ABSTRACT
Cyclin-dependent kinase 5 (Cdk5) is a multifunctional neuronal protein kinase that is required for neurite outgrowth and cortical lamination and that plays an important role in dopaminergic signaling in the neostriatum through phosphorylation of Thr-75 of DARPP-32 (dopamine and cAMP-regulated phosphoprotein, molecular mass 32 kDa). Casein kinase 1 (CK1) has been implicated in a variety of cellular functions such as DNA repair, circadian rhythm, and intracellular trafficking. In the neostriatum, CK1 has been found to phosphorylate Ser-137 of DARPP-32. However, first messengers for the regulation of Cdk5 or CK1 have remained unknown. Here we report that both Cdk5 and CK1 are regulated by metabotropic glutamate receptors (mGluRs) in neostriatal neurons. (S)-3,5-dihydroxyphenylglycine (DHPG), an agonist for group I mGluRs, increased Cdk5 and CK1 activities in neostriatal slices, leading to the enhanced phosphorylation of Thr-75 and Ser-137 of DARPP-32, respectively. The effect of DHPG on Thr-75, but not on Ser-137, was blocked by a Cdk5-specific inhibitor, butyrolactone. In contrast, the effects of DHPG on both Thr-75 and Ser-137 were blocked by CK1-7 and IC261, specific inhibitors of CK1, suggesting that activation of Cdk5 by mGluRs requires CK1 activity. In support of this possibility, the DHPG-induced increase in Cdk5 activity, measured in extracts of neostriatal slices, was abolished by CK1-7 and IC261. Treatment of acutely dissociated neurons with DHPG enhanced voltage-dependent Ca(2+) currents. This enhancement was eliminated by either butyrolactone or CK1-7 and was absent in DARPP-32 knockout mice. Together these results indicate that a CK1-Cdk5-DARPP-32 cascade may be involved in the regulation by mGluR agonists of Ca(2+) channels.
Subject(s)
Cyclin-Dependent Kinases/metabolism , Methoxyhydroxyphenylglycol/analogs & derivatives , Neostriatum/physiology , Neurons/physiology , Protein Kinases/metabolism , Receptors, Metabotropic Glutamate/physiology , Animals , Calcium Channels/physiology , Casein Kinases , Cyclin-Dependent Kinase 5 , Dopamine and cAMP-Regulated Phosphoprotein 32 , Enzyme Inhibitors/pharmacology , In Vitro Techniques , Kinetics , Male , Membrane Potentials/physiology , Methoxyhydroxyphenylglycol/pharmacology , Mice , Mice, Inbred C57BL , Neostriatum/drug effects , Nerve Tissue Proteins/metabolism , Patch-Clamp Techniques , Phosphoproteins/metabolism , Phosphorylation , Phosphoserine , Phosphothreonine , Receptors, Metabotropic Glutamate/drug effectsABSTRACT
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.
Subject(s)
Neurodegenerative Diseases/metabolism , Neurodegenerative Diseases/pathology , Protein Kinases/metabolism , tau Proteins/metabolism , Aged , Aged, 80 and over , Alzheimer Disease/metabolism , Alzheimer Disease/pathology , Amyotrophic Lateral Sclerosis/metabolism , Amyotrophic Lateral Sclerosis/pathology , Antibody Specificity , Brain/enzymology , Brain/pathology , Casein Kinases , Female , Humans , Immunohistochemistry , Inclusion Bodies/chemistry , Inclusion Bodies/enzymology , Lewy Bodies/chemistry , Lewy Bodies/enzymology , Male , Middle Aged , Neurofibrillary Tangles/chemistry , Neurofibrillary Tangles/enzymology , Neuroglia/chemistry , Neuroglia/enzymology , Parkinson Disease/metabolism , Parkinson Disease/pathology , Protein Kinases/analysis , Protein Kinases/immunology , tau Proteins/analysis , tau Proteins/immunologyABSTRACT
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.
