G2Cdb::Gene report

Gene id
Gene symbol
Homo sapiens
casein kinase 1, epsilon
G00000161 (Mus musculus)

Databases (7)

ENSG00000100181 (Ensembl human gene)
1454 (Entrez Gene)
504 (G2Cdb plasticity & disease)
CSNK1E (GeneCards)
600863 (OMIM)
Marker Symbol
HGNC:2453 (HGNC)
Protein Sequence
P49674 (UniProt)

Synonyms (1)


Literature (78)

Pubmed - human_disease

  • Suggestive evidence for association of the circadian genes PERIOD3 and ARNTL with bipolar disorder.

    Nievergelt CM, Kripke DF, Barrett TB, Burg E, Remick RA, Sadovnick AD, McElroy SL, Keck PE, Schork NJ and Kelsoe JR

    Department of Psychiatry, University of California, San Diego, La Jolla, California 92093-0603, USA.

    Bipolar affective disorder (BPAD) is suspected to arise in part from malfunctions of the circadian system, a system that enables adaptation to a daily and seasonally cycling environment. Genetic variations altering functions of genes involved with the input to the circadian clock, in the molecular feedback loops constituting the circadian oscillatory mechanism itself, or in the regulatory output systems could influence BPAD as a result. Several human circadian system genes have been identified and localized recently, and a comparison with linkage hotspots for BPAD has revealed some correspondences. We have assessed evidence for linkage and association involving polymorphisms in 10 circadian clock genes (ARNTL, CLOCK, CRY2, CSNK1epsilon, DBP, GSK3beta, NPAS2, PER1, PER2, and PER3) to BPAD. Linkage analysis in 52 affected families showed suggestive evidence for linkage to CSNK1epsilon. This finding was not substantiated in the association study. Fifty-two SNPs in 10 clock genes were genotyped in 185 parent proband triads. Single SNP TDT analyses showed no evidence for association to BPAD. However, more powerful haplotype analyses suggest two candidates deserving further studies. Haplotypes in ARNTL and PER3 were found to be significantly associated with BPAD via single-gene permutation tests (PG = 0.025 and 0.008, respectively). The most suggestive haplotypes in PER3 showed a Bonferroni-corrected P-value of PGC = 0.07. These two genes have previously been implicated in circadian rhythm sleep disorders and affective disorders. With correction for the number of genes considered and tests conducted, these data do not provide statistically significant evidence for association. However, the trends for ARNTL and PER3 are suggestive of their involvement in bipolar disorder and warrant further study in a larger sample.

    Funded by: Intramural NIH HHS; NCRR NIH HHS: M01 RR000827, M01 RR00827; NHLBI NIH HHS: HL054998-09, HL064777-06, HL069758-03, HL070137-01A1, HL071123, HL074730-02, HL61280, P50 HL054998, R01 HL070137, R01 HL071123, R01 HL074730, R56 HL071123, R56 HL071123-04, U01 HL064777, U01 HL069758; NIA NIH HHS: AG023122-01, AG12364, AG15763, R01 AG012364, R01 AG015763, U19 AG023122; NIDA NIH HHS: DA13769; NIMH NIH HHS: K08 MH067959, MH059567-05A2, MH067959, MH068503-01A1, MH47612, MH59567, MH68503, R01 MH059567, R01 MH068503, U01 MH46274, UO1 MH46280, UO1 MH46282; PHS HHS: HLMH065571-02

    American journal of medical genetics. Part B, Neuropsychiatric genetics : the official publication of the International Society of Psychiatric Genetics 2006;141B;3;234-41

Pubmed - other

  • PER2 variantion is associated with depression vulnerability.

    Lavebratt C, Sjöholm LK, Partonen T, Schalling M and Forsell Y

    Department of Molecular Medicine and Surgery, Karolinska Institutet at Karolinska University Hospital Solna, Stockholm, Sweden.

    The circadian clock is driven by transcription-translation feedback loops and regulates rhythms that approximate the 24-hr day-night cycle or light-dark transitions. Disruptions of the circadian rhythms are common in depressed patients, expressed for example as sleep disturbances. Genetic variations in core circadian genes may in part explain these abnormalities. To investigate whether genetic variation in core circadian genes associates with vulnerability to depression, we genotyped 18 genes in a Swedish population based sample. Genetic variations indicative of association with depression, or with winter depression in our previous study, were tested for association to depression in a second Swedish depression-control sample set. PER2 genetic variation was associated with depression vulnerability, and this genetic risk did not seem to require exposure to potential sleep disturbance factors such as negative life event or financial strain that are known to increase the risk for depression. Polymorphisms in the circadian genes NPAS2, ARNTL, and RORA were also suggested to contribute to depression vulnerability. The findings we report for PER2, ARNTL, and RORA are supported by at least two of the three sample sets. In conclusion, genetic variation in PER2 is associated with depression vulnerability a Swedish population-based sample. More studies are needed to determine if this is the case also for NPAS2, ARNTL, and RORA.

    American journal of medical genetics. Part B, Neuropsychiatric genetics : the official publication of the International Society of Psychiatric Genetics 2010;153B;2;570-581

  • A coordinated phosphorylation by Lats and CK1 regulates YAP stability through SCF(beta-TRCP).

    Zhao B, Li L, Tumaneng K, Wang CY and Guan KL

    Department of Pharmacology and Moores Cancer Center, University of California at San Diego, La Jolla, California 92093-0815, USA.

    The Yes-associated protein (YAP) transcription coactivator is a key regulator of organ size and a candidate human oncogene. YAP is inhibited by the Hippo pathway kinase cascade, at least in part via phosphorylation of Ser 127, which results in YAP 14-3-3 binding and cytoplasmic retention. Here we report that YAP is phosphorylated by Lats on all of the five consensus HXRXXS motifs. Phosphorylation of Ser 381 in one of them primes YAP for subsequent phosphorylation by CK1delta/epsilon in a phosphodegron. The phosphorylated phosphodegron then recruits the SCF(beta-TRCP) E3 ubiquitin ligase, which catalyzes YAP ubiquitination, ultimately leading to YAP degradation. The phosphodegron-mediated degradation and the Ser 127 phosphorylation-dependent translocation coordinately suppress YAP oncogenic activity. Our study identified CK1delta/epsilon as new regulators of YAP and uncovered an intricate mechanism of YAP regulation by the Hippo pathway via both S127 phosphorylation-mediated spatial regulation (nuclear-cytoplasmic shuttling) and the phosphodegron-mediated temporal regulation (degradation).

    Genes & development 2010;24;1;72-85

  • Testing the circadian gene hypothesis in prostate cancer: a population-based case-control study.

    Zhu Y, Stevens RG, Hoffman AE, Fitzgerald LM, Kwon EM, Ostrander EA, Davis S, Zheng T and Stanford JL

    Department of Epidemiology, Yale University School of Medicine, New Haven, Connecticut 06520, USA. yong.zhu@yale.edu

    Circadian genes are responsible for maintaining the ancient adaptation of a 24-hour circadian rhythm and influence a variety of cancer-related biological pathways, including the regulation of sex hormone levels. However, few studies have been undertaken to investigate the role of circadian genes in the development of prostate cancer, the most common cancer type among men (excluding nonmelanoma skin cancer). The current genetic association study tested the circadian gene hypothesis in relation to prostate cancer by genotyping a total of 41 tagging and amino acid-altering single nucleotide polymorphisms (SNP) in 10 circadian-related genes in a population-based case-control study of Caucasian men (n = 1,308 cases and 1,266 controls). Our results showed that at least one SNP in nine core circadian genes (rs885747 and rs2289591 in PER1; rs7602358 in PER2; rs1012477 in PER3; rs1534891 in CSNK1E; rs12315175 in CRY1; rs2292912 in CRY2; rs7950226 in ARNTL; rs11133373 in CLOCK; and rs1369481, rs895521, and rs17024926 in NPAS2) was significantly associated with susceptibility to prostate cancer (either overall risk or risk of aggressive disease), and the risk estimate for four SNPs in three genes (rs885747 and rs2289591 in PER1, rs1012477 in PER3, and rs11133373 in CLOCK) varied by disease aggressiveness. Further analyses of haplotypes were consistent with these genotyping results. Findings from this candidate gene association study support the hypothesis of a link between genetic variants in circadian genes and prostate cancer risk, warranting further confirmation and mechanistic investigation of circadian biomarkers in prostate tumorigenesis.

    Funded by: NCI NIH HHS: R01 CA056678, R01 CA056678-05, R01 CA082664, R01 CA082664-03, R01 CA092579, R01 CA092579-05, R01 CA122676, R01 CA56678, R03 CA110937; NIGMS NIH HHS: T32 GM007814

    Cancer research 2009;69;24;9315-22

  • Association study of 21 circadian genes with bipolar I disorder, schizoaffective disorder, and schizophrenia.

    Mansour HA, Talkowski ME, Wood J, Chowdari KV, McClain L, Prasad K, Montrose D, Fagiolini A, Friedman ES, Allen MH, Bowden CL, Calabrese J, El-Mallakh RS, Escamilla M, Faraone SV, Fossey MD, Gyulai L, Loftis JM, Hauser P, Ketter TA, Marangell LB, Miklowitz DJ, Nierenberg AA, Patel J, Sachs GS, Sklar P, Smoller JW, Laird N, Keshavan M, Thase ME, Axelson D, Birmaher B, Lewis D, Monk T, Frank E, Kupfer DJ, Devlin B and Nimgaonkar VL

    Department of Psychiatry, University of Pittsburgh School of Medicine, Western Psychiatric Institute and Clinic, Pittsburgh, PA 15213, USA.

    Objective: Published studies suggest associations between circadian gene polymorphisms and bipolar I disorder (BPI), as well as schizoaffective disorder (SZA) and schizophrenia (SZ). The results are plausible, based on prior studies of circadian abnormalities. As replications have not been attempted uniformly, we evaluated representative, common polymorphisms in all three disorders.

    Methods: We assayed 276 publicly available 'tag' single nucleotide polymorphisms (SNPs) at 21 circadian genes among 523 patients with BPI, 527 patients with SZ/SZA, and 477 screened adult controls. Detected associations were evaluated in relation to two published genome-wide association studies (GWAS).

    Results: Using gene-based tests, suggestive associations were noted between EGR3 and BPI (p = 0.017), and between NPAS2 and SZ/SZA (p = 0.034). Three SNPs were associated with both sets of disorders (NPAS2: rs13025524 and rs11123857; RORB: rs10491929; p < 0.05). None of the associations remained significant following corrections for multiple comparisons. Approximately 15% of the analyzed SNPs overlapped with an independent study that conducted GWAS for BPI; suggestive overlap between the GWAS analyses and ours was noted at ARNTL.

    Conclusions: Several suggestive, novel associations were detected with circadian genes and BPI and SZ/SZA, but the present analyses do not support associations with common polymorphisms that confer risk with odds ratios greater than 1.5. Additional analyses using adequately powered samples are warranted to further evaluate these results.

    Funded by: NCATS NIH HHS: UL1 TR000005; NCRR NIH HHS: M01 RR000056, M01 RR00056, UL1 RR024153; NIMH NIH HHS: K23 MH072995, MH30915, MH45156, MH56242, MH63420, P20 MH-66054, P20 MH066054, P30 MH030915, P50 MH045156, R01 MH056242, R01 MH060952, R01 MH063420, R01MH060952-06, R10 MH056242

    Bipolar disorders 2009;11;7;701-10

  • Genetical genomic determinants of alcohol consumption in rats and humans.

    Tabakoff B, Saba L, Printz M, Flodman P, Hodgkinson C, Goldman D, Koob G, Richardson HN, Kechris K, Bell RL, Hübner N, Heinig M, Pravenec M, Mangion J, Legault L, Dongier M, Conigrave KM, Whitfield JB, Saunders J, Grant B, Hoffman PL and WHO/ISBRA Study on State and Trait Markers of Alcoholism

    Department of Pharmacology, University of Colorado, Denver, Aurora, CO, USA. boris.tabakoff@ucdenver.edu

    Background: We have used a genetical genomic approach, in conjunction with phenotypic analysis of alcohol consumption, to identify candidate genes that predispose to varying levels of alcohol intake by HXB/BXH recombinant inbred rat strains. In addition, in two populations of humans, we assessed genetic polymorphisms associated with alcohol consumption using a custom genotyping array for 1,350 single nucleotide polymorphisms (SNPs). Our goal was to ascertain whether our approach, which relies on statistical and informatics techniques, and non-human animal models of alcohol drinking behavior, could inform interpretation of genetic association studies with human populations.

    Results: In the HXB/BXH recombinant inbred (RI) rats, correlation analysis of brain gene expression levels with alcohol consumption in a two-bottle choice paradigm, and filtering based on behavioral and gene expression quantitative trait locus (QTL) analyses, generated a list of candidate genes. A literature-based, functional analysis of the interactions of the products of these candidate genes defined pathways linked to presynaptic GABA release, activation of dopamine neurons, and postsynaptic GABA receptor trafficking, in brain regions including the hypothalamus, ventral tegmentum and amygdala. The analysis also implicated energy metabolism and caloric intake control as potential influences on alcohol consumption by the recombinant inbred rats. In the human populations, polymorphisms in genes associated with GABA synthesis and GABA receptors, as well as genes related to dopaminergic transmission, were associated with alcohol consumption.

    Conclusion: Our results emphasize the importance of the signaling pathways identified using the non-human animal models, rather than single gene products, in identifying factors responsible for complex traits such as alcohol consumption. The results suggest cross-species similarities in pathways that influence predisposition to consume alcohol by rats and humans. The importance of a well-defined phenotype is also illustrated. Our results also suggest that different genetic factors predispose alcohol dependence versus the phenotype of alcohol consumption.

    Funded by: Howard Hughes Medical Institute; NHLBI NIH HHS: HL35018, P01 HL035018; NIAAA NIH HHS: AA006420, AA013162, AA013517-INIA, AA013522-INIA, AA016649-INIA, AA016663-INIA, AA16922, K01 AA016922, P50 AA006420, P60 AA006420, R01 AA013162, R24 AA013162, R24 AA015512, U01 AA013517, U01 AA013522, U01 AA016649, U01 AA016663, U24 AA013517, U24 AA013522; NIDDK NIH HHS: R01 DK100340; Wellcome Trust

    BMC biology 2009;7;70

  • CKIepsilon/delta-dependent phosphorylation is a temperature-insensitive, period-determining process in the mammalian circadian clock.

    Isojima Y, Nakajima M, Ukai H, Fujishima H, Yamada RG, Masumoto KH, Kiuchi R, Ishida M, Ukai-Tadenuma M, Minami Y, Kito R, Nakao K, Kishimoto W, Yoo SH, Shimomura K, Takao T, Takano A, Kojima T, Nagai K, Sakaki Y, Takahashi JS and Ueda HR

    Comparative Systems Biology Team, Genomic Science Center, RIKEN, 1-7-22, Suehiro-cho, Tsurumi, Yokohama 230-0045, Japan.

    A striking feature of the circadian clock is its flexible yet robust response to various environmental conditions. To analyze the biochemical processes underlying this flexible-yet-robust characteristic, we examined the effects of 1,260 pharmacologically active compounds in mouse and human clock cell lines. Compounds that markedly (>10 s.d.) lengthened the period in both cell lines, also lengthened it in central clock tissues and peripheral clock cells. Most compounds inhibited casein kinase Iepsilon (CKIepsilon) or CKIdelta phosphorylation of the PER2 protein. Manipulation of CKIepsilon/delta-dependent phosphorylation by these compounds lengthened the period of the mammalian clock from circadian (24 h) to circabidian (48 h), revealing its high sensitivity to chemical perturbation. The degradation rate of PER2, which is regulated by CKIepsilon/delta-dependent phosphorylation, was temperature-insensitive in living clock cells, yet sensitive to chemical perturbations. This temperature-insensitivity was preserved in the CKIepsilon/delta-dependent phosphorylation of a synthetic peptide in vitro. Thus, CKIepsilon/delta-dependent phosphorylation is likely a temperature-insensitive period-determining process in the mammalian circadian clock.

