HRR25, a putative protein kinase from budding yeast: association with repair of damaged DNA.

In simple eukaryotes, protein kinases regulate mitotic and meiotic cell cycles, the response to polypeptide pheromones, and the initiation of nuclear DNA synthesis. The protein HRR25 from the budding yeast Saccharomyces cerevisiae was defined by the mutation hrr25-1. This mutation resulted in sensitivity to continuous expression of the HO double-strand endonuclease, to methyl methanesulfonate, and to x-irradiation. Homozygotes of hrr25-1 were unable to sporulate and disruption and deletion of HRR25 interfered with mitotic and meiotic cell division. Sequence analysis revealed two distinctive regions in the protein. The NH2-terminus of HRR25 contains the hallmark features of protein kinases, whereas the COOH-terminus is rich in proline and glutamine. Mutations in HRR25 at conserved residues found in all protein kinases inactivated the gene, and these mutants exhibited the hrr25 null phenotypes. Taken together, the hrr25 mutant phenotypes and the features of the gene product indicate that HRR25 is a distinctive member of the protein kinase superfamily.

Genetically identified protein kinases in yeast. II: DNA metabolism and meiosis.

Genetic analysis of protein kinases in Saccharomyces cerevisiae has revealed protein phosphorylation as a key regulatory mechanism both in the mitotic cell cycle and in meiosis. This article reviews genetically identified protein kinases that are associated with DNA metabolism and the meiotic pathway.

Genetically identified protein kinases in yeast. I: Transcription, translation, transport and mating.

Studies from a wide array of different fields using Saccharomyces cerevisiae as an experimental organism have uncovered protein phosphorylation as a recurrent theme in the regulation of diverse cellular activities. Protein kinases in yeast regulate a variety of processes; this article discusses several genetically identified protein kinases and the roles that these kinases play in cell growth and development.

Prenylated isoforms of yeast casein kinase I, including the novel Yck3p, suppress the gcs1 blockage of cell proliferation from stationary phase.

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.

A structural basis for substrate specificities of protein Ser/Thr kinases: primary sequence preference of casein kinases I and II, NIMA, phosphorylase kinase, calmodulin-dependent kinase II, CDK5, and Erk1.

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.

Budding and fission yeast casein kinase I isoforms have dual-specificity protein kinase activity.

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.

p53 is phosphorylated in vitro and in vivo by the delta and epsilon isoforms of casein kinase 1 and enhances the level of casein kinase 1 delta in response to topoisomerase-directed drugs.

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.

Profile of human macrophage transcripts: insights into macrophage biology and identification of novel chemokines.

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.

Casein kinase 1 delta is associated with pathological accumulation of tau in several neurodegenerative diseases.

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.

Casein kinase 1 delta mRNA is upregulated in Alzheimer disease brain.

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.

Increased regional brain concentrations of ceruloplasmin in neurodegenerative disorders.

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.

Altered guanosine and guanine concentrations in rabbit striatum following increased dopamine turnover.

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).

Effects of enhanced striatal dopamine turnover in vivo on glutathione oxidation.

In Parkinson's disease (PD), a compensatory increase in dopamine (DA) turnover occurs in the remaining nigrostriatal dopaminergic neurons, resulting in greater exposure of each neuron to hydrogen peroxide (H2O2) derived from oxidative deamination of DA. The formation of oxyradicals from H2O2 is regarded as a mechanism that could contribute to the progression of PD, and incubation of rat striatal synaptosomes with levodopa (LD) results in an increase in oxidized glutathione (GSSG), indicative of oxidant stress. The present study was undertaken to determine whether striatal GSSG levels increase in response to administration of LD in vivo. Acute and repeated (3-week) treatment of normal rats with LD at doses of up to 100 mg/kg did not increase striatal GSSG despite marked increase in DA turnover. These results suggest that intact striatum may possess increased defense capacity against oxidant stress generated by increased DA turnover as compared with isolated synaptosomes.

The budding yeast HRR25 gene product is a casein kinase I isoform.

The Saccharomyces cerevisiae HRR25 gene was identified as a regulator of DNA strand-break repair. HRR25 encodes a protein kinase that is closely related to bovine casein kinase I (CKI). CKI is a ubiquitous multipotential protein kinase. Rabbit polyclonal antibodies that recognize and immunoprecipitate Hrr25p have been generated and an immune complex protein kinase assay has been developed. The reaction depends upon HRR25 and shows that Hrr25p uses casein as a substrate. The identity between Hrr25p and bovine CKI suggests that Hrr25p is a yeast isoform of the CKI family and that CKIs may play a role in regulating DNA metabolism.

Monoamine changes in the brain of BALB/c mice following sub-lethal infection with Nocardia asteroides (GUH-2).

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.

The beta-adrenergic receptor kinase interacts with the amino terminus of the G protein beta subunit.

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.