Subject(s)
Alzheimer Disease/metabolism , Alzheimer Disease/physiopathology , Brain/metabolism , Brain/physiopathology , Protein Kinases/genetics , Protein Kinases/metabolism , RNA, Messenger/metabolism , Up-Regulation/physiology , Aged , Alzheimer Disease/pathology , Blotting, Western , Brain/pathology , Casein Kinases , Humans , Protein Kinases/analysis , Reverse Transcriptase Polymerase Chain Reaction , Transcription, Genetic/physiologyABSTRACT
BALB/c mice injected intravenously with a single, sub-lethal dose of Nocardia asteroides GUH-2 develop several levodopa responsive movement disorders. These included headshake, stooped posture, bradykinesia, and hesitation to forward movement. The changes in monoamine levels in the brain of these mice were determined. There was a significant loss of dopamine with greatly increased dopamine turnover in the neostriatum 7 to 29 days after infection. These effects were specific for dopaminergic neurons since minimal changes were found in neostriatal norepinephrine and serotonin even though serotonin turnover was increased. Changes in monoamine metabolism were not limited to the neostriatum. There were reduced levels of serotonin and norepinephrine with increased serotonin turnover in the cerebellum. One year after infection, dopamine metabolism had returned to near normal levels, but many of the movement disorders persisted. Specific changes in neurochemistry did not always appear to correspond with these impairments. Nevertheless, these data are similar to those reported in MPTP treated BALB/c mice.
Subject(s)
Biogenic Monoamines/metabolism , Brain/metabolism , Brain/microbiology , Movement Disorders/metabolism , Nocardia Infections/metabolism , Nocardia asteroides , 3,4-Dihydroxyphenylacetic Acid/metabolism , Animals , Brain/pathology , Cerebellum/metabolism , Dopamine/metabolism , Female , Homovanillic Acid/metabolism , Hydroxyindoleacetic Acid/metabolism , Mice , Mice, Inbred BALB C , Movement Disorders/etiology , Movement Disorders/microbiology , Neostriatum/metabolism , Nocardia Infections/pathology , Nocardia asteroides/isolation & purification , Nocardia asteroides/pathogenicity , Norepinephrine/metabolism , Serotonin/metabolism , Survival RateABSTRACT
Members of the casein kinase-1 family of protein kinases play an essential role in cell regulation and disease pathogenesis. Unlike most protein kinases, they appear to function as constitutively active enzymes. As a result, selective pharmacological inhibitors can play an important role in dissection of casein kinase-1-dependent processes. To address this need, new small molecule inhibitors of casein kinase-1 acting through ATP-competitive and ATP-noncompetitive mechanisms were isolated on the basis of in vitro screening. Here we report the crystal structure of 3-[(2,4,6-trimethoxyphenyl) methylidenyl]-indolin-2-one (IC261), an ATP-competitive inhibitor with differential activity among casein kinase-1 isoforms, in complex with the catalytic domain of fission yeast casein kinase-1 refined to a crystallographic R-factor of 22.4% at 2.8 A resolution. The structure reveals that IC261 stabilizes casein kinase-1 in a conformation midway between nucleotide substrate liganded and nonliganded conformations. We propose that adoption of this conformation by casein kinase-1 family members stabilizes a delocalized network of side chain interactions and results in a decreased dissociation rate of inhibitor.
Subject(s)
Indoles/chemistry , Indoles/pharmacology , Phloroglucinol/analogs & derivatives , Protein Kinase Inhibitors , Casein Kinases , Computer Simulation , Crystallography, X-Ray , Enzyme Inhibitors/pharmacology , Humans , Hydrogen Bonding , Inhibitory Concentration 50 , Kinetics , Models, Molecular , Molecular Sequence Data , Mutation , Oxindoles , Peptide Library , Phloroglucinol/chemistry , Phloroglucinol/pharmacology , Phosphotransferases/metabolism , Protein Binding/drug effects , Protein Conformation/drug effects , Protein Isoforms/chemistry , Protein Structure, Tertiary , Schizosaccharomyces/enzymology , Static ElectricityABSTRACT
Alzheimer's Disease (AD) is a progressive neurodegenerative disorder involving select neurons of the hippocampus, neocortex, and other regions of the brain. Markers of end stage disease include fibrillar lesions, which accumulate hyperphosphorylated tau protein polymerized into filaments, and granulovacuolar lesions, which appear primarily within the hippocampus. The mechanism by which only select populations of neurons develop these lesions as well as the relationship between them is unknown. To address these questions, we have turned to AD tissue to search for enzymes specifically involved in tau hyperphosphorylation. Recently, we showed that the principal phosphotransferases associated with AD brain-derived tau filaments are members of the casein kinase-1 (CK1) family of protein kinases. Here we report the distribution of three CK1 isoforms (Ckialpha, Ckidelta, and Ckiepsilon) in AD and control brains using immunohistochemistry and Western analysis. In addition to colocalizing with elements of the fibrillar pathology, CK1 is found within the matrix of granulovacuolar degeneration bodies. Furthermore, levels of all CK1 isoforms are elevated in the CA1 region of AD hippocampus relative to controls, with one isoform, Ckidelta, being elevated >30-fold. We propose that overexpression of this protein kinase family plays a key role in the hyperphosphorylation of tau and in the formation of AD-related pathology.