    Funded by: Howard Hughes Medical Institute; NIMH NIH HHS: P50 MH074924, P50 MH074924-05

    Proceedings of the National Academy of Sciences of the United States of America 2009;106;37;15744-9

  • A common variant in DRD3 receptor is associated with autism spectrum disorder.

    de Krom M, Staal WG, Ophoff RA, Hendriks J, Buitelaar J, Franke B, de Jonge MV, Bolton P, Collier D, Curran S, van Engeland H and van Ree JM

    Department of Neuroscience and Pharmacology and Department of Child and Adolescent Psychiatry, Rudolph Magnus Institute of Neuroscience, University Medical Center Utrecht, Heidelberglaan 100, Utrecht, The Netherlands.

    Background: The presence of specific and common genetic etiologies for autism spectrum disorders (ASD) and attention-deficit/hyperactivity disorder (ADHD) was investigated for 132 candidate genes in a two-stage design-association study.

    Methods: 1,536 single nucleotide polymorphisms (SNPs) covering these candidate genes were tested in ASD (n = 144) and ADHD (n = 110) patients and control subjects (n = 404) from The Netherlands. A second stage was performed with those SNPs from Stage I reaching a significance threshold for association of p < .01 in an independent sample of ASD patients (n = 128) and controls (n = 124) from the United Kingdom and a Dutch ADHD (n = 150) and control (n = 149) sample.

    Results: No shared association was found between ASD and ADHD. However, in the first and second ASD samples and in a joint statistical analysis, a significant association between SNP rs167771 located in the DRD3 gene was found (joint analysis uncorrected: p = 3.11 x 10(-6); corrected for multiple testing and potential stratification: p = .00162).

    Conclusions: The DRD3 gene is related to stereotyped behavior, liability to side effects of antipsychotic medication, and movement disorders and may therefore have important clinical implications for ASD.

    Biological psychiatry 2009;65;7;625-30

  • Casein kinase I delta/epsilon phosphorylates topoisomerase IIalpha at serine-1106 and modulates DNA cleavage activity.

    Grozav AG, Chikamori K, Kozuki T, Grabowski DR, Bukowski RM, Willard B, Kinter M, Andersen AH, Ganapathi R and Ganapathi MK

    Clinical Pharmacology Program, Taussig Cancer Institute, Cleveland, OH 44195, USA.

    We previously reported that phosphorylation of topoisomerase (topo) IIalpha at serine-1106 (Ser-1106) regulates enzyme activity and sensitivity to topo II-targeted drugs. In this study we demonstrate that phosphorylation of Ser-1106, which is flanked by acidic amino acids, is regulated in vivo by casein kinase (CK) Idelta and/or CKIepsilon, but not by CKII. The CKI inhibitors, CKI-7 and IC261, reduced Ser-1106 phosphorylation and decreased formation of etoposide-stabilized topo II-DNA cleavable complex. In contrast, the CKII inhibitor, 5,6-dichlorobenzimidazole riboside, did not affect etoposide-stabilized topo II-DNA cleavable complex formation. Since, IC261 specifically targets the Ca(2+)-regulated isozymes, CKIdelta and CKIepsilon, we examined the effect of down-regulating these enzymes on Ser-1106 phosphorylation. Down-regulation of these isozymes with targeted si-RNAs led to hypophosphorylation of the Ser-1106 containing peptide. However, si-RNA-mediated down-regulation of CKIIalpha and alpha' did not alter Ser-1106 phosphorylation. Furthermore, reduced phosphorylation of Ser-1106, observed in HRR25 (CKIdelta/epsilon homologous gene)-deleted Saccharomyces cerevisiae cells transformed with human topo IIalpha, was enhanced following expression of human CKIepsilon. Down-regulation of CKIdelta and CKIepsilon also led to reduced formation of etoposide stabilized topo II-DNA cleavable complex. These results provide strong support for an essential role of CKIdelta/epsilon in phosphorylating Ser-1106 in human topo IIalpha and in regulating enzyme function.

    Funded by: NCI NIH HHS: R01 CA 117928, R01 CA 74939

    Nucleic acids research 2009;37;2;382-92

  • Casein kinase I epsilon (CKIvarepsilon) N408 allele is very rare in the Brazilian population and is not involved in susceptibility to circadian rhythm sleep disorders.

    Castro RM, Barbosa AA, Pedrazzoli M and Tufik S

    Psychobiology Department, Universidade Federal de São Paulo, UNIFESP, Brazil. castro.rosamaria@gmail.com

    Circadian rhythms are regulated by clock proteins through post-translational modifications. Indeed, Casein kinase I epsilon (CKIvarepsilon) promotes reversible phosphorylation of PER proteins, and a deficiency in this phosphorylation has been implicated in human sleep disorders. Here, we investigated the CKIvarepsilon S408N polymorphism in a Brazilian population sample. The N408 allele was previously described to be much less frequent in individuals with Delayed Sleep-Phase Syndrome (DSPS), than in the general Japanese population, suggesting a protective function for the allele against the disease. We found that this polymorphism is very rare in the Brazilian population (1.37%), indicating that it has no influence on susceptibility to circadian rhythm sleep disorders. Therefore, it is necessary to account for adaptative influences in genetic background, analyzing different groups with different photoperiods, to validate the effects of this and other polymorphisms on sleep and circadian disorders.

    Behavioural brain research 2008;193;1;156-7

  • Correlation between circadian gene variants and serum levels of sex steroids and insulin-like growth factor-I.

    Chu LW, Zhu Y, Yu K, Zheng T, Chokkalingam AP, Stanczyk FZ, Gao YT and Hsing AW

    Division of Cancer Epidemiology and Genetics, National Cancer Institute, NIH, 6120 Executive Boulevard, MSC 7234, Bethesda, MD 20892, USA. chulisa@mail.nih.gov

    A variety of biological processes, including steroid hormone secretion, have circadian rhythms, which are influenced by nine known circadian genes. Previously, we reported that certain variants in circadian genes were associated with risk for prostate cancer. To provide some biological insight into these findings, we examined the relationship of five variants of circadian genes, including NPAS2 (rs2305160:G > A), PER1 (rs2585405:G > C), CSNK1E (rs1005473:A > C), PER3 (54-bp repeat length variant), and CRY2 (rs1401417:G > C), with serum levels of sex steroids and insulin-like growth factor (IGF)-I and IGF-binding protein 3 (IGFBP3) in 241 healthy elderly Chinese men (mean age of 71.5). Age-adjusted and waist-to-hip ratio-adjusted ANOVA followed by likelihood ratio tests (LRT) showed that the NPAS2 variant A allele was associated with lower free and bioavailable testosterone (P(LRT) = 0.02 and 0.01, respectively) compared with the GG genotype. In addition, the PER1 variant was associated with higher serum levels of sex hormone-binding globulin levels (Ptrend = 0.03), decreasing 5alpha-androstane-3alpha, 17beta-diol glucuronide levels (Ptrend = 0.02), and decreasing IGFBP3 levels (Ptrend = 0.05). Furthermore, the CSNK1E variant C allele was associated with higher testosterone to dihydrotestosterone ratios (P(LRT) = 0.01) compared with the AA genotype, whereas the longer PER3 repeat was associated with higher serum levels of IGF-I (P(LRT) = 0.03) and IGF-I to IGFBP3 ratios (P(LRT) = 0.04). The CRY2 polymorphism was not associated with any biomarkers analyzed. Our findings, although in need of confirmation, suggest that variations in circadian genes are associated with serum hormone levels, providing biological support for the role of circadian genes in hormone-related cancers.

    Cancer epidemiology, biomarkers & prevention : a publication of the American Association for Cancer Research, cosponsored by the American Society of Preventive Oncology 2008;17;11;3268-73

  • Clock genes may influence bipolar disorder susceptibility and dysfunctional circadian rhythm.

    Shi J, Wittke-Thompson JK, Badner JA, Hattori E, Potash JB, Willour VL, McMahon FJ, Gershon ES and Liu C

    Department of Psychiatry, University of Chicago, Chicago, Illinois 60637, USA.

    Several previous studies suggest that dysfunction of circadian rhythms may increase susceptibility to bipolar disorder (BP). We conducted an association study of five circadian genes (CRY2, PER1-3, and TIMELESS) in a family collection of 36 trios and 79 quads (Sample I), and 10 circadian genes (ARNTL, ARNTL2, BHLHB2, BHLHB3, CLOCK, CRY1, CSNK1D, CSNK1E, DBP, and NR1D1) in an extended family collection of 70 trios and 237 quads (Sample II), which includes the same 114 families but not necessarily the same individuals as Sample I. In Sample II, the Sibling-Transmission Disequilibrium Test (sib-tdt) analysis showed nominally significant association of BP with three SNPs within or near the CLOCK gene (rs534654, P = 0.0097; rs6850524, P = 0.012; rs4340844, P = 0.015). In addition, SNPs in the ARNTL2, CLOCK, DBP, and TIMELESS genes and haplotypes in the ARNTL, CLOCK, CSNK1E, and TIMELESS genes showed suggestive evidence of association with several circadian phenotypes iden 1f40 tified in BP patients. However, none of these associations reached gene-wide or experiment-wide significance after correction for multiple-testing. A multi-locus interaction between rs6442925 in the 5' upstream of BHLHB2, rs1534891 in CSNK1E, and rs534654 near the 3' end of the CLOCK gene, however, is significantly associated with BP (P = 0.00000172). It remains significant after correcting for multiple testing using the False Discovery Rate method. Our results indicate an interaction between three circadian genes in susceptibility to bipolar disorder.

    Funded by: Intramural NIH HHS; NIMH NIH HHS: MH065560-02, MH61613-05A1, R01 MH059535, R01 MH059535-08, R01 MH061613, R01 MH065560, R01 MH065560-05, T32 MH020065, T32 MH020065-06, T32 MH200065; Wellcome Trust: 076113

    American journal of medical genetics. Part B, Neuropsychiatric genetics : the official publication of the International Society of Psychiatric Genetics 2008;147B;7;1047-55

  • Association study between casein kinase 1 epsilon gene and methamphetamine dependence.

    Kotaka T, Ujike H, Morita Y, Kishimoto M, Okahisa Y, Inada T, Harano M, Komiyama T, Hori T, Yamada M, Sekine Y, Iwata N, Iyo M, Sora I, Ozaki N and Kuroda S

    Department of Neuropsychiatry, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama, Japan.

    Casein kinase 1 epsilon (CKIepsilon) is a component of the DARPP-32 in second-messenger pathway. CKIepsilon phosphorylates and activates DARPP-32, a key molecule in various complex signaling pathways, including dopamine and glutamine signaling, which have both been demonstrated to be main pathways in substance dependence. A recent clinical study showed that rs135745, a noncoding single nucleotide polymorphism of the 3'-untranslated region of the CSNK1E gene, was associated with the intensity of the subjective response to an oral amphetamine dose in normal volunteers. Differences in sensitivity to the drug should affect development of dependence to it. Hence, we genotyped rs135745 of the CSNK1E (MIM 600863) gene in 215 patients with methamphetamine dependence and 274 age- and gender-matched normal controls. No significant differences in genotype and allele frequencies were observed between the patients with methamphetamine dependence and controls. There was also no significant association between rs135745 and the clinical characteristics of methamphetamine dependence and co-morbid methamphetamine psychosis (e.g., age of first consumption, latency of psychosis, prognosis of psychosis after therapy, spontaneous relapse of psychotic symptoms, and poly-substance abuse status). The present findings suggest that having a genetic variant of the CSNK1E gene did not affect susceptibility to methamphetamine dependence or psychosis, at least in a Japanese population.

    Annals of the New York Academy of Sciences 2008;1139;43-8

  • Genetic susceptibility to heroin addiction: a candidate gene association study.

    Levran O, Londono D, O'Hara K, Nielsen DA, Peles E, Rotrosen J, Casadonte P, Linzy S, Randesi M, Ott J, Adelson M and Kreek MJ

    The Laboratory of the Biology of Addictive Diseases, The Rockefeller University, New York, NY, USA. levrano@rockefeller.edu

    Heroin addiction is a chronic complex disease with a substantial genetic contribution. This study was designed to identify genetic variants that are associated with susceptibility to develop heroin addiction by analyzing 1350 variants in 130 candidate genes. All subjects had Caucasian ancestry. The sample consisted of 412 former severe heroin addicts in methadone treatment, and 184 healthy controls with no history of drug abuse. Nine variants, in six genes, showed the lowest nominal P values in the association tests (P < 0.01). These variants were in noncoding regions of the genes encoding the mu (OPRM1; rs510769 and rs3778151), kappa (OPRK1; rs6473797) and delta (OPRD1; rs2236861, rs2236857 and rs3766951) opioid receptors; the neuropeptide galanin (GAL; rs694066); the serotonin receptor subtype 3B (HTR3B; rs3758987) and the casein kinase 1 isoform epsilon (CSNK1E; rs1534891). Several haplotypes and multilocus genotype patterns showed nominally significant associations (e.g. OPRM1; P = 0.0006 and CSNK1E; P = 0.0007). Analysis of a combined effect of OPRM1 and OPRD1 showed that rs510769 and rs2236861 increase the risk of heroin addiction (P = 0.0005). None of these associations remained significant after adjustment for multiple testing. This study suggests the involvement of several genes and variants in heroin addiction, which is worthy of future study.

    Funded by: NCRR NIH HHS: UL1 RR024143, UL1-RR024143; NIDA NIH HHS: K05 DA000049, K05 DA000049-22, K05 DA000049-23, K05 DA000049-24, K05 DA000049-25, K05 DA000049-26, K05 DA000049-27, K05 DA000049-28, K05 DA000049-29, K05 DA000049-30, P60 DA005130, P60 DA005130-229010; PHS HHS: P60-05130, R01-44292

    Genes, brain, and behavior 2008;7;7;720-9

  • Association of genetic variation in genes implicated in the beta-catenin destruction complex with risk of breast cancer.

    Wang X, Goode EL, Fredericksen ZS, Vierkant RA, Pankratz VS, Liu-Mares W, Rider DN, Vachon CM, Cerhan JR, Olson JE and Couch FJ

    Department of Laboratory Medicine and Pathology, Mayo Clinic, Stabile 2-42, 200 First Street Southwest, Rochester, MN 55905, USA.

    Aberrant Wnt/beta-catenin signaling leading to nuclear accumulation of the oncogene product beta-catenin is observed in a wide spectrum of human malignancies. The destruction complex in the Wnt/beta-catenin pathway is critical for regulating the level of beta-catenin in the cytoplasm and in the nucleus. Here, we report a comprehensive study of the contribution of genetic variation in six genes encoding the beta-catenin destruction complex (APC, AXIN1, AXIN2, CSNK1D, CSNK1E, and GSK3B) to breast cancer using a Mayo Clinic Breast Cancer Case-Control Study. A total of 79 candidate functional and tagging single nucleotide polymorphisms (SNP) were genotyped in 798 invasive cases and 843 unaffected controls. Of these, rs454886 in the APC tumor suppressor gene was associated with increased breast cancer risk (per allele odds ratio, 1.23; 95% confidence intervals, 1.05-1.43; P(trend) = 0.01). In addition, five SNPs in AXIN2 were associated with increased risk of breast cancer (P(trend) < 0.05). Haplotype-based tests identified significant associations between specific haplotypes in APC and AXIN2 (P < or = 0.03) and breast cancer risk. Further characterization of the APC and AXIN2 variants suggested that AXIN2 rs4791171 was significantly associated with risk in premenopausal (P(trend) = 0.0002) but not in postmenopausal women. The combination of our findings and numerous genetic and functional studies showing that APC and AXIN2 perform crucial tumor suppressor functions suggest that further investigation of the contribution of AXIN2 and APC SNPs to breast cancer risk are needed.

