 family appears to consist of three main branches. We predict that the branch containing the hhp genes encodes nuclear kinases involved in the regulation of DNA metabolism.
Asunto(s)
Genes Fúngicos , Isoenzimas/biosíntesis , Familia de Multigenes , Filogenia , Proteínas Quinasas/biosíntesis , Schizosaccharomyces/enzimología , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Caseína Quinasas , Mapeo Cromosómico , Cromosomas Fúngicos , Clonación Molecular/métodos , ADN Complementario/aislamiento & purificación , Biblioteca de Genes , Isoenzimas/química , Isoenzimas/genética , Mamíferos , Datos de Secuencia Molecular , Proteínas Quinasas/química , Proteínas Quinasas/genética , Schizosaccharomyces/genéticaRESUMEN
The structure of TPK1delta, a truncated variant of the cAMP-dependent protein kinase catalytic subunit from Saccharomyces cerevisiae, was determined in an unliganded state at 2.8 A resolution and refined to a crystallographic R-factor of 19.4%. Comparison of this structure to that of its fully liganded mammalian homolog revealed a highly conserved protein fold comprised of two globular lobes. Within each lobe, root mean square deviations in Calpha positions averaged approximately equals 0.9 A. In addition, a phosphothreonine residue was found in the C-terminal domain of each enzyme. Further comparison of the two structures suggests that a trio of conformational changes accompanies ligand-binding. The first consists of a 14.7 degrees rigid-body rotation of one lobe relative to the other and results in closure of the active site cleft. The second affects only the glycine-rich nucleotide binding loop, which moves approximately equals 3 A to further close the active site and traps the nucleotide substrate. The third is localized to a C-terminal segment that makes direct contact with ligands and the ligand-binding cleft. In addition to resolving the conformation of unliganded enzyme, the model shows that the salient features of the cAMP-dependent protein kinase are conserved over long evolutionary distances.