Subject(s)
Alzheimer Disease/enzymology , Cytoplasmic Granules/enzymology , Neurofibrillary Tangles/enzymology , Protein Kinases/metabolism , Vacuoles/enzymology , Aged , Aged, 80 and over , Alkaline Phosphatase/metabolism , Alzheimer Disease/pathology , Antibodies, Monoclonal , Antibody Specificity , Blotting, Western , Brain/enzymology , Brain/ultrastructure , Casein Kinases , Cytoplasmic Granules/ultrastructure , Female , Humans , Immunohistochemistry , Isoenzymes/immunology , Isoenzymes/metabolism , Male , Middle Aged , Phosphorylation , Protein Kinases/immunology , Vacuoles/ultrastructure , tau Proteins/metabolismABSTRACT
UNLABELLED: Eukaryotic cells respond to DNA damage and S phase replication blocks by arresting cell-cycle progression through the DNA structure checkpoint pathways. In Schizosaccharomyces pombe, the Chk1 kinase is essential for mitotic arrest and is phosphorylated after DNA damage. During S phase, the Cds1 kinase is activated in response to DNA damage and DNA replication blocks. The response of both Chk1 and Cds1 requires the six 'checkpoint Rad' proteins (Rad1, Rad3, Rad9, Rad17, Rad26 and Hus1). We demonstrate that DNA damage-dependent phosphorylation of Chk1 is also cell-cycle specific, occurring primarily in late S phase and G2, but not during M/G1 or early S phase. We have also isolated and characterized a temperature-sensitive allele of rad3. Rad3 functions differently depending on which checkpoint pathway is activated. Following DNA damage, rad3 is required to initiate but not maintain the Chk1 response. When DNA replication is inhibited, rad3 is required for both initiation and maintenance of the Cds1 response. We have identified a strong genetic interaction between rad3 and cds1, and biochemical evidence shows a physical interaction is possible between Rad3 and Cds1, and between Rad3 and Chk1 in vitro. Together, our results highlight the cell-cycle specificity of the DNA structure-dependent checkpoint response and identify distinct roles for Rad3 in the different checkpoint responses. KEYWORDS: ATM/ATR/cell-cycle checkpoints/Chk1/Rad3
Subject(s)
Adenosine Triphosphatases/metabolism , DNA Helicases/metabolism , Interphase/physiology , Protein Kinases/metabolism , Protein Serine-Threonine Kinases , Adenosine Triphosphatases/genetics , Amino Acid Sequence , Checkpoint Kinase 1 , Checkpoint Kinase 2 , DNA Damage , DNA Helicases/genetics , DNA Replication , G2 Phase/physiology , Gene Dosage , Hydroxyurea/pharmacology , Molecular Sequence Data , Mutation , Phosphorylation , Protein Binding , Radiation Tolerance , S Phase/physiology , Saccharomyces cerevisiae Proteins , Schizosaccharomyces pombe Proteins , Selection, Genetic , Suppression, Genetic , Ultraviolet RaysABSTRACT
Parkinson's disease (PD) is characterized by decreased striatal dopamine, but serotonin (5-HT) is also reduced. Because 5-HT decreases following a single levodopa injection, levodopa has been suggested to contribute to PD's serotonergic deficits. However, in a recent study, rat striatal serotonin levels were reported to increase following 15-day levodopa administration. To address this issue, we administered levodopa (50 mg/kg) to rabbits for 5 days, then measured serotonin, its precursors tryptophan and 5-hydroxytryptophan (5-HTP), and its major metabolite 5-hydroxyindole-acetic acid (5-HIAA) in striatum and CSF. Striatal serotonin and tryptophan were unchanged, while 5-HTP and 5-HIAA increased 4- and 7-fold, respectively. CSF 5-HTP and 5-HIAA were also significantly increased. In levodopa-treated animals, 5-HTP concentrations were moderately correlated (r = 0.679) between striatum and CSF, while weak correlations were present between striatal and CSF concentrations of both serotonin and 5-HIAA. These results suggest that repeated levodopa treatment increases striatal serotonin turnover without changing serotonin content. However, levodopa-induced alterations in striatal serotonin metabolism may not be accurately reflected by measurement of serotonin and 5-HIAA in CSF.