    Funded by: NCI NIH HHS: R01 CA128978, R01 CA128978-01A2, R25 CA092049

    Cancer epidemiology, biomarkers & prevention : a publication of the American Association for Cancer Research, cosponsored by the American Society of Preventive Oncology 2008;17;8;2101-8

  • Casein Kinase I epsilon positively regulates the Akt pathway in breast cancer cell lines.

    Modak C and Bryant P

    Department of Developmental and Cell Biology, University of California Irvine, Mc Gaugh Hall Room 4340, Irvine, CA 92697, USA.

    The Akt pathway is very important in both development and cancer. Here we show that, expression of Casein Kinase I epsilon (CKIepsilon) causes up-regulation of the Akt pathway despite normal protein expression of the pathway inhibitor phosphate and tensin homologue deleted on chromosome ten (PTEN). Conversely, we show that a CKIepsilon/delta-specific inhibitor can inhibit Akt phosphorylation at both Thr308 and Ser473 and drastically reduce phosphorylation of the Akt target Glycogen Synthase Kinase 3beta (GSK3beta). These conclusions were confirmed between MCF7 cells transiently transfected with CKIepsilon and Hs578T cells which already express endogenous CKIepsilon. The results suggest that CKIepsilon is a new positive regulator of the Akt pathway. Here we propose that, rather than inhibiting PTEN function, CKIepsilon positively regulates Akt possibly by inhibiting Protein Phosphatase 2A (PP2A).

    Funded by: NCI NIH HHS: CA91043, R01 CA091043, R01 CA091043-05

    Biochemical and biophysical research communications 2008;368;3;801-7

  • Setting clock speed in mammals: the CK1 epsilon tau mutation in mice accelerates circadian pacemakers by selectively destabilizing PERIOD proteins.

    Meng QJ, Logunova L, Maywood ES, Gallego M, Lebiecki J, Brown TM, Sládek M, Semikhodskii AS, Glossop NR, Piggins HD, Chesham JE, Bechtold DA, Yoo SH, Takahashi JS, Virshup DM, Boot-Handford RP, Hastings MH and Loudon AS

    Faculty of Life Sciences, University of Manchester, Stopford Building, Oxford Road, Manchester M13 9PT, UK.

    The intrinsic period of circadian clocks is their defining adaptive property. To identify the biochemical mechanisms whereby casein kinase1 (CK1) determines circadian period in mammals, we created mouse null and tau mutants of Ck1 epsilon. Circadian period lengthened in CK1epsilon-/-, whereas CK1epsilon(tau/tau) shortened circadian period of behavior in vivo and suprachiasmatic nucleus firing rates in vitro, by accelerating PERIOD-dependent molecular feedback loops. CK1epsilon(tau/tau) also accelerated molecular oscillations in peripheral tissues, revealing its global role in circadian pacemaking. CK1epsilon(tau) acted by promoting degradation of both nuclear and cytoplasmic PERIOD, but not CRYPTOCHROME, proteins. Together, these whole-animal and biochemical studies explain how tau, as a gain-of-function mutation, acts at a specific circadian phase to promote degradation of PERIOD proteins and thereby accelerate the mammalian clockwork in brain and periphery.

    Funded by: Biotechnology and Biological Sciences Research Council: BB/D004357/1, BB/E022553/1, S18856; Howard Hughes Medical Institute; Medical Research Council: G0900414, MC_U105170643

    Neuron 2008;58;1;78-88

  • Casein kinase I epsilon does not rescue double-time function in Drosophila despite evolutionarily conserved roles in the circadian clock.

    Sekine T, Yamaguchi T, Hamano K, Young MW, Shimoda M and Saez L

    Division of Insect Sciences, National Institute of Agrobiological Sciences, Ohwashi 1-2, Tsukuba, Ibaraki 305-8634, Japan.

    Double-time (dbt) is a casein kinase gene involved in cell survival, proliferation, and circadian rhythms in the fruit fly, Drosophila melanogaster. Genetic and biochemical studies have shown that dbt and its mammalian ortholog casein kinase I epsilon (hckI epsilon) regulate the circadian phosphorylation of period (per), thus controlling per subcellular localization and stability. Mutations in these kinases can shorten the circadian period in both mammals and Drosophila. Since similar activities in circadian clock have been described for these kinases, we investigated whether the expression of mammalian casein kinase I can replace the activity of dbt in flies. Global expression of the full-length dbt rescued lethality of the null mutant dbt revVIII and rescued flies showed normal locomotor activity rhythms. Global expression of dbt also restored the locomotor activity rhythm of the arrhythmic genotype, dbt ar/dbt revVIII. In contrast, global expression of hckI epsilon or hckI alpha did not rescue lethality or locomotor activity of dbt mutants. Furthermore dbt overexpression in wild-type clock cells had only a small effect on period length, whereas hckI epsilon expression in clock cells greatly lengthened period to ~30.5 hours and increased the number of arrhythmic flies. These results indicate that hckI epsilon cannot replace the activity of dbt in flies despite the high degree of similarity in primary sequence and kinase function. Moreover, expression of hck Iepsilon in flies appears to interfere with dbt activity. Thus, caution should be used in interpreting assays that measure activity of mammalian casein kinase mutants in Drosophila, or that employ vertebrate CKI in studies of dPER phosphorylations.

    Funded by: NIGMS NIH HHS: GM54339

    Journal of biological rhythms 2008;23;1;3-15

  • Inhibition of casein kinase 1-epsilon induces cancer-cell-selective, PERIOD2-dependent growth arrest.

    Yang WS and Stockwell BR

    Department of Biological Sciences, Columbia University, Fairchild Center, Amsterdam Avenue, New York, NY 10027, USA.

    Background: Kinases are under extensive investigation as targets for drug development. Discovering novel kinases whose inhibition induces cancer-cell-selective lethality would be of value. Recent advances in RNA interference have enabled the realization of this goal.

    Results: We screened 5,760 short hairpin RNA clones targeting the human kinome to detect human kinases on which cancer cells are more dependent than normal cells. We employed a two-step screening strategy using human sarcoma cell lines and human fibroblast-derived isogenic cell lines, and found that short hairpin RNAs targeting CSNK1E, a clock gene that regulates circadian rhythms, can induce selective growth inhibition in engineered tumor cells. Analysis of gene-expression data revealed that CSNK1E is overexpressed in several cancer tissue samples examined compared to non-tumorigenic normal tissue, suggesting a positive role of CSNK1E in neogenesis or maintenance. Treatment with IC261, a kinase domain inhibitor of casein kinase 1-epsilon (CK1epsilon), a protein product of CSNK1E, showed a similar degree of cancer-cell-selective growth inhibition. In a search for substrates of CK1epsilon that mediate IC261-induced growth inhibition, we discovered that knocking down PER2, another clock gene involved in circadian rhythm control, rescues IC261-induced growth inhibition.

    Conclusion: We identified CK1epsilon as a potential target for developing anticancer reagents with a high therapeutic index. These data support the hypothesis that circadian clock genes can control the cell cycle and cell survival signaling, and emphasize a central role of CK1epsilon and PERIOD2 in linking these systems.

    Funded by: NCI NIH HHS: R01 CA097061, R01CA097061

    Genome biology 2008;9;6;R92

  • Variants in circadian genes and prostate cancer risk: a population-based study in China.

    Chu LW, Zhu Y, Yu K, Zheng T, Yu H, Zhang Y, Sesterhenn I, Chokkalingam AP, Danforth KN, Shen MC, Stanczyk FZ, Gao YT and Hsing AW

    Cancer Prevention Fellowship Program, Office of Preventive Oncology, Division of Cancer Prevention, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892, USA. chulisa@mail.nih.gov

    Circadian genes influence a variety of biological processes that are important in prostate tumorigenesis including metabolism. To determine if variants in circadian genes alter prostate cancer risk, we genotyped five variants in five circadian genes in a population-based case-control study conducted in China (187 cases and 242 controls). These variants included CRY2 rs1401417:G>C, CSNK1E rs1005473:A>C, NPAS2 rs2305160:G>A, PER1 rs2585405:G>C and PER3 54-bp repeat length variant. Men with the cryptochrome 2 (CRY2)-variant C allele had 1d09 a significant 1.7-fold increased prostate cancer risk (95% confidence interval (CI), 1.1-2.7) relative to those with the GG genotype. This risk increased to 4.1-fold (95% CI, 2.2-8.0) in men who also had greater insulin resistance (IR) as compared to men with the GG genotype and less IR. In contrast, among men with less IR, the NPAS2-variant A allele was associated with decreased prostate cancer risk (odds ratio=0.5, 95% CI, 0.3-1.0) as compared to the GG genotype. Our findings, although in need of confirmation, suggest that variations in circadian genes may alter prostate cancer risk and some biological processes may modify this effect.

    Prostate cancer and prostatic diseases 2008;11;4;342-8

  • Phosphorylation at Ser244 by CK1 determines nuclear localization and substrate targeting of PKD2.

    von Blume J, Knippschild U, Dequiedt F, Giamas G, Beck A, Auer A, Van Lint J, Adler G and Seufferlein T

    Department of Internal Medicine I, University of Ulm, Ulm, Germany.

    Protein kinase D2 (PKD2), a member of the PKD family of serine/threonine kinases, is localized in various subcellular compartments including the nucleus where the kinase accumulates upon activation of G-protein-coupled receptors. We define three critical post-translational modifications required for nuclear accumulation of PKD2 in response to activation of the CCK2 receptor (CCK2R): phosphorylation at Ser706 and Ser710 within the activation loop by PKC eta leading to catalytic activity and phosphorylation at Ser244 within the zinc-finger domain, which is crucial for blocking nuclear export of active PKD2 by preventing its interaction with the Crm-1 export machinery. We identify CK1delta and epsilon as upstream activated kinases by CCK2R that phosphorylate PKD2 at Ser244. Moreover, nuclear accumulation of active PKD2 is a prerequisite for efficient phosphorylation of its nuclear substrate, HDAC7. Only nuclear, active PKD2 mediates CCK2R-induced HDAC7 phosphorylation and Nur77 expression. Thus, we define a novel, compartment-specific signal transduction pathway downstream of CCK2R that phosphorylates PKD2 at three specific sites, results in nuclear accumulation of the active kinase and culminates in efficient phosphorylation of nuclear PKD2 substrates in human gastric cancer cells.

    The EMBO journal 2007;26;22;4619-33

  • Proteomics analysis of protein kinases by target class-selective prefractionation and tandem mass spectrometry.

    Wissing J, Jänsch L, Nimtz M, Dieterich G, Hornberger R, Kéri G, Wehland J and Daub H

    Department of Cell Biology, Helmholtz Centre for Infection Research (HZI), Inhoffenstrasse 7, 38124 Braunschweig, Germany.

    Protein kinases constitute a large superfamily of enzymes with key regulatory functions in nearly all signal transmission processes of eukaryotic cells. However, due to their relatively low abundance compared with the vast majority of cellular proteins, currently available proteomics techniques do not permit the comprehensive biochemical characterization of protein kinases. To address these limitations, we have developed a prefractionation strategy that uses a combination of immobilized low molecular weight inhibitors for the selective affinity capture of protein kinases. This approach resulted in the direct purification of cell type-specific sets of expressed protein kinases, and more than 140 different members of this enzyme family could be detected by LC-MS/MS. Furthermore the enrichment technique combined with phosphopeptide fractionation led to the identification of more than 200 different phosphorylation sites on protein kinases, which often remain occluded in global phosphoproteome analysis. As the phosphorylation states of protein kinases can provide a readout for the signaling activities within a cellular system, kinase-selective phosphoproteomics based on the procedures described here has the potential to become an important tool in signal transduction analysis.

    Molecular & cellular proteomics : MCP 2007;6;3;537-47

  • Modeling of a human circadian mutation yields insights into clock regulation by PER2.

    Xu Y, Toh KL, Jones CR, Shin JY, Fu YH and Ptácek LJ

    Department of Neurology, University of California, San Francisco, San Francisco, CA 94158, USA.

    Circadian rhythms are endogenous oscillations of physiological and behavioral phenomena with period length of approximately 24 hr. A mutation in human Period 2 (hPER2), a gene crucial for resetting the central clock in response to light, is associated with familial advanced sleep phase syndrome (FASPS), an autosomal dominant condition with early morning awakening and early sleep times. The FASPS hPER2 S662G mutation resulted in PER2 being hypophosphorylated by casein kinase I (CKI) in vitro. We generated transgenic mice carrying the FASPS hPER2 S662G mutation and faithfully recapitulate the human phenotype. We show that phosphorylation at S662 leads to increased PER2 transcription and suggest that phosphorylation at another site leads to PER2 degradation. Altering CKIdelta dosage modulates the S662 phenotype demonstrating that CKIdelta can regulate period through PER2 in vivo. Modeling a naturally occurring human variant in mice has yielded novel insights into PER2 regulation.

    Funded by: NHLBI NIH HHS: R01 HL059596; NIMH NIH HHS: P50 MH074924

    Cell 2007;128;1;59-70

  • Large-scale mapping of human protein-protein interactions by mass spectrometry.

    Ewing RM, Chu P, Elisma F, Li H, Taylor P, Climie S, McBroom-Cerajewski L, Robinson MD, O'Connor L, Li M, Taylor R, Dharsee M, Ho Y, Heilbut A, Moore L, Zhang S, Ornatsky O, Bukhman YV, Ethier M, Sheng Y, Vasilescu J, Abu-Farha M, Lambert JP, Duewel HS, Stewart II, Kuehl B, Hogue K, Colwill K, Gladwish K, Muskat B, Kinach R, Adams SL, Moran MF, Morin GB, Topaloglou T and Figeys D

    Protana, Toronto, Ontario, Canada.

    Mapping protein-protein interactions is an invaluable tool for understanding protein function. Here, we report the first large-scale study of protein-protein interactions in human cells using a mass spectrometry-based approach. The study maps protein interactions for 338 bait proteins that were selected based on known or suspected disease and functional associations. Large-scale immunoprecipitation of Flag-tagged versions of these proteins followed by LC-ESI-MS/MS analysis resulted in the identification of 24,540 potential protein interactions. False positives and redundant hits were filtered out using empirical criteria and a calculated interaction confidence score, producing a data set of 6463 interactions between 2235 distinct proteins. This data set was further cross-validated using previously published and predicted human protein interactions. In-depth mining of the data set shows that it represents a valuable source of novel protein-protein interactions with relevance to human diseases. In addition, via our preliminary analysis, we report many novel protein interactions and pathway associations.

    Molecular systems biology 2007;3;89

  • Global, in vivo, and site-specific phosphorylation dynamics in signaling networks.

    Olsen JV, Blagoev B, Gnad F, Macek B, Kumar C, Mortensen P and Mann M

    Center for Experimental BioInformatics, Department of Biochemistry and Molecular Biology, University of Southern Denmark, DK-5230 Odense, Denmark.