Subject(s)
Corpus Striatum/metabolism , Levodopa/pharmacology , Serotonin/metabolism , 5-Hydroxytryptophan/cerebrospinal fluid , 5-Hydroxytryptophan/metabolism , Animals , Hydroxyindoleacetic Acid/cerebrospinal fluid , Hydroxyindoleacetic Acid/metabolism , Levodopa/administration & dosage , Male , Rabbits , Serotonin/cerebrospinal fluid , Tryptophan/cerebrospinal fluid , Tryptophan/metabolismABSTRACT
High throughput partial sequencing of randomly selected cDNA clones has proven to be a powerful tool for examining the relative abundance of mRNAs and for the identification of novel gene products. Because of the important role played by macrophages in immune and inflammatory responses, we sequenced over 3000 randomly selected cDNA clones from a human macrophage library. These sequences represent a molecular inventory of mRNAs from macrophages and provide a catalog of highly expressed transcripts. Two of the most abundant clones encode recently identified CC chemokines. Macrophage-derived chemokine (MDC) plays a complex role in immunoregulation and is a potent chemoattractant for dendritic cells, T cells, and natural killer cells. The chemokine receptor CCR4 binds MDC with high affinity and also responds by calcium flux and chemotaxis. CCR4 has been shown to be expressed by Th2 type T cells. Recent studies also implicate MDC as a major component of the host defense against human immunodeficiency virus.
Subject(s)
Chemokines/biosynthesis , Chemokines/genetics , DNA, Complementary/analysis , Macrophages/metabolism , RNA, Messenger/metabolism , Base Sequence , Cloning, Molecular , DNA, Complementary/genetics , HumansABSTRACT
The significance of guanine nucleotides and nucleosides in neurodegenerative disorders is suggested by recent reports that these molecules enhance neurite branching and astrocyte proliferation. The objective of this study was to investigate the influence of increased dopamine metabolism, produced by 5-day treatment of rabbits with reserpine (2 mg/kg) or levodopa (LD) (50 mg/kg), on striatal concentrations of guanosine, guanine, and their metabolites. Reserpine treatment decreased striatal guanosine by 41% and increased guanine by 50%, while LD decreased guanosine by 48% (all p < 0.01 vs. vehicle-treated controls). LD also increased guanine by 22% (not statistically significant). Xanthine and uric acid concentrations were unchanged. Because of the neurotrophic properties of guanosine and guanine, changes in striatal concentrations of these purines secondary to increased dopamine (DA) turnover may have relevance for survival of remaining dopaminergic neurons in Parkinson's disease (PD).
Subject(s)
Corpus Striatum/metabolism , Dopamine Agents/pharmacology , Dopamine/metabolism , Guanine/metabolism , Guanosine/metabolism , Reserpine/pharmacology , Animals , Corpus Striatum/cytology , Levodopa/pharmacology , Male , Neurons/drug effects , RabbitsABSTRACT
The protein kinase activity tightly associated with paired helical filaments (PHFs) purified from the brain tissue of individuals with Alzheimer's disease has been characterized in vitro. The activity is shown to phosphorylate casein, an exogenous substrate, with a maximal velocity of approximately 2 nmol/min/mg, suggesting it comprises a significant component of the total protein in the PHF preparation. On the basis of substrate selectivity, isoquinoline sulfonamide inhibitor selectivity, in-gel renaturation assays, and western analysis, the activity consists of closely related members of the alpha branch of the casein kinase 1 family of protein kinases. Because of its tight association with PHFs and its phosphate-directed substrate selectivity, casein kinase 1 is positioned to participate in the pathological hyperphosphorylation of tau protein that is observed in neurodegenerative diseases such as Alzheimer's disease.
Subject(s)
Alzheimer Disease/enzymology , Alzheimer Disease/pathology , Brain/enzymology , Brain/pathology , Neurofibrils/pathology , Protein Kinases/metabolism , tau Proteins/physiology , Adult , Animals , Blotting, Western , Casein Kinases , Humans , Isoenzymes/metabolism , Isoquinolines/pharmacology , Mice , Mice, Inbred BALB C , Middle Aged , Neurofibrils/metabolism , Substrate Specificity , tau Proteins/chemistryABSTRACT
Desensitization of G protein-coupled receptors involves phosphorylation of the receptors by G protein-coupled receptor kinases, such as the beta-adrenergic receptor kinase (beta ARK). beta ARK activity depends upon its translocation from the cytoplasm to the membrane. The beta gamma subunits of G proteins bind to beta ARK and recruit the kinase to the membrane. The G beta gamma binding domain is localized to a carboxyl terminal region of beta ARK but the beta ARK binding domain of G beta gamma is not known. We used the yeast two-hybrid assay to characterize the interaction between G beta and beta ARK. We demonstrate an interaction between the carboxyl terminus of beta ARK and G beta 2. The strength of this interaction is increased when the VP16 transactivation domain is placed on the carboxyl end of G beta 2, indicating that an accessible G beta 2 amino terminus is important for its interaction with beta ARK. In addition, we show that amino acids 1 to 145 of G beta 2 are sufficient for beta ARK binding.
Subject(s)
Cyclic AMP-Dependent Protein Kinases/metabolism , GTP-Binding Proteins/chemistry , GTP-Binding Proteins/metabolism , Amino Acid Sequence , Animals , Binding Sites , Cattle , Cloning, Molecular , Cyclic AMP-Dependent Protein Kinases/chemistry , Kinetics , Macromolecular Substances , Peptide Fragments/chemistry , Recombinant Fusion Proteins/chemistry , Recombinant Fusion Proteins/metabolism , Saccharomyces cerevisiae , Transcriptional Activation , beta-Adrenergic Receptor KinasesSubject(s)
Cell Division , DNA/metabolism , Protein Kinases/metabolism , Amino Acid Sequence , Animals , Casein Kinases , DNA Damage , Humans , Molecular Sequence Data , Protein Kinases/chemistry , Protein Kinases/genetics , Saccharomyces cerevisiae/cytology , Saccharomyces cerevisiae/enzymology , Saccharomyces cerevisiae/genetics , Schizosaccharomyces/cytology , Schizosaccharomyces/enzymology , Sequence Homology, Amino AcidABSTRACT
The p53 tumour suppressor protein plays a key role in the integration of stress signals. Multi-site phosphorylation of p53 may play an integral part in the transmission of these signals and is catalysed by many different protein kinases including an unidentified p53-N-terminus-targeted protein kinase (p53NK) which phosphorylates a group of sites at the N-terminus of the protein. In this paper, we present evidence that the delta and epsilon isoforms of casein kinase 1 (CK1delta and CK1epsilon) show identical features to p53NK and can phosphorylate p53 both in vitro and in vivo. Recombinant, purified glutathione S-transferase (GST)-CK1delta and GST-CK1epsilon fusion proteins each phosphorylate p53 in vitro at serines 4, 6 and 9, the sites recognised by p53NK. Furthermore, p53NK (i) co-purifies with CK1delta/epsilon, (ii) shares identical kinetic properties to CK1delta/epsilon, and (iii) is inhibited by a CK1delta/epsilon-specific inhibitor (IC261). In addition, CK1delta is also present in purified preparations of p53NK as judged by immunoanalysis using a CK1delta-specific monoclonal antibody. Treatment of murine SV3T3 cells with IC261 specifically blocked phosphorylation in vivo of the CK1delta/epsilon phosphorylation sites in p53, indicating that p53 interacts physiologically with CK1delta and/or CK1epsilon. Similarly, over-expression of a green fluorescent protein (GFP)-CK1delta fusion protein led to hyper-phosphorylation of p53 at its N-terminus. Treatment of MethAp53ts cells with the topoisomerase-directed drugs etoposide or camptothecin led to increases in both CK1delta-mRNA and -protein levels in a manner dependent on the integrity of p53. These data suggest that p53 is phosphorylated by CK1delta and CK1epsilon and additionally that there may be a regulatory feedback loop involving p53 and CK1delta.