    Cell signaling mechanisms often transmit information via posttranslational protein modifications, most importantly reversible protein phosphorylation. Here we develop and apply a general mass spectrometric technology for identification and quantitation of phosphorylation sites as a function of stimulus, time, and subcellular location. We have detected 6,600 phosphorylation sites on 2,244 proteins and have determined their temporal dynamics after stimulating HeLa cells with epidermal growth factor (EGF) and recorded them in the Phosida database. Fourteen percent of phosphorylation sites are modulated at least 2-fold by EGF, and these were classified by their temporal profiles. Surprisingly, a majority of proteins contain multiple phosphorylation sites showing different kinetics, suggesting that they serve as platforms for integrating signals. In addition to protein kinase cascades, the targets of reversible phosphorylation include ubiquitin ligases, guanine nucleotide exchange factors, and at least 46 different transcriptional regulators. The dynamic phosphoproteome provides a missing link in a global, integrative view of cellular regulation.

    Cell 2006;127;3;635-48

  • An opposite role for tau in circadian rhythms revealed by mathematical modeling.

    Gallego M, Eide EJ, Woolf MF, Virshup DM and Forger DB

    Huntsman Cancer Institute, Department of Oncological Sciences, and Department of Pediatrics, University of Utah, Salt Lake City, UT 84112, USA.

    Biological clocks with a period of approximately 24 h (circadian) ex 1f40 ist in most organisms and time a variety of functions, including sleep-wake cycles, hormone release, bioluminescence, and core body temperature fluctuations. Much of our understanding of the clock mechanism comes from the identification of specific mutations that affect circadian behavior. A widely studied mutation in casein kinase I (CKI), the CKIepsilon(tau) mutant, has been shown to cause a loss of kinase function in vitro, but it has been difficult to reconcile this loss of function with the current model of circadian clock function. Here we show that mathematical modeling predicts the opposite, that the kinase mutant CKIepsilon(tau) increases kinase activity, and we verify this prediction experimentally. CKIepsilon(tau) is a highly specific gain-of-function mutation that increases the in vivo phosphorylation and degradation of the circadian regulators PER1 and PER2. These findings experimentally validate a mathematical modeling approach to a complex biological function, clarify the role of CKI in the clock, and demonstrate that a specific mutation can be both a gain and a loss of function depending on the substrate.

    Funded by: NCI NIH HHS: P30 CA042014, P30CA42014; NIGMS NIH HHS: R01 GM060387

    Proceedings of the National Academy of Sciences of the United States of America 2006;103;28;10618-23

  • Negative regulation of LRP6 function by casein kinase I epsilon phosphorylation.

    Swiatek W, Kang H, Garcia BA, Shabanowitz J, Coombs GS, Hunt DF and Virshup DM

    Department of Oncological Sciences, Huntsman Cancer Institute, University of Utah, Salt Lake City, Utah 84112-5550, USA.

    Wnt signaling acts in part through the low density lipoprotein receptor-related transmembrane proteins LRP5 and LRP6 to regulate embryonic development and stem cell proliferation. Up-regulated signaling is associated with many forms of cancer. Casein kinase I epsilon (CKIepsilon) is a known component of the Wnt-beta-catenin signaling pathway. We find that CKIepsilon binds to LRP5 and LRP6 in vitro and in vivo and identify three CKIepsilon-specific phosphorylation sites in LRP6. Two of the identified phosphorylation sites, Ser1420 and Ser1430, influence Wnt signaling in vivo, since LRP6 with mutation of these sites is a more potent activator of both beta-catenin accumulation and Lef-1 reporter activity. Whereas Wnt3a regulates CKIepsilon kinase activity, LRP6 does not, placing CKIepsilon upstream of LRP6. Mutation of LRP6 Ser1420 and Ser1430 to alanine strengthens its interaction with axin, suggesting a mechanism by which CKIepsilon may negatively regulate Wnt signaling. The role of CKIepsilon is therefore more complex than was previously appreciated. Generation of active CKIepsilon may induce a negative feedback loop by phosphorylation of sites on LRP5/6 that modulate axin binding and hence beta-catenin degradation.

    Funded by: NCI NIH HHS: P01 CA73992, P30 CA42014; NIGMS NIH HHS: R01 GM37537

    The Journal of biological chemistry 2006;281;18;12233-41

  • Association between the casein kinase 1 epsilon gene region and subjective response to D-amphetamine.

    Veenstra-VanderWeele J, Qaadir A, Palmer AA, Cook EH and de Wit H

    Institute of Juvenile Research, Department of Psychiatry, University of Illinois at Chicago, Chicago, IL 60637, USA.

    Animal models suggest that the casein kinase 1 epsilon gene (Csnk1e) contributes to variability in stimulant response. Csnk1e is a key component in the Darpp-32 (Dopamine-And-cAMP-Regulated-Phosphoprotein-32 kDa) second messenger pathway and has been implicated in previous pharmacological and pharmacogenetic studies in mice. Mice bred for methamphetamine sensitivity showed linkage to the region of chromosome 15 that contains Csnk1e and also showed a 10-fold increase in expression of Csnk1e. We used a double-blind, crossover design in healthy human volunteers to test association between polymorphisms in the CSNK1E region and subjective response to placebo, 10, or 20 mg of oral D-amphetamine. Repeated-measures ANOVA was used to analyze interactions between genotype and drug response. The primary outcome measure, subjects' ratings of whether they felt a drug effect (Drug Effects Questionnaire (DEQ)), revealed a significant effect (p=0.010) at one single-nucleotide polymorphism (rs135745). Subjects with more copies of the rs135745 C allele were more sensitive to the low dose of D-amphetamine (p=0.001), which corresponded to a leftward shift in the dose-response curve. These findings demonstrate the successful translation of pharmacogenetic results from mice to humans.

    Funded by: NCRR NIH HHS: RR00055; NIDA NIH HHS: DA02812

    Neuropsychopharmacology : official publication of the American College of Neuropsychopharmacology 2006;31;5;1056-63

  • Casein kinase-1 isoforms differentially associate with neurofibrillary and granulovacuolar degeneration lesions.

    Kannanayakal TJ, Tao H, Vandre DD and Kuret J

    Center for Molecular Neurobiology, Ohio State University College of Medicine, Columbus, OH 43210, USA.

    Alzheimer's Disease (AD) is characterized by the appearance of neurofibrillary and granulovacuolar lesions in the brains of affected individuals. The former is composed of hyperphosphorylated aggregates of the microtubule-associated protein tau. The latter is poorly characterized but reacts strongly with anti-phosphoepi 1563 tope antibodies indicating that it too accumulates phosphoproteins. Both lesions react strongly with antibodies directed against members of the casein kinase-1 family of phosphotransferases, a group of closely related protein kinases that frequently function in tandem with the ubiquitin modification system. To determine whether individual members of the casein kinase-1 family differentially associate with AD lesions, hippocampal sections isolated from late stage cases of AD were subjected to double-label fluorescence immunohistochemistry using a panel of selective anti-casein kinase 1 antibodies and small-molecule fluorochromes thioflavin S and thiazin red. The resultant colocalization patterns revealed that the alpha CK1 isoform strongly correlated with thioflavin S and thiazin red fluorescence, indicating that it preferentially associated with neurofibrillary lesions. In contrast, the delta isoform staining pattern was dominated by colocalization with granulovacuolar degeneration bodies. These findings suggest that granulovacuolar and neurofibrillary lesions occupy separate populations of neurons, and implicate CK1 isoforms in the generation of lesion-associated phosphoepitopes. They also suggest a nexus between the phosphorylation and ubiquitination modifications found in both lesions.

    Funded by: NIA NIH HHS: AG010161, AG018254, AG14452, AG16756; NIMH NIH HHS: MH/NS31862

    Acta neuropathologica 2006;111;5;413-21

  • Dysbindin structural homologue CK1BP is an isoform-selective binding partner of human casein kinase-1.

    Yin H, Laguna KA, Li G and Kuret J

    Center for Molecular Neurobiology and Department of Molecular and Cellular Biochemistry, The Ohio State University College of Medicine, Columbus, Ohio 43210, USA.

    Casein kinase-1 is a family of ubiquitous eukaryotic protein kinases that frequently function in tandem with the ubiquitin modification system to modulate protein turnover and trafficking. In Alzheimer's disease, these enzymes colocalize with ubiquitinated lesions, including neurofibrillary tangles and granulovacuolar degeneration bodies, suggesting they also play a role in disease pathogenesis. To identify binding partners that potentially regulate or recruit these enzymes toward disease lesions, a Sos-recruitment yeast two-hybrid screen was performed with human Ckidelta (the casein kinase-1 isoform most closely linked to granulovacuolar degeneration bodies) and a human brain cDNA library. All interacting clones contained a single open reading frame termed casein kinase-1 binding protein (CK1BP). On the basis of sequence alignments, CK1BP was a structural homologue of the acidic domain of dysbindin, a component of the dystrophin-associated protein complex and the biogenesis of lysosome-related organelles complex-1. CK1BP interacted with full-length Ckidelta, the isolated Ckidelta catalytic domain, Ckigamma2, -gamma3, and -epsilon in the yeast two-hybrid system, and bound Ckidelta and -epsilon in pulldown assays but did not interact with Ckialpha. Interaction with the Ckidelta catalytic domain led to concentration-dependent inhibition of protein kinase activity in the presence of protein substrates tau and alpha-synuclein. Although intact dysbindin did not bind any CK1 isoform, deletion of its coiled-coil domain yielded a protein fragment that behaved much like CK1BP in two-hybrid screens. These data suggest that the acidic domain of dysbindin and its paralogs in humans may function to recruit casein kinase-1 isoforms to protein complexes involved in multiple biological functions.

    Funded by: NIA NIH HHS: AG14452

    Biochemistry 2006;45;16;5297-308

  • Casein kinase I epsilon associates with and phosphorylates the tight junction protein occludin.

    McKenzie JA, Riento K and Ridley AJ

    Ludwig Institute for Cancer Research, Royal Free and University College, School of Medicine, 91 Riding Street, London WIW 7BS, UK.

    Occludin is an integral-membrane protein that contributes to tight junction function. We have identified casein kinase I epsilon (CKI epsilon) as a binding partner for the C-terminal cytoplasmic domain of occludin by yeast two-hybrid screening. CKI epsilon phosphorylated occludin and co-localised and co-immunoprecipitated with occludin from human endothelial cells. Amino acids 265-318 of occludin were sufficient for CKI epsilon binding and phosphorylation. Deletion of the C-terminal 48 amino acids of occludin increased CKI epsilon binding and phosphorylation, suggesting that this region inhibits CKI epsilon binding. These data identify CKI epsilon as a novel occludin kinase that may be important for the regulation of occludin.

    FEBS letters 2006;580;9;2388-94

  • The PITSLRE/CDK11p58 protein kinase promotes centrosome maturation and bipolar spindle formation.

    Petretti C, Savoian M, Montembault E, Glover DM, Prigent C and Giet R

    CNRS UMR 6061 Université de Rennes I, Equipe Labellisée Ligue Nationale Contre le Cancer, IFR140 GFAS, Faculté de Médecine, France.

    The CDK11 (cyclin-dependent kinase 11) gene has an internal ribosome entry site (IRES), allowing the expression of two protein kinases. The longer 110-kDa isoform is expressed at constant levels during the cell cycle and the shorter 58-kDa isoform is expressed only during G2 and M phases. By means of RNA interference (RNAi), we show that the CDK11 gene is required for mitotic spindle formation. CDK11 RNAi leads to mitotic checkpoint activation. Mitotic cells are arrested with short or monopolar spindles. gamma-Tubulin as well as Plk1 and Aurora A protein kinase levels are greatly reduced at centrosomes, resulting in microtubule nucleation defects. We show that the mitotic CDK11(p58) isoform, but not the CDK11(p110) isoform, associates with mitotic centrosomes and rescues the phenotypes resulting from CDK11 RNAi. This work demonstrates for the first time the role of CDK11(p58) in centrosome maturation and bipolar spindle morphogenesis.

    EMBO reports 2006;7;4;418-24

  • Casein kinase Iepsilon down-regulates phospho-Akt via PTEN, following genotoxic stress-induced apoptosis in hematopoietic cells.

    Okamura A, Iwata N, Tamekane A, Yakushijin K, Nishikawa S, Hamaguchi M, Fukui C, Yamamoto K and Matsui T

    Hematology/Oncology, Department of Medicine, Kobe University Graduate School of Medicine 7-5-2, Chuo-ku, Kobe 650-0017, Japan.

    Here, we show a functional role of casein kinase I (CKI) epsilon in hematopoietic cell survival through the modification of phosphatidylinositol 3-kinase (PI3K)/Akt signaling. Introduction of wild-type (WT)-CKIepsilon into interleukin-3 (IL-3)-dependent 32D cells increased the sensitivity to genotoxic stresses, such as gamma-irradiation, etoposide, and IL-3 deprivation, whereas kinase-negative (KN)-CKIepsilon suppressed it. Contrary to KN-CKIepsilon, WT-CKIepsilon attenuated the IL-3-induced activation of Akt with the increase of PTEN activity. Similarly, the increase of Akt activation, as well as PTEN inactivation, was accompanied both by a decrease of CKIepsilon expression induced by all-trans retinoic acid and by the addition of a specific inhibitor for CKIepsilon in HL-60 cells. CKIepsilon seems to activate PTEN by physical interaction. These results suggest that the CKIepsilon-induced down-regulation of PI3K/Akt signaling through PTEN lead to amplified sensitivity to apoptosis. Thus, the suppression of CKIepsilon in many human leukemia cell lines may play a role in the cell immortalization.

    Life sciences 2006;78;14;1624-9

  • Towards a proteome-scale map of the human protein-protein interaction network.

    Rual JF, Venkatesan K, Hao T, Hirozane-Kishikawa T, Dricot A, Li N, Berriz GF, Gibbons FD, Dreze M, Ayivi-Guedehoussou N, Klitgord N, Simon C, Boxem M, Milstein S, Rosenberg J, Goldberg DS, Zhang LV, Wong SL, Franklin G, Li S, Albala JS, Lim J, Fraughton C, Llamosas E, Cevik S, Bex C, Lamesch P, Sikorski RS, Vandenhaute J, Zoghbi HY, Smolyar A, Bosak S, Sequerra R, Doucette-Stamm L, Cusick ME, Hill DE, Roth FP and Vidal M

    Center for Cancer Systems Biology and Department of Cancer Biology, Dana-Farber Cancer Institute, Harvard Medical School, 44 Binney Street, Boston, Massachusetts 02115, USA.

    Systematic mapping of protein-protein interactions, or 'interactome' mapping, was initiated in model organisms, starting with defined biological processes and then expanding to the scale of the proteome. Although far from complete, such maps have revealed global topological and dynamic features of interactome networks that relate to known biological properties, suggesting that a human interactome map will provide insight into development and disease mechanisms at a systems level. Here we describe an initial version of a proteome-scale map of human binary protein-protein interactions. Using a stringent, high-throughput yeast two-hybrid system, we tested pairwise interactions among the products of approximately 8,100 currently available Gateway-cloned open reading frames and detected approximately 2,800 interactions. This data set, called CCSB-HI1, has a verification rate of approximately 78% as revealed by an independent co-affinity purification assay, and correlates significantly with other biological attributes. The CCSB-HI1 data set increases by approximately 70% the set of available binary interactions within the tested space and reveals more than 300 new connections to over 100 disease-associated proteins. This work represents an important step towards a systematic and comprehensive human interactome project.

    Funded by: NCI NIH HHS: R33 CA132073; NHGRI NIH HHS: P50 HG004233, R01 HG001715, RC4 HG006066, U01 HG001715; NHLBI NIH HHS: U01 HL098166

    Nature 2005;437;7062;1173-8

  • SCFbeta-TRCP controls clock-dependent transcription via casein kinase 1-dependent degradation of the mammalian period-1 (Per1) protein.