Subject(s)
Isoenzymes/metabolism , Protein Kinases/metabolism , Topoisomerase II Inhibitors , Tumor Suppressor Protein p53/metabolism , Animals , COS Cells , Camptothecin/pharmacology , Casein Kinases , Enzyme Inhibitors/pharmacology , Etoposide/pharmacology , Gene Expression Regulation, Enzymologic/drug effects , Mice , Phosphopeptides/analysis , Phosphorylation , RNA, Messenger/genetics , RatsABSTRACT
The rad3 gene of Schizosaccharomyces pombe is required for checkpoint pathways that respond to DNA damage and replication blocks. We report the complete rad3 gene sequence and show that rad3 is the homologue of Saccharomyces cerevisiae ESR1 (MEC1/SAD3) and Drosophila melanogaster mei-41 checkpoint genes. This establishes Rad3/Mec1 as the only conserved protein which is required for all the DNA structure checkpoints in both yeast model systems. Rad3 is an inessential member of the 'lipid kinase' subclass of kinases which includes the ATM protein defective in ataxia telangiectasia patients. Mutational analysis indicates that the kinase domain is required for Rad3 function, and immunoprecipitation of overexpressed Rad3 demonstrates an associated protein kinase activity. The previous observation that rad3 mutations can be rescued by a truncated clone lacking the kinase domain may be due to intragenic complementation. Consistent with this, biochemical data suggest that Rad3 exists in a complex containing multiple copies of Rad3. We have identified a novel human gene (ATR) whose product is closely related to Rad3/Esr1p/Mei-41. ATR can functionally complement esr1-1 radiation sensitivity in S. cerevisiae. Together, the structural conservation and functional complementation suggest strongly that the mechanisms underlying the DNA structure checkpoints are conserved throughout evolution.
Subject(s)
Adenosine Triphosphatases/genetics , DNA Helicases/genetics , Genes, Fungal , Protein Serine-Threonine Kinases , Schizosaccharomyces/cytology , Schizosaccharomyces/genetics , Adenosine Triphosphatases/chemistry , Adenosine Triphosphatases/metabolism , Amino Acid Sequence , Animals , Ataxia Telangiectasia Mutated Proteins , Base Sequence , Cell Cycle Proteins/chemistry , Cell Cycle Proteins/metabolism , Cloning, Molecular , DNA Damage , DNA Helicases/chemistry , DNA Helicases/metabolism , DNA Primers , DNA Replication , Dose-Response Relationship, Radiation , Drosophila melanogaster/genetics , Genetic Complementation Test , Humans , Kinetics , Molecular Sequence Data , Mutagenesis , Oligodeoxyribonucleotides , Phosphotransferases/metabolism , Phylogeny , Recombinant Proteins/chemistry , Recombinant Proteins/metabolism , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae Proteins , Schizosaccharomyces/radiation effects , Sequence Deletion , Sequence Homology, Amino AcidABSTRACT
We have developed a method to study the primary sequence specificities of protein kinases by using an oriented degenerate peptide library. We report here the substrate specificities of eight protein Ser/Thr kinases. All of the kinases studied selected distinct optimal substrates. The identified substrate specificities of these kinases, together with known crystal structures of protein kinase A, CDK2, Erk2, twitchin, and casein kinase I, provide a structural basis for the substrate recognition of protein Ser/Thr kinases. In particular, the specific selection of amino acids at the +1 and -3 positions to the substrate serine/threonine can be rationalized on the basis of sequences of protein kinases. The identification of optimal peptide substrates of CDK5, casein kinases I and II, NIMA, calmodulin-dependent kinases, Erk1, and phosphorylase kinase makes it possible to predict the potential in vivo targets of these kinases.