    Shirogane T, Jin J, Ang XL and Harper JW

    Department of Pathology, Harvard Medical School, Boston, Massachusetts 02115, USA.

    Circadian rhythms are controlled by the periodic accumulation of Period proteins, which act as transcriptional repressors of Clock-dependent genes. Period genes are themselves Clock targets, thereby establishing a negative transcriptional feedback circuit controlling circadian periodicity. Previous data have implicated the CK1epsilon isolog Doubletime (Dbt) and the F-box protein Slimb in the regulation of Drosophila Period (Per) through an unknown mechanism. In this work, we have identified components of the machinery involved in regulating the abundance of human Per1 in tissue culture cells. CK1epsilon and CK1gamma2 were found to bind to Per1 and to promote its degradation in an in vivo degradation assay. Per1 turnover was blocked by a dominant negative version of the Cul1 protein, a component of the SCF (Skp1-Cul1-F-box protein) ubiquitin ligase. We screened a panel of F-box proteins for those that would associate with Per1 in a CK1epsilon-dependent manner, and we identified beta-TRCP1 and beta-TRCP2, isologs of the Drosophila Slimb protein. RNA interference against beta-transducin repeat-containing protein (beta-TRCP) stabilizes endogenous and exogenous Per1. beta-TRCP associates with sequences near the N terminus of Per1 in a region distinct from the previously characterized CK1epsilon-binding site. beta-TRCP and CK1epsilon promote Per1 ubiquitination in vitro. Finally, RNA interference against beta-TRCP greatly decreases Clock-dependent gene expression in tissue culture cells, indicating that beta-TRCP controls endogenous Per1 activity and the circadian clock by directly targeting Per1 for degradation.

    Funded by: NIA NIH HHS: AG11085, R01 AG011085; NINDS NIH HHS: F31 NS054507-01

    The Journal of biological chemistry 2005;280;29;26863-72

  • Chromosome-wide mapping of estrogen receptor binding reveals long-range regulation requiring the forkhead protein FoxA1.

    Carroll JS, Liu XS, Brodsky AS, Li W, Meyer CA, Szary AJ, Eeckhoute J, Shao W, Hestermann EV, Geistlinger TR, Fox EA, Silver PA and Brown M

    Department of Medical Oncology, Dana-Farber Cancer Institute, Harvard Medical School, 44 Binney Street, Boston, Massachusetts 02115, USA.

    Estrogen plays an essential physiologic role in reproduction and a pathologic one in breast cancer. The completion of the human genome has allowed the identification of the expressed regions of protein-coding genes; however, little is known concerning the organization of their cis-regulatory elements. We have mapped the association of the estrogen receptor (ER) with the complete nonrepetitive sequence of human chromosomes 21 and 22 by combining chromatin immunoprecipitation (ChIP) with tiled microarrays. ER binds selectively to a limited number of sites, the majority of which are distant from the transcription start sites of regulated genes. The unbiased sequence interrogation of the genuine chromatin binding sites suggests that direct ER binding requires the presence of Forkhead factor binding in close proximity. Furthermore, knockdown of FoxA1 expression blocks the association of ER with chromatin and estrogen-induced gene expression demonstrating the necessity of FoxA1 in mediating an estrogen response in breast cancer cells.

    Funded by: NCI NIH HHS: F32 CA108380-02; NHGRI NIH HHS: K22 HG02488-01A1; NIDDK NIH HHS: T90 DK070078-01

    Cell 2005;122;1;33-43

  • Control of mammalian circadian rhythm by CKIepsilon-regulated proteasome-mediated PER2 degradation.

    Eide EJ, Woolf MF, Kang H, Woolf P, Hurst W, Camacho F, Vielhaber EL, Giovanni A and Virshup DM

    Huntsman Cancer Institute, University of Utah, Salt Lake City, UT 84112, USA.

    The mammalian circadian regulatory proteins PER1 and PER2 undergo a daily cycle of accumulation followed by phosphorylation and degradation. Although phosphorylation-regulated proteolysis of these inhibitors is postulated to be essential for the function of the clock, inhibition of this process has not yet been shown to alter mammalian circadian rhythm. We have developed a cell-based model of PER2 degradation. Murine PER2 (mPER2) hyperphosphorylation induced by the cell-permeable protein phosphatase inhibitor calyculin A is rapidly followed by ubiquitination and degradation by the 26S proteasome. Proteasome-mediated degradation is critically important in the circadian clock, as proteasome inhibitors cause a significant lengthening of the circadian period in Rat-1 cells. CKIepsilon (casein kinase Iepsilon) has been postulated to prime PER2 for degradation. Supporting this idea, CKIepsilon inhibition also causes a significant lengthening of circadian period in synchronized Rat-1 cells. CKIepsilon inhibition also slows the degradation of PER2 in cells. CKIepsilon-mediated phosphorylation of PER2 recruits the ubiquitin ligase adapter protein beta-TrCP to a specific site, and dominant negative beta-TrCP blocks phosphorylation-dependent degradation of mPER2. These results provide a biochemical mechanism and functional relevance for the observed phosphorylation-degradation cycle of mammalian PER2. Cell culture-based biochemical assays combined with measurement of cell-based rhythm complement genetic studies to elucidate basic mechanisms controlling the mammalian clock.

    Funded by: NCI NIH HHS: P04CA42104; NIGMS NIH HHS: R01 GM060387, R01 GM60387

    Molecular and cellular biology 2005;25;7;2795-807

  • A second protein kinase CK1-mediated step negatively regulates Wnt signalling by disrupting the lymphocyte enhancer factor-1/beta-catenin complex.

    Hämmerlein A, Weiske J and Huber O

    ABDA - Federal Union of German Associations of Pharmacists, Jägerstr. 49/50, 10117, Berlin, Germany.

    Deregulated activation of the canonical Wnt signalling pathway leads to stabilization of beta-catenin and is critically involved in carcinogenesis by an inappropriate induction of lymphocyte enhancer factor (LEF-1)/beta-catenin-dependent transcription of Wnt target genes. Phosphorylation of the pathway components beta-catenin, Dishevelled, Axin and APC (adenomatous polyposis coli) by glycogen synthase kinase-3beta, CK1 and CK2 is of central importance in the regulation of the beta-catenin destruction complex. Here, we identify CK1 and CK2 as major kinases that directly bind to and phosphorylate LEF-1 inducing distinct, kinase-specific changes in the LEF-1/DNA complex. Moreover, CK1-dependent phosphorylation in contrast to CK2 disrupts the association of beta-catenin and LEF-1 but does not impair DNA binding of LEF-1. Sequential phosphorylation assays revealed that for efficient disruption of the LEF-1/beta-catenin complex, beta-catenin also has to be phosphorylated. Consistent with these observations, CK1-dependent phosphorylation inhibits, whereas CK2 activates LEF-1/beta-catenin transcriptional activity in reporter gene assays. These data are in line with a negative regulatory function of CK1 in the Wnt signalling pathway, where CK1 in addition to the beta-catenin destruction complex at a second level acts as a negative regulator of the LEF-1/beta-catenin transcription complex, thereby protecting cells from development of cancer.

    Cellular and molecular life sciences : CMLS 2005;62;5;606-18

  • A missense variation in human casein kinase I epsilon gene that induces functional alteration and shows an inverse association with circadian rhythm sleep disorders.

    Takano A, Uchiyama M, Kajimura N, Mishima K, Inoue Y, Kamei Y, Kitajima, Shibui K, Katoh M, Watanabe T, Hashimotodani Y, Nakajima T, Ozeki Y, Hori T, Yamada N, Toyoshima R, Ozaki N, Okawa M, Nagai K, Takahashi K, Isojima Y, Yamauchi T and Ebisawa T

    Division of Protein Metabolism, Institute for Protein Research, Osaka University, Osaka, Japan.

    Recent studies have shown that functional variations in clock genes, which generate circadian rhythms through interactive positive/negative feedback loops, contribute to the development of circadian rhythm sleep disorders in humans. Another potential candidate for rhythm disorder susceptibility is casein kinase I epsilon (CKIepsilon), which phosphorylates clock proteins and plays a pivotal role in the circadian clock. To determine whether variations in CKIepsilon induce vulnerability to human circadian rhythm sleep disorders, such as delayed sleep phase syndrome (DSPS) and non-24-h sleep-wake syndrome (N-24), we analyzed all of the coding exons of the human CKIepsilon gene. One of the variants identified encoded an amino-acid substitution S408N, eliminating one of the putative autophosphorylation sites in the carboxyl-terminal extension of CKIepsilon. The N408 allele was less common in both DSPS (p = 0.028) and N-24 patients (p = 0.035) compared to controls. When DSPS and N-24 subjects were combined, based on an a priori prediction of a common mechanism underlying both DSPS and N-24, the inverse association between the N408 allele and rhythm disorders was highly significant (p = 0.0067, odds ratio = 0.42, 95% confidence interval: 0.22-0.79). In vitro kinase assay revealed that CKIepsilon with the S408N variation was approximately 1.8-fold more active than wild-type CKIepsilon. These results indicate that the N408 allele in CKIepsilon plays a protective role in the development of DSPS and N-24 through alteration of the enzyme activity.

    Neuropsychopharmacology : official publication of the American College of Neuropsychopharmacology 2004;29;10;1901-9

  • The status, quality, and expansion of the NIH full-length cDNA project: the Mammalian Gene Collection (MGC).

    Gerhard DS, Wagner L, Feingold EA, Shenmen CM, Grouse LH, Schuler G, Klein SL, Old S, Rasooly R, Good P, Guyer M, Peck AM, Derge JG, Lipman D, Collins FS, Jang W, Sherry S, Feolo M, Misquitta L, Lee E, Rotmistrovsky K, Greenhut SF, Schaefer CF, Buetow K, Bonner TI, Haussler D, Kent J, Kiekhaus M, Furey T, Brent M, Prange C, Schreiber K, Shapiro N, Bhat NK, Hopkins RF, Hsie F, Driscoll T, Soares MB, Casavant TL, Scheetz TE, Brown-stein MJ, Usdin TB, Toshiyuki S, Carninci P, Piao Y, Dudekula DB, Ko MS, Kawakami K, Suzuki Y, Sugano S, Gruber CE, Smith MR, Simmons B, Moore T, Waterman R, Johnson SL, Ruan Y, Wei CL, Mathavan S, Gunaratne PH, Wu J, Garcia AM, Hulyk SW, Fuh E, Yuan Y, Sneed A, Kowis C, Hodgson A, Muzny DM, McPherson J, Gibbs RA, Fahey J, Helton E, Ketteman M, Madan A, Rodrigues S, Sanchez A, Whiting M, Madari A, Young AC, Wetherby KD, Granite SJ, Kwong PN, Brinkley CP, Pearson RL, Bouffard GG, Blakesly RW, Green ED, Dickson MC, Rodriguez AC, Grimwood J, Schmutz J, Myers RM, Butterfield YS, Griffith M, Griffith OL, Krzywinski MI, Liao N, Morin R, Morrin R, Palmquist D, Petrescu AS, Skalska U, Smailus DE, Stott JM, Schnerch A, Schein JE, Jones SJ, Holt RA, Baross A, Marra MA, Clifton S, Makowski KA, Bosak S, Malek J and MGC Project Team

    The National Institutes of Health's Mammalian Gene Collection (MGC) project was designed to generate and sequence a publicly accessible cDNA resource containing a complete open reading frame (ORF) for every human and mouse gene. The project initially used a random strategy to select clones from a large number of cDNA libraries from diverse tissues. Candidate clones were chosen based on 5'-EST sequences, and then fully sequenced to high accuracy and analyzed by algorithms developed for this project. Currently, more than 11,000 human and 10,000 mouse genes are represented in MGC by at least one clone with a full ORF. The random selection approach is now reaching a saturation point, and a transition to protocols targeted at the missing transcripts is now required to complete the mouse and human collections. Comparison of the sequence of the MGC clones to reference genome sequences reveals that most cDNA clones are of very high sequence quality, although it is likely that some cDNAs may carry missense variants as a consequence of experimental artifact, such as PCR, cloning, or reverse transcriptase errors. Recently, a rat cDNA component was added to the project, and ongoing frog (Xenopus) and zebrafish (Danio) cDNA projects were expanded to take advantage of the high-throughput MGC pipeline.

    Funded by: PHS HHS: N01-C0-12400

    Genome research 2004;14;10B;2121-7

  • Mechanism of phosphorylation-dependent binding of APC to beta-catenin and its role in beta-catenin degradation.

    Ha NC, Tonozuka T, Stamos JL, Choi HJ and Weis WI

    Department of Structural Biology, Stanford University School of Medicine, Stanford, CA 94043, USA.

    The transcriptional coactivator beta-catenin mediates Wnt growth factor signaling. In the absence of a Wnt signal, casein kinase 1 (CK1) and glycogen synthase kinase-3beta (GSK-3beta) phosphorylate cytosolic beta-catenin, thereby flagging it for recognition and destruction by the ubiquitin/proteosome machinery. Phosphorylation occurs in a multiprotein complex that includes the kinases, beta-catenin, axin, and the Adenomatous Polyposis Coli (APC) protein. The role of APC in this process is poorly understood. CK1epsilon and GSK-3beta phosphorylate APC, which increases its affinity for beta-catenin. Crystal structures of phosphorylated and nonphosphorylated APC bound to beta-catenin reveal a phosphorylation-dependent binding motif generated by mutual priming of CK1 and GSK-3beta substrate sequences. Axin is shown to act as a scaffold for substrate phosphorylation by these kinases. Phosphorylated APC and axin bind to the same surface of, and compete directly for, beta-catenin. The structural and biochemical data suggest a novel model for how APC functions in beta-catenin degradation.

    Funded by: NIGMS NIH HHS: GM56169

    Molecular cell 2004;15;4;511-21

  • Large-scale characterization of HeLa cell nuclear phosphoproteins.

    Beausoleil SA, Jedrychowski M, Schwartz D, Elias JE, Villén J, Li J, Cohn MA, Cantley LC and Gygi SP

    Department of Cell Biology, Harvard Medical School, Boston, MA 02115, USA.

    Determining the site of a regulatory phosphorylation event is often essential for elucidating specific kinase-substrate relationships, providing a handle for understanding essential signaling pathways and ultimately allowing insights into numerous disease pathologies. Despite intense research efforts to elucidate mechanisms of protein phosphorylation regulation, efficient, large-scale identification and characterization of phosphorylation sites remains an unsolved problem. In this report we describe an application of existing technology for the isolation and identification of phosphorylation sites. By using a strategy based on strong cation exchange chromatography, phosphopeptides were enriched from the nuclear fraction of HeLa cell lysate. From 967 proteins, 2,002 phosphorylation sites were determined by tandem MS. This unprecedented large collection of sites permitted a detailed accounting of known and unknown kinase motifs and substrates.

    Funded by: NHGRI NIH HHS: HG00041, K22 HG000041, T32 HG000041; NIGMS NIH HHS: GM67945, GMS6203, R01 GM056203, R01 GM067945

    Proceedings of the National Academy of Sciences of the United States of America 2004;101;33;12130-5

  • Casein kinase Iepsilon plays a functional role in the transforming growth factor-beta signaling pathway.

    Waddell DS, Liberati NT, Guo X, Frederick JP and Wang XF

    Department of Pharmacology and Cancer Biology, Duke University Medical Center, Durham, North Carolina 27710, USA.

    The transforming growth factor-beta (TGF-beta) signaling pathway is known to be involved in a wide range of biological events, including development, cellular differentiation, apoptosis, and oncogenesis. The TGF-beta signal is mediated by ligand binding to the type II receptor, leading to the recruitment and activation of the type I receptor, and subsequent activation of a family of intracellular signal transducing proteins called Smads. Here we report a regulatory role for casein kinase Iepsilon (CKIepsilon) in the TGF-beta signaling cascade. We find that CKIepsilon binds to all Smads and the cytoplasmic domains of the type I and type II receptors both in vitro and in vivo. The interaction of CKIepsilon with the type I and type II receptors is independent of TGF-beta stimulation, whereas the CKIepsilon/Smad interaction is transiently disrupted by ligand treatment. Additionally, CKIepsilon is able to phosphorylate the receptor-activated Smads (Smads 1-3 and 5) and the type II receptor in vitro. Transcriptional reporter assays reveal that transient overexpression of wild type CKIepsilon dramatically reduces basal reporter activity but enhances TGF-beta-stimulated transcription. Furthermore, overexpression of a kinase-dead mutant of CKIepsilon inhibits both basal and ligand-induced transcription, whereas inhibition of endogenous CKI catalytic activity with IC261 blocks only TGF-beta-stimulated reporter activity. Finally, knocking down CKIepsilon protein levels results in a significant increase in basal and TGF-beta-induced transcription. These results suggest that CKIepsilon plays a ligand-dependent, differential, and dual regulatory role within the TGF-beta signaling pathway.

    Funded by: NCI NIH HHS: CA75368, CA83770

    The Journal of biological chemistry 2004;279;28;29236-46

  • Involvement of casein kinase Iepsilon in cytokine-induced granulocytic differentiation.

    Okamura A, Iwata N, Nagata A, Tamekane A, Shimoyama M, Gomyo H, Yakushijin K, Urahama N, Hamaguchi M, Fukui C, Chihara K, Ito M and Matsui T

    Hematology/Oncology, Department of Medicine, Kobe University School of Medicine, Kobe, Japan.

    Two closely related casein kinase I (CKI) isoforms, CKIdelta and CKIepsilon, are ubiquitously expressed in many human tissues, but their specific biologic function remains to be clarified. Here, we provide the first evidence that CKIepsilon is involved in hematopoietic cell differentiation. CKIepsilon, but not CKIdelta, was down-regulated along with human granulocytic differentiation. The specific down-regulation was observed in granulocyte colony-stimulating factor (G-CSF)-induced cell differentiation of murine interleukin-3 (IL-3)-dependent myeloid progenitor 32D cells. Introduction of wild-type (WT)-CKIepsilon into 32D cells inhibited the G-CSF-induced cell differentiation, whereas kinase-negative (KN)-CKIepsilon promoted the differentiation. Neither WT- nor KN-CKIepsilon affected IL-3-dependent cell growth. Moreover, introduction of WT- or KN-CKIdelta did not affect the cytokine-induced cell growth and differentiation. While G-CSF-induced activation of signal transducers and activators of transcription 3 (STAT3) was sustained by KN-CKIepsilon, STAT3 activation was attenuated by WT-CKIepsilon. This may be explained by the fact that the suppressor of cytokine signaling 3 (SOCS3) was stabilized by its physical association with CKIepsilon. Such stabilization by CKIepsilon was also seen in IL-3-induced beta-catenin. The stabilization of downstream components of cytokine and Wnt signaling by CKIepsilon might be critical for integration of several intracellular signaling pathways to a cell-specific biologic response in hematopoietic cell self-renewal.

    Blood 2004;103;8;2997-3004

  • Association of CPI-17 with protein kinase C and casein kinase I.

    Zemlickova E, Johannes FJ, Aitken A and Dubois T

    University of Edinburgh, Division of Biomedical and Clinical Laboratory Sciences, Hugh Robson Building, George Square, Edinburgh EH8 9XD, UK.

    The protein kinase C-potentiated inhibitor protein of 17kDa, called CPI-17, specifically inhibits myosin light chain phosphatase (MLCP). Phosphorylation of Thr-38 in vivo highly potentiates the ability of CPI-17 to inhibit MLCP. Thr-38 has been shown to be phosphorylated in vitro by a number of protein kinases including protein kinase C (PKC), Rho-associated coiled-coil kinase (ROCK), and protein kinase N (PKN). In this study we have focused on the association of protein kinases with CPI-17. Using affinity chromatography and Western blot analysis, we found interaction with all PKC isotypes and casein kinase I isoforms, CKIalpha and CKI. By contrast, ROCK and PKN did not associate with CPI-17, suggesting that PKC may be the relevant kinase that phosphorylates Thr-38 in vivo. CPI-17 interacted with the cysteine-rich domain of PKC and was phosphorylated by all PKC isotypes. We previously found that CPI-17 co-purified with casein kinase I in brain suggesting they are part of a complex and we now show that CPI-17 associates with the kinase domain of CKI isoforms.

    Biochemical and biophysical research communications 2004;316;1;39-47

  • Regulation of casein kinase I epsilon activity by Wnt signaling.

    Swiatek W, Tsai IC, Klimowski L, Pepler A, Barnette J, Yost HJ and Virshup DM

    Department of Oncological Sciences and the Center for Children, Huntsman Cancer Institute, 2000 Circle of Hope, Salt Lake City, UT 84112, USA.

    The Wnt/beta-catenin signaling pathway is important in both development and cancer. Casein kinase Iepsilon (CKIepsilon) is a positive regulator of the canonical Wnt pathway. CKIepsilon itself can be regulated in vitro by inhibitory autophosphorylation, and recent data suggest that in vivo kinase activity can be regulated by extracellular stimuli. We show here that the phosphorylation state and kinase activity of CKIepsilon are directly regulated by Wnt signaling. Coexpression of XWnt-8 or addition of soluble Wnt-3a ligand led to a significant and rapid increase in the activity of endogenous CKIepsilon. The increase in CKIepsilon activity is the result of decreased inhibitory autophosphorylation because it is abolished by preincubation of immunoprecipitated kinase with ATP. Furthermore, mutation of CKIepsilon inhibitory autophosphorylation sites creates a kinase termed CKIepsilon(MM2) that is significantly more active than CKIepsilon and is not activated further upon Wnt stimulation. Autoinhibition of CKIepsilon is biologically relevant because CKIepsilon(MM2) is more effective than CKIepsilon at activating transcription from a Lef1-dependent promoter. Finally, CKIepsilon(MM2) expression in Xenopus embryos induces both axis duplication and additional developmental abnormalities. The data suggest that Wnt signaling activates CKIepsilon by causing transient dephosphorylation of critical inhibitory sites present in the carboxyl-terminal domain of the kinase. Activation of the Wnt pathway may therefore stimulate a cellular phosphatase to dephosphorylate and activate CKIepsilon

    Funded by: NCI NIH HHS: R01 CA 80809

    The Journal of biological chemistry 2004;279;13;13011-7

  • Complete sequencing and characterization of 21,243 full-length human cDNAs.

    Ota T, Suzuki Y, Nishikawa T, Otsuki T, Sugiyama T, Irie R, Wakamatsu A, Hayashi K, Sato H, Nagai K, Kimura K, Makita H, Sekine M, Obayashi M, Nishi T, Shibahara T, Tanaka T, Ishii S, Yamamoto J, Saito K, Kawai Y, Isono Y, Nakamura Y, Nagahari K, Murakami K, Yasuda T, Iwayanagi T, Wagatsuma M, Shiratori A, Sudo H, Hosoiri T, Kaku Y, Kodaira H, Kondo H, Sugawara M, Takahashi M, Kanda K, Yokoi T, Furuya T, Kikkawa E, Omura Y, Abe K, Kamihara K, Katsuta N, Sato K, Tanikawa M, Yamazaki M, Ninomiya K, Ishibashi T, Yamashita H, Murakawa K, Fujimori K, Tanai H, Kimata M, Watanabe M, Hiraoka S, Chiba Y, Ishida S, Ono Y, Takiguchi S, Watanabe S, Yosida M, Hotuta T, Kusano J, Kanehori K, Takahashi-Fujii A, Hara H, Tanase TO, Nomura Y, Togiya S, Komai F, Hara R, Takeuchi K, Arita M, Imose N, Musashino K, Yuuki H, Oshima A, Sasaki N, Aotsuka S, Yoshikawa Y, Matsunawa H, Ichihara T, Shiohata N, Sano S, Moriya S, Momiyama H, Satoh N, Takami S, Terashima Y, Suzuki O, Nakagawa S, Senoh A, Mizoguchi H, Goto Y, Shimizu F, Wakebe H, Hishigaki H, Watanabe T, Sugiyama A, Takemoto M, Kawakami B, Yamazaki M, Watanabe K, Kumagai A, Itakura S, Fukuzumi Y, Fujimori Y, Komiyama M, Tashiro H, Tanigami A, Fujiwara T, Ono T, Yamada K, Fujii Y, Ozaki K, Hirao M, Ohmori Y, Kawabata A, Hikiji T, Kobatake N, Inagaki H, Ikema Y, Okamoto S, Okitani R, Kawakami T, Noguchi S, Itoh T, Shigeta K, Senba T, Matsumura K, Nakajima Y, Mizuno T, Morinaga M, Sasaki M, Togashi T, Oyama M, Hata H, Watanabe M, Komatsu T, Mizushima-Sugano J, Satoh T, Shirai Y, Takahashi Y, Nakagawa K, Okumura K, Nagase T, Nomura N, Kikuchi H, Masuho Y, Yamashita R, Nakai K, Yada T, Nakamura Y, Ohara O, Isogai T and Sugano S

    Helix Research Institute, 1532-3 Yana, Kisarazu, Chiba 292-0812, Japan.

    As a base for human transcriptome and functional genomics, we created the "full-length long Japan" (FLJ) collection of sequenced human cDNAs. We determined the entire sequence of 21,243 selected clones and found that 14,490 cDNAs (10,897 clusters) were unique to the FLJ collection. About half of them (5,416) seemed to be protein-coding. Of those, 1,999 clusters had not been predicted by computational methods. The distribution of GC content of nonpredicted cDNAs had a peak at approximately 58% compared with a peak at approximately 42%for predicted cDNAs. Thus, there seems to be a slight bias against GC-rich transcripts in current gene prediction procedures. The rest of the cDNAs unique to the FLJ collection (5,481) contained no obvious open reading frames (ORFs) and thus are candidate noncoding RNAs. About one-fourth of them (1,378) showed a clear pattern of splicing. The distribution of GC content of noncoding cDNAs was narrow and had a peak at approximately 42%, relatively low compared with that of protein-coding cDNAs.

    Nature genetics 2004;36;1;40-5

  • Direct association between mouse PERIOD and CKIepsilon is critical for a functioning circadian clock.

    Lee C, Weaver DR and Reppert SM

    Department of Neurobiology, LRB-728, University of Massachusetts Medical School, 364 Plantation Street, Worcester, MA 01605, USA.

    The mPER1 and mPER2 proteins have important roles in the circadian clock mechanism, whereas mPER3 is expendable. Here we examine the posttranslational regulation of mPER3 in vivo in mouse liver and compare it to the other mPER proteins to define the salient features required for clock function. Like mPER1 and mPER2, mPER3 is phosphorylated, changes cellular location, and interacts with other clock proteins in a time-dependent manner. Consistent with behavioral data from mPer2/3 and mPer1/3 double-mutant mice, either mPER1 or mPER2 alone can sustain rhythmic posttranslational events. However, mPER3 is unable to sustain molecular rhythmicity in mPer1/2 double-mutant mice. Indeed, mPER3 is always cytoplasmic and is not phosphorylated in the livers of mPer1-deficient mice, suggesting that mPER3 is regulated by mPER1 at a posttranslational level. In vitro studies with chimeric proteins suggest that the inability of mPER3 to support circadian clock function results in part from lack of direct and stable interaction with casein kinase Iepsilon (CKIepsilon). We thus propose that the CKIepsilon-binding domain is critical not only for mPER phosphorylation but also for a functioning circadian clock.

    Funded by: NINDS NIH HHS: NS 39303, R01 NS039303

    Molecular and cellular biology 2004;24;2;584-94

  • A genome annotation-driven approach to cloning the human ORFeome.

    Collins JE, Wright CL, Edwards CA, Davis MP, Grinham JA, Cole CG, Goward ME, Aguado B, Mallya M, Mokrab Y, Huckle EJ, Beare DM and Dunham I

    The Wellcome Trust Sanger Institute, Wellcome Trust Genome Campus, Hinxton, Cambridge, CB10 1SA, UK.

    We have developed a systematic approach to generating cDNA clones containing full-length open reading frames (ORFs), exploiting knowledge of gene structure from genomic sequence. Each ORF was amplified by PCR from a pool of primary cDNAs, cloned and confirmed by sequencing. We obtained clones representing 70% of genes on human chromosome 22, whereas searching available cDNA clone collections found at best 48% from a single collection and 60% for all collections combined.

    Genome biology 2004;5;10;R84

  • Polo-like kinase 1 regulates Nlp, a centrosome protein involved in microtubule nucleation.

    Casenghi M, Meraldi P, Weinhart U, Duncan PI, Körner R and Nigg EA

    Department of Cell Biology, Max Planck Institute of Biochemistry, Am Klopferspitz 18a, D-82152, Martinsried, Germany.

    In animal cells, most microtubules are nucleated at centrosomes. At the onset of mitosis, centrosomes undergo a structural reorganization, termed maturation, which leads to increased microtubule nucleation activity. Centrosome maturation is regulated by several kinases, including Polo-like kinase 1 (Plk1). Here, we identify a centrosomal Plk1 substrate, termed Nlp (ninein-like protein), whose properties suggest an important role in microtubule organization. Nlp interacts with two components of the gamma-tubulin ring complex and stimulates microtubule nucleation. Plk1 phosphorylates Nlp and disrupts both its centrosome association and its gamma-tubulin interaction. Overexpression of an Nlp mutant lacking Plk1 phosphorylation sites severely disturbs mitotic spindle formation. We propose that Nlp plays an important role in microtubule organization during interphase, and that the activation of Plk1 at the onset of mitosis triggers the displacement of Nlp from the centrosome, allowing the establishment of a mitotic scaffold with enhanced microtubule nucleation activity.

    Developmental cell 2003;5;1;113-25

  • Casein kinase I epsilon enhances the binding of Dvl-1 to Frat-1 and is essential for Wnt-3a-induced accumulation of beta-catenin.

    Hino S, Michiue T, Asashima M and Kikuchi A

    Department of Biochemistry, Graduate School of Biomedical Sciences, Hiroshima University, 1-2-3, Kasumi, Minami-ku, Japan.

    We demonstrate that Dvl-1, casein kinase I epsilon (CKI epsilon), and Frat-1 activate the Wnt signaling pathway cooperatively. The amino acid region 228-250 of Dvl-1 was necessary for its binding to Frat-1, and the interaction of Dvl-1 with Frat-1 was enhanced by CKI epsilon. Coexpression of Dvl-1 and Frat-1 caused accumulation of beta-catenin synergistically in L cells. Both proteins also activated the transcriptional activity of T-cell factor-4 (Tcf-4) synergistically in human embryonic kidney 293 cells, but coexpression of Dvl-1-(Delta 228-250), which lacks the amino acid region 228-250 from Dvl-1, and Frat-1 did not. Dvl-1, but not Dvl-1-(Delta 228-250), acted synergistically with CKI epsilon to activate Tcf-4. Depletion of CKI epsilon by double-stranded RNA interference in HeLa S3 cells led to the inhibition of Wnt-3a-induced phosphorylation of Dvl and the binding of Dvl-1 to Frat-1. Furthermore, depletion of CKI epsilon reduced the Wnt-3a-induced accumulation of beta-catenin, although it did not affect the basal level of beta-catenin. These results indicate that CKI epsilon-dependent phosphorylation of Dvl enhances the formation of a complex of Dvl-1 with Frat-1 and that this complex leads to the activation of the Wnt signaling pathway.