Subject(s)
CDC2-CDC28 Kinases , Cell Cycle Proteins , Mitogen-Activated Protein Kinases , Protein Serine-Threonine Kinases/chemistry , Protein Serine-Threonine Kinases/metabolism , Amino Acid Sequence , Caenorhabditis elegans Proteins , Calcium-Calmodulin-Dependent Protein Kinase Type 2 , Calcium-Calmodulin-Dependent Protein Kinases/chemistry , Calcium-Calmodulin-Dependent Protein Kinases/metabolism , Calmodulin-Binding Proteins/chemistry , Calmodulin-Binding Proteins/metabolism , Casein Kinase II , Casein Kinases , Crystallography, X-Ray , Cyclic AMP-Dependent Protein Kinases/chemistry , Cyclic AMP-Dependent Protein Kinases/metabolism , Cyclin-Dependent Kinase 2 , Cyclin-Dependent Kinase 5 , Cyclin-Dependent Kinases/chemistry , Cyclin-Dependent Kinases/metabolism , Databases, Factual , Mitogen-Activated Protein Kinase 1 , Mitogen-Activated Protein Kinase 3 , Models, Molecular , Muscle Proteins/chemistry , Muscle Proteins/metabolism , NIMA-Related Kinase 1 , NIMA-Related Kinases , Oligopeptides/chemistry , Oligopeptides/metabolism , Phosphopeptides/chemistry , Phosphopeptides/isolation & purification , Phosphorylase Kinase/metabolism , Protein Conformation , Protein Kinases/chemistry , Protein Kinases/metabolism , Substrate SpecificityABSTRACT
Ceruloplasmin (CP), the major plasma anti-oxidant and copper transport protein, is synthesized in several tissues, including the brain. We compared regional brain concentrations of CP and copper between subjects with Alzheimer's disease (AD, n = 12), Parkinson's disease (PD, n = 14), Huntington's disease (HD, n = 11), progressive supranuclear palsy (PSP, n = 11), young adult normal controls (YC, n = 6) and elderly normal controls (EC, n = 7). Mean CP concentrations were significantly increased vs. EC (P < 0.05) in AD hippocampus, entorhinal cortex, frontal cortex, and putamen. PD hippocampus, frontal, temporal, and parietal cortices, and HD hippocampus, parietal cortex, and substantia nigra. Immunocytochemical staining for CP in AD hippocampus revealed marked staining within neurons, astrocytes, and neuritic plaques. Increased CP concentrations in brain in these disorders may indicate a localized acute phase-type response and/or a compensatory increase to oxidative stress.
Subject(s)
Alzheimer Disease/physiopathology , Brain/physiology , Ceruloplasmin/metabolism , Copper/metabolism , Nerve Degeneration/physiology , Adult , Aged , Aged, 80 and over , Alzheimer Disease/metabolism , Alzheimer Disease/pathology , Brain/metabolism , Brain/pathology , Case-Control Studies , Cell Count , Hippocampus/pathology , Humans , Huntington Disease/metabolism , Huntington Disease/pathology , Huntington Disease/physiopathology , Middle Aged , Neurons/pathology , Parkinson Disease/metabolism , Parkinson Disease/pathology , Parkinson Disease/physiopathology , Supranuclear Palsy, Progressive/metabolism , Supranuclear Palsy, Progressive/pathology , Supranuclear Palsy, Progressive/physiopathologyABSTRACT
The Escherichia coli tet-repressor (TetR) operator system was used to develop a variation of the yeast two-hybrid assay in which disruptions of protein-protein interactions can be identified by a positive selection. This assay, designated the "split-hybrid system," contains a two-component reporter. The first component contains LexA binding sites upstream of the TetR gene and the second contains TetR operator binding sites upstream of HIS3. Interaction of one protein fused to the LexA DNA binding domain with a second protein fused to the VP16 activation domain results in TetR expression. TetR subsequently binds to the tet operators, blocking the expression of HIS3 and preventing yeast growth in media lacking histidine. The utility of the split-hybrid system was analyzed by examining the phosphorylation-dependent interaction of CREB and its coactivator CREB binding protein (CBP). CREB and CBP associate through an interaction that depends upon CREB phosphorylation at Ser-133. Mutation of this phosphorylation site prevents yeast growth in the standard two-hybrid assay but allows growth in the split-hybrid strains. The split-hybrid system was used to identify other CREB mutations that disrupt its association with CBP. These mutations localized around the site of CREB phosphorylation, indicating that only a small portion of the CREB activation domain is required for CBP interaction. The yeast split-hybrid system should be useful in identifying mutations, proteins, peptides, and drugs that disrupt protein-protein interactions.