    The Journal of biological chemistry 2003;278;16;14066-73

  • Centrosomal anchoring of the protein kinase CK1delta mediated by attachment to the large, coiled-coil scaffolding protein CG-NAP/AKAP450.

    Sillibourne JE, Milne DM, Takahashi M, Ono Y and Meek DW

    Biomedical Research Centre, Ninewells Hospital and Medical School, University of Dundee, Scotland DD1 9SY, UK.

    Protein kinase CK1 (formerly termed casein kinase I) is ubiquitous in eukaryotic cells and comprises a family of as many as 14 isoforms (including splice variants) in mammalian cells. Mammalian CK1delta and CK1epsilon, which are highly related to each other, are enriched at the centrosomes in interphase cells and at the spindle during mitosis. In the present study we have isolated, using the yeast two-hybrid system, a 182 amino acid residue fragment of the centrosomal and golgi N-kinase anchoring protein (CG-NAP, also known as AKAP450), which specifically interacts with CK1delta and CK1epsilon, but not with other CK1 isoforms. The 182 amino acid residue CG-NAP fragment, or full length CG-NAP, co-immunoprecipitates with CK1delta and CK1epsilon from mammalian cells. Consistent with this association, endogenous CG-NAP/AKAP450 and CK1delta co-localize in cells. Moreover, when expressed in the presence of CK1delta the 182 amino acid residue CG-NAP fragment adopts the same sub-cellular localization as CK1delta. Strikingly, attachment of the CG-NAP fragment to the plasma membrane is sufficient to re-localize a significant level of CK1delta to the membrane. These findings support a model in which sub-cellular localization of CK1delta/epsilon molecules at the centrosome is mediated, at least in part, through the action of CG-NAP/AKAP450 and provide a potential mechanism by which the contribution to cell cycle progression by CK1delta/epsilon may be regulated.

    Journal of molecular biology 2002;322;4;785-97

  • Centrosomal proteins CG-NAP and kendrin provide microtubule nucleation sites by anchoring gamma-tubulin ring complex.

    Takahashi M, Yamagiwa A, Nishimura T, Mukai H and Ono Y

    Biosignal Research Center, Kobe University, Japan.

    Microtubule assembly is initiated by the gamma-tubulin ring complex (gamma-TuRC). In yeast, the microtubule is nucleated from gamma-TuRC anchored to the amino-terminus of the spindle pole body component Spc110p, which interacts with calmodulin (Cmd1p) at the carboxy-terminus. However, mammalian protein that anchors gamma-TuRC remains to be elucidated. A giant coiled-coil protein, CG-NAP (centrosome and Golgi localized PKN-associated protein), was localized to the centrosome via the carboxyl-terminal region. This region was found to interact with calmodulin by yeast two-hybrid screening, and it shares high homology with the carboxyl-terminal region of another centrosomal coiled-coil protein, kendrin. The amino-terminal region of either CG-NAP or kendrin indirectly associated with gamma-tubulin through binding with gamma-tubulin complex protein 2 (GCP2) and/or GCP3. Furthermore, endogenous CG-NAP and kendrin were coimmunoprecipitated with each other and with endogenous GCP2 and gamma-tubulin, suggesting that CG-NAP and kendrin form complexes and interact with gamma-TuRC in vivo. These proteins were localized to the center of microtubule asters nucleated from isolated centrosomes. Pretreatment of the centrosomes by antibody to CG-NAP or kendrin moderately inhibited the microtubule nucleation; moreover, the combination of these antibodies resulted in stronger inhibition. These results imply that CG-NAP and kendrin provide sites for microtubule nucleation in the mammalian centrosome by anchoring gamma-TuRC.

    Molecular biology of the cell 2002;13;9;3235-45

  • The ankyrin repeat protein Diversin recruits Casein kinase Iepsilon to the beta-catenin degradation complex and acts in both canonical Wnt and Wnt/JNK signaling.

    Schwarz-Romond T, Asbrand C, Bakkers J, Kühl M, Schaeffer HJ, Huelsken J, Behrens J, Hammerschmidt M and Birchmeier W

    Max Delbrueck-Center for Molecular Medicine, D-13092 Berlin, Germany.

    Wnt signals control decisive steps in development and can induce the formation of tumors. Canonical Wnt signals control the formation of the embryonic axis, and are mediated by stabilization and interaction of beta-catenin with Lef/Tcf transcription factors. An alternative branch of the Wnt pathway uses JNK to establish planar cell polarity in Drosophila and gastrulation movements in vertebrates. We describe here the vertebrate protein Diversin that interacts with two components of the canonical Wnt pathway, Casein kinase Iepsilon (CKIepsilon) and Axin/Conductin. Diversin recruits CKIepsilon to the beta-catenin degradation complex that consists of Axin/Conductin and GSK3beta and allows efficient phosphorylation of beta-catenin, thereby inhibiting beta-catenin/Tcf signals. Morpholino-based gene ablation in zebrafish shows that Diversin is crucial for axis formation, which depends on beta-catenin signaling. Diversin is also involved in JNK activation and gastrulation movements in zebrafish. Diversin is distantly related to Diego of Drosophila, which functions only in the pathway that controls planar cell polarity. Our data show that Diversin is an essential component of the Wnt-signaling pathway and acts as a molecular switch, which suppresses Wnt signals mediated by the canonical beta-catenin pathway and stimulates signaling via JNK.

    Genes & development 2002;16;16;2073-84

  • Casein kinase I and casein kinase II differentially regulate axin function in Wnt and JNK pathways.

    Zhang Y, Qiu WJ, Chan SC, Han J, He X and Lin SC

    Department of Biochemistry, Hong Kong University of Science and Technology, Clear Water Bay, Kowloon, Hong Kong, China.

    Axin uses different combinations of functional domains in down-regulation of the Wnt pathway and activation of the MEKK1/JNK pathway. We are interested in the elucidation of the functional switch of Axin. In the present study, we show that the Wnt activator CKIepsilon, but not CKIIalpha, Frat1, LRP5, or LRP6, inhibited Axin-mediated JNK activation. We also found that both CKIalpha and CKIepsilon interacted with Axin, whereas CKIIalpha did not bind to Axin and had no effect on Axin-mediated JNK activity even though CKIIalpha has also been suggested to be an activator for the Wnt pathway. The COOH-terminal region and the MEKK1-interacting domain of Axin are important for CKIalpha-Axin and CKIepsilon-Axin interaction. We further demonstrated that CKIepsilon and CKIalpha binding to Axin excluded MEKK1 binding, indicating that a competitive physical occupancy may underlie the inhibitory effect. Moreover, our data indicated that CKIepsilon kinase activity plays an additive role in this effect. Taken together, we have demonstrated that CKI and CKII exhibit differential effects on Axin-MEKK1 interaction and Axin-mediated JNK activation. Furthermore, our data suggest that CKI may provide a possible switch mechanism for Axin function in the regulation of Wnt and JNK pathways.

    The Journal of biological chemistry 2002;277;20;17706-12

  • The circadian regulatory proteins BMAL1 and cryptochromes are substrates of casein kinase Iepsilon.

    Eide EJ, Vielhaber EL, Hinz WA and Virshup DM

    Department of Oncological Sciences, Huntsman Cancer Institute Center for Children, University of Utah School of Medicine, Salt Lake City, Utah 84112, USA.

    The serine/threonine protein kinase casein kinase I epsilon (CKIepsilon) is a key regulator of metazoan circadian rhythm. Genetic and biochemical data suggest that CKIepsilon binds to and phosphorylates the PERIOD proteins. However, the PERIOD proteins interact with a variety of circadian regulators, suggesting the possibility that CKIepsilon may interact with and phosphorylate additional clock components as well. We find that CRY1 and BMAL1 are phosphoproteins in cultured cells. Mammalian PERIOD proteins act as a scaffold with distinct domains that simultaneously bind CKIepsilon and mCRY1 and mCRY2 (mCRY). mCRY is phosphorylated by CKIepsilon only when both proteins are bound to mammalian PERIOD proteins. BMAL1 is also a substrate for CKIepsilon in vitro, and CKIepsilon kinase activity positively regulates BMAL1-dependent transcription from circadian promoters in reporter assays. We conclude that CKIepsilon phosphorylates multiple circadian substrates and may exert its effects on circadian rhythm in part by a direct effect on BMAL1-dependent transcription.

    Funded by: NCI NIH HHS: P30 CA042014, P30CA42014, R01CA71074; NIGMS NIH HHS: R01 GM060387-04

    The Journal of biological chemistry 2002;277;19;17248-54

  • Axin-mediated CKI phosphorylation of beta-catenin at Ser 45: a molecular switch for the Wnt pathway.

    Amit S, Hatzubai A, Birman Y, Andersen JS, Ben-Shushan E, Mann M, Ben-Neriah Y and Alkalay I

    The Lautenberg Center for Immunology, The Hebrew University-Hadassah Medical School, Jerusalem 91120, Israel.

    The Wnt pathway controls numerous developmental processes via the beta-catenin-TCF/LEF transcription complex. Deregulation of the pathway results in the aberrant accumulation of beta-catenin in the nucleus, often leading to cancer. Normally, cytoplasmic beta-catenin associates with APC and axin and is continuously phosphorylated by GSK-3beta, marking it for proteasomal degradation. Wnt signaling is considered to prevent GSK-3beta from phosphorylating beta-catenin, thus causing its stabilization. However, the Wnt mechanism of action has not been resolved. Here we study the regulation of beta-catenin phosphorylation and degradation by the Wnt pathway. Using mass spectrometry and phosphopeptide-specific antibodies, we show that a complex of axin and casein kinase I (CKI) induces beta-catenin phosphorylation at a single site: serine 45 (S45). Immunopurified axin and recombinant CKI phosphorylate beta-catenin in vitro at S45; CKI inhibition suppresses this phosphorylation in vivo. CKI phosphorylation creates a priming site for GSK-3beta and is both necessary and sufficient to initiate the beta-catenin phosphorylation-degradation cascade. Wnt3A signaling and Dvl overexpression suppress S45 phosphorylation, thereby precluding the initiation of the cascade. Thus, a single, CKI-dependent phosphorylation event serves as a molecular switch for the Wnt pathway.

    Genes & development 2002;16;9;1066-76

  • Control of intracellular dynamics of mammalian period proteins by casein kinase I epsilon (CKIepsilon) and CKIdelta in cultured cells.

    Akashi M, Tsuchiya Y, Yoshino T and Nishida E

    Department of Biophysics, Graduate School of Science, Kyoto University, Sakyo-ku, Kyoto 606-8502, Japan.

    Recent studies have shown that casein kinase I epsilon (CKIepsilon) is an essential regulator of the mammalian circadian clock. However, the detailed mechanisms by which CKIepsilon regulates each component of the circadian negative-feedback loop have not been fully defined. We show here that mPer proteins, negative limbs of the autoregulatory loop, are specific substrates for CKIepsilon and CKIdelta. The CKI phosphorylation of mPer1 and mPer3 proteins results in their rapid degradation, which is dependent on the ubiquitin-proteasome pathway. Moreover, CKIepsilon and CKIdelta are able to induce nuclear translocation of mPer3, which requires its nuclear localization signal. The mutation in potential phosphorylation sites on mPer3 decreased the extent of both nuclear translocation and degradation of mPer3 that are stimulated by CKIepsilon. CKIepsilon and CKIdelta affected the inhibitory effect of mPer proteins on the transcriptional activity of BMAL1-CLOCK, but the inhibitory effect of mCry proteins on the activity of BMAL1-CLOCK was unaffected. These results suggest that CKIepsilon and CKIdelta regulate the mammalian circadian autoregulatory loop by controlling both protein turnover and subcellular localization of mPer proteins.

    Molecular and cellular biology 2002;22;6;1693-703

  • Casein kinase I phosphorylates and destabilizes the beta-catenin degradation complex.

    Gao ZH, Seeling JM, Hill V, Yochum A and Virshup DM

    Department of Oncological Sciences, Huntsman Cancer Institute, 2000 East North Campus Drive, University of Utah, Salt Lake City, UT 84112-5550, USA.

    Wnt signaling plays a key role in cell proliferation and development. Recently, casein kinase I (CKI) and protein phosphatase 2A (PP2A) have emerged as positive and negative regulators of the Wnt pathway, respectively. However, it is not clear how these two enzymes with opposing functions regulate Wnt signaling. Here we show that both CKI delta and CKI epsilon interacted directly with Dvl-1, and that CKI phosphorylated multiple components of the Wnt-regulated beta-catenin degradation complex in vitro, including Dvl-1, adenomatous polyposis coli (APC), axin, and beta-catenin. Comparison of peptide maps from in vivo and in vitro phosphorylated beta-catenin and axin suggests that CKI phosphorylates these proteins in vivo as well. CKI abrogated beta-catenin degradation in Xenopus egg extracts. Notably, CKI decreased, whereas inhibition of CKI increased, the association of PP2A with the beta-catenin degradation complex in vitro. Additionally, inhibition of CKI in vivo stabilized the beta-catenin degradation complex, suggesting that CKI actively destabilizes the complex in vivo. The ability of CKI to induce secondary body axes in Xenopus embryos was reduced by the B56 regulatory subunit of PP2A, and kinase-dead CKI epsilon acted synergistically with B56 in inhibiting Wnt signaling. The data suggest that CKI phosphorylates and destabilizes the beta-catenin degradation complex, likely through the dissociation of PP2A, providing a mechanism by which CKI stabilizes beta-catenin and propagates the Wnt signal.

    Funded by: NCI NIH HHS: 2P30CA42014, P30 CA042014, R01 CA080809, R01CA71074, R01CA80809

    Proceedings of the National Academy of Sciences of the United States of America 2002;99;3;1182-7

  • Axin-dependent phosphorylation of the adenomatous polyposis coli protein mediated by casein kinase 1epsilon.

    Rubinfeld B, Tice DA and Polakis P

    Department of Molecular Oncology, Genentech, Inc., South San Francisco, California 94080, USA.

    Axin and the adenomatous polyposis coli protein (APC) interact to down-regulate the proto-oncogene beta-catenin. We show that transposition of an axin-binding site can confer beta-catenin regulatory activity to a fragment of APC normally lacking this activity. The fragment containing the axin-binding site also underwent hyperphosphorylation when coexpressed with axin. The phosphorylation did not require glycogen synthase kinase 3beta but instead required casein kinase 1epsilon, which bound directly to axin. Mutation of conserved serine residues in the beta-catenin regulatory motifs of APC interfered with both axin-dependent phosphorylation and phosphorylation by CKIepsilon and impaired the ability of APC to regulate beta-catenin. These results suggest that the axin-dependent phosphorylation of APC is mediated in part by CKIepsilon and is involved in the regulation of APC function.

    The Journal of biological chemistry 2001;276;42;39037-45

  • Phosphorylation of bid by casein kinases I and II regulates its cleavage by caspase 8.

    Desagher S, Osen-Sand A, Montessuit S, Magnenat E, Vilbois F, Hochmann A, Journot L, Antonsson B and Martinou JC

    CNRS UPR 9023, Montpellier, France. desagher@montp.inserm.fr

    Bid plays an essential role in Fas-mediated apoptosis of the so-called type II cells. In these cells, following cleavage by caspase 8, the C-terminal fragment of Bid translocates to mitochondria and triggers the release of apoptogenic factors, thereby inducing cell death. Here we report that Bid is phosphorylated by casein kinase I (CKI) and casein kinase II (CKII). Inhibition of CKI and CKII accelerated Fas-mediated apoptosis and Bid cleavage, whereas hyperactivity of the kinases delayed apoptosis. When phosphorylated, Bid was insensitive to caspase 8 cleavage in vitro. Moreover, a mutant of Bid that cannot be phosphorylated was found to be more toxic than wild-type Bid. Together, these data indicate that phosphorylation of Bid represents a new mechanism whereby cells control apoptosis.

    Molecular cell 2001;8;3;601-11

  • An hPer2 phosphorylation site mutation in familial advanced sleep phase syndrome.

    Toh KL, Jones CR, He Y, Eide EJ, Hinz WA, Virshup DM, Ptácek LJ and Fu YH

    Department of Human Genetics, University of Utah, Salt Lake City, UT 84112, USA.

    Familial advanced sleep phase syndrome (FASPS) is an autosomal dominant circadian rhythm variant; affected individuals are "morning larks" with a 4-hour advance of the sleep, temperature, and melatonin rhythms. Here we report localization of the FASPS gene near the telomere of chromosome 2q. A strong candidate gene (hPer2), a human homolog of the period gene in Drosophila, maps to the same locus. Affected individuals have a serine to glycine mutation within the casein kinase Iepsilon (CKIepsilon) binding region of hPER2, which causes hypophosphorylation by CKIepsilon in vitro. Thus, a variant in human sleep behavior can be attributed to a missense mutation in a clock component, hPER2, which alters the circadian period.

    Funded by: NCRR NIH HHS: M01-RR00064; NHLBI NIH HHS: HL/HD 59596

    Science (New York, N.Y.) 2001;291;5506;1040-3

  • The centrosomal protein C-Nap1 is required for cell cycle-regulated centrosome cohesion.

    Mayor T, Stierhof YD, Tanaka K, Fry AM and Nigg EA

    Department of Molecular Biology, Sciences II, University of Geneva, CH-1211 Geneva, Switzerland.

    Duplicating centrosomes are paired during interphase, but are separated at the onset of mitosis. Although the mechanisms controlling centrosome cohesion and separation are important for centrosome function throughout the cell cycle, they remain poorly understood. Recently, we have proposed that C-Nap1, a novel centrosomal protein, is part of a structure linking parental centrioles in a cell cycle-regulated manner. To test this model, we have performed a detailed structure-function analysis on C-Nap1. We demonstrate that antibody-mediated interference with C-Nap1 function causes centrosome splitting, regardless of the cell cycle phase. Splitting occurs between parental centrioles and is not dependent on the presence of an intact microtubule or microfilament network. Centrosome splitting can also be induced by overexpression of truncated C-Nap1 mutants, but not full-length protein. Antibodies raised against different domains of C-Nap1 prove that this protein dissociates from spindle poles during mitosis, but reaccumulates at centrosomes at the end of cell division. Use of the same antibodies in immunoelectron microscopy shows that C-Nap1 is confined to the proximal end domains of centrioles, indicating that a putative linker structure must contain additional proteins. We conclude that C-Nap1 is a key component of a dynamic, cell cycle-regulated structure that mediates centriole-centriole cohesion.

    The Journal of cell biology 2000;151;4;837-46

  • Cloning and characterization of rat casein kinase 1epsilon.

    Takano A, Shimizu K, Kani S, Buijs RM, Okada M and Nagai K

    Division of Protein Metabolism, Institute for Protein Research, Osaka University, 3-2 Yamado-Oka, Suita, Osaka, Japan. atsuko@protein.osaka-u.ac.jp

    Genes differentially expressed in the subjective day and night in the rat suprachiasmatic nucleus (SCN) were surveyed by differential display. A gene homologous to human casein kinase 1epsilon (CK1epsilon) was isolated, which initially appeared to be expressed in the suprachiasmatic nucleus (SCN) in a circadian manner. We here describe the cDNA cloning of the rat CK1epsilon and characterization of the protein products. The rCK1epsilon is predominantly expressed in the brain including the SCN, binds and phosphorylates mPer1, mPer2, and mPer3 in vitro, and translocates mPer1 and mPer3, but not mPer2, to the cell nucleus depending on its kinase activity when coexpressed with these Per proteins in COS-7 cells.

    FEBS letters 2000;477;1-2;106-12

  • Nuclear entry of the circadian regulator mPER1 is controlled by mammalian casein kinase I epsilon.

    Vielhaber E, Eide E, Rivers A, Gao ZH and Virshup DM

    Department of Oncological Sciences, Huntsman Cancer Institute, University of Utah, Salt Lake City 84132, USA.

    The molecular oscillator that keeps circadian time is generated by a negative feedback loop. Nuclear entry of circadian regulatory proteins that inhibit transcription from E-box-containing promoters appears to be a critical component of this loop in both Drosophila and mammals. The Drosophila double-time gene product, a casein kinase I epsilon (CKIepsilon) homolog, has been reported to interact with dPER and regulate circadian cycle length. We find that mammalian CKIepsilon binds to and phosphorylates the murine circadian regulator mPER1. Unlike both dPER and mPER2, mPER1 expressed alone in HEK 293 cells is predominantly a nuclear protein. Two distinct mechanisms appear to retard mPER1 nuclear entry. First, coexpression of mPER2 leads to mPER1-mPER2 heterodimer formation and cytoplasmic colocalization. Second, coexpression of CKIepsilon leads to masking of the mPER1 nuclear localization signal and phosphorylation-dependent cytoplasmic retention of both proteins. CKIepsilon may regulate mammalian circadian rhythm by controlling the rate at which mPER1 enters the nucleus.

    Funded by: NCI NIH HHS: 3P30 CA42014, P30 CA042014, R01 CA71074

    Molecular and cellular biology 2000;20;13;4888-99

  • Phosphorylation and destabilization of human period I clock protein by human casein kinase I epsilon.

    Keesler GA, Camacho F, Guo Y, Virshup D, Mondadori C and Yao Z

    Department of Neuroscience, Aventis Pharmaceuticals Inc., Bridgewater, NJ 08807, USA. george.keesler@aventis.com

    Period (PER), a central component of the circadian clock in Drosophila, undergoes daily oscillation in abundance and phosphorylation state. Here we report that human casein kinase I epsilon (hCKI epsilon) can phosphorylate human PER I (hPER I). Purified recombinant hCKI epsilon (but not a kinase negative mutant of hCKI epsilon, hCKI epsilon-K38R) phosphorylated hPER I in vitro. When co-transfected with wild-type hCKI epsilon, in 293T cells, hPER I showed a significant increase in phosphorylation as evidenced by a shift in molecular mass. Furthermore, phosphorylation of hPER I by hCKI epsilon caused a decrease in protein stability in hPER I. Whereas phosphorylated hPER I had a half-life of approximately 12 h, unphosphorylated hPER I remained stable in the cell for > 24 h. hPER I protein could also be co-immunoprecipitated with transfected hCKI epsilon as well as endogenous hCKI epsilon, indicating physical association between hPER I and hCKI epsilon proteins in vivo.

    Neuroreport 2000;11;5;951-5

  • The DNA sequence of human chromosome 22.

    Dunham I, Shimizu N, Roe BA, Chissoe S, Hunt AR, Collins JE, Bruskiewich R, Beare DM, Clamp M, Smink LJ, Ainscough R, Almeida JP, Babbage A, Bagguley C, Bailey J, Barlow K, Bates KN, Beasley O, Bird CP, Blakey S, Bridgeman AM, Buck D, Burgess J, Burrill WD, O'Brien KP et al.

    Sanger Centre, Wellcome Trust Genome Campus, Hinxton, Cambridge, UK. id1@sanger.ac.uk

    Knowledge of the complete genomic DNA sequence of an organism allows a systematic approach to defining its genetic components. The genomic sequence provides access to the complete structures of all genes, including those without known function, their control elements, and, by inference, the proteins they encode, as well as all other biologically important sequences. Furthermore, the sequence is a rich and permanent source of information for the design of further biological studies of the organism and for the study of evolution through cross-species sequence comparison. The power of this approach has been amply demonstrated by the determination of the sequences of a number of microbial and model organisms. The next step is to obtain the complete sequence of the entire human genome. Here we report the sequence of the euchromatic part of human chromosome 22. The sequence obtained consists of 12 contiguous segments spanning 33.4 megabases, contains at least 545 genes and 134 pseudogenes, and provides the first view of the complex chromosomal landscapes that will be found in the rest of the genome.

    Nature 1999;402;6761;489-95

  • Identification of inhibitory autophosphorylation sites in casein kinase I epsilon.

    Gietzen KF and Virshup DM

    Division of Molecular Biology, Department of Oncological Sciences, Huntsman Cancer Institute, Salt Lake City, Utah 84132, USA.

    Casein kinase I epsilon (CKIepsilon) is a widely expressed protein kinase implicated in the regulation of diverse cellular processes including DNA replication and repair, nuclear trafficking, and circadian rhythm. CKIepsilon and the closely related CKIdelta are regulated in part through autophosphorylation of their carboxyl-terminal extensions, resulting in down-regulation of enzyme activity. Treatment of CKIepsilon with any of several serine/threonine phosphatases causes a marked increase in kinase activity that is self-limited. To identify the sites of inhibitory autophosphorylation, a series of carboxyl-terminal deletion mutants was constructed by site-directed mutagenesis. Truncations that elim 1f40 inated specific phosphopeptides present in the wild-type kinase were used to guide construction of specific serine/threonine to alanine mutants. Amino acids Ser-323, Thr-325, Thr-334, Thr-337, Ser-368, Ser-405, Thr-407, and Ser-408 in the carboxyl-terminal tail of CKIepsilon were identified as probable in vivo autophosphorylation sites. A recombinant CKIepsilon protein with serine and threonine to alanine mutations eliminating these autophosphorylation sites was 8-fold more active than wild-type CKIepsilon using IkappaBalpha as a substrate. The identified autophosphorylation sites do not conform to CKI substrate motifs identified in peptide substrates.

    Funded by: NCI NIH HHS: CA42014, CA71074

    The Journal of biological chemistry 1999;274;45;32063-70

  • Casein kinase iepsilon in the wnt pathway: regulation of beta-catenin function.

    Sakanaka C, Leong P, Xu L, Harrison SD and Williams LT

    Chiron Corporation, Emeryville, CA 94608, USA.

    Wnt and its intracellular effector beta-catenin regulate developmental and oncogenic processes. Using expression cloning to identify novel components of the Wnt pathway, we isolated casein kinase Iepsilon (CKIepsilon). CKIepsilon mimicked Wnt in inducing a secondary axis in Xenopus, stabilizing beta-catenin, and stimulating gene transcription in cells. Inhibition of endogenous CKIepsilon by kinase-defective CKIepsilon or CKIepsilon antisense-oligonucleotides attenuated Wnt signaling. CKIepsilon was in a complex with axin and other downstream components of the Wnt pathway, including Dishevelled. CKIepsilon appears to be a positive regulator of the pathway and a link between upstream signals and the complexes that regulate beta-catenin.

    Proceedings of the National Academy of Sciences of the United States of America 1999;96;22;12548-52

  • The Drosophila clock gene double-time encodes a protein closely related to human casein kinase Iepsilon.

    Kloss B, Price JL, Saez L, Blau J, A, Wesley CS and Young MW

    Laboratory of Genetics and National Science Foundation Science and Technology Center for Biological Timing, The Rockefeller University, New York, New York 10021, USA.

    The cloning of double-time (dbt) is reported. DOUBLETIME protein (DBT) is most closely related to human casein kinase Iepsilon. dbtS and dbtL mutations, which alter period length of Drosophila circadian rhythms, produce single amino acid changes in conserved regions of the predicted kinase. dbtP mutants, which eliminate rhythms of per and tim expression and constitutively overproduce hypophosphorylated PER proteins, abolish most dbt expression. dbt mRNA appears to be expressed in the same cell types as are per and tim and shows no evident oscillation in wild-type heads. DBT is capable of binding to PER in vitro and in Drosophila cells, suggesting that a physical association of PER and DBT regulates PER phosphorylation and accumulation in vivo.

    Funded by: NIGMS NIH HHS: GM 54339; NIMH NIH HHS: MH 56895

    Cell 1998;94;1;97-107

  • Regulation of casein kinase I epsilon and casein kinase I delta by an in vivo futile phosphorylation cycle.

    Rivers A, Gietzen KF, Vielhaber E and Virshup DM

    Division of Molecular Biology and Genetics, Department of Oncological Sciences, Huntsman Cancer Institute, University of Utah, Salt Lake City, Utah 84132, USA.

    Casein kinase I delta (CKIdelta) and casein kinase I epsilon (CKIepsilon) have been implicated in the response to DNA damage, but the understanding of how these kinases are regulated remains incomplete. In vitro, these kinases rapidly autophosphorylate, predominantly on their carboxyl-terminal extensions, and this autophosphorylation markedly inhibits kinase activity (Cegielska, A., Gietzen, K. F., Rivers, A., and Virshup, D. M. (1998) J. Biol. Chem. 273, 1357-1364). However, we now report that while these kinases are able to autophosphorylate in vivo, they are actively maintained in the dephosphorylated, active state by cellular protein phosphatases. Treatment of cells with the cell-permeable serine/threonine phosphatase inhibitors okadaic acid or calyculin A leads to rapid increases in kinase intramolecular autophosphorylation. Since CKI autophosphorylation decreases kinase activity, this dynamic autophosphorylation/dephosphorylation cycle provides a mechanism for kinase regulation in vivo.

    Funded by: NCI NIH HHS: R01 CA71074

    The Journal of biological chemistry 1998;273;26;15980-4

  • Autoinhibition of casein kinase I epsilon (CKI epsilon) is relieved by protein phosphatases and limited proteolysis.

    Cegielska A, Gietzen KF, Rivers A and Virshup DM

    Department of Oncological Sciences, Huntsman Cancer Institute, Salt Lake City, Utah, USA.

    Casein kinase I epsilon (CKI epsilon) is a member of the CKI gene family, members of which are involved in the control of SV40 DNA replication, DNA repair, and cell metabolism. The mechanisms that regulate CKI epsilon activity and substrate specificity are not well understood. We report that CKI epsilon, which contains a highly phosphorylated 123-amino acid carboxyl-terminal extension not present in CKI alpha, is substantially less active than CKI alpha in phosphorylating a number of substrates including SV40 large T antigen and is unable to inhibit the initiation of SV40 DNA replication. Two mechanisms for the activation of CKI epsilon have been identified. First, limited tryptic digestion of CKI epsilon produces a protease-resistant amino-terminal 39-kDa core kinase with several-fold enhanced activity. Second, phosphatase treatment of CKI epsilon activates CKI epsilon 5-20-fold toward T antigen. Similar treatment of a truncated form of CKI epsilon produced only a 2-fold activation. Notably, this activation was transient; reautophosphorylation led to a rapid down-regulation of the kinase within 5 min. Phosphatase treatment also activated CKI epsilon toward the novel substrates I kappa B alpha and Ets-1. These mechanisms may serve to regulate CKI epsilon and related forms of CKI in the cell, perhaps in response to DNA damage.

    Funded by: NCI NIH HHS: 3P30 CA42014, CA71074

    The Journal of biological chemistry 1998;273;3;1357-64

  • Construction and characterization of a full length-enriched and a 5'-end-enriched cDNA library.

    Suzuki Y, Yoshitomo-Nakagawa K, Maruyama K, Suyama A and Sugano S

    International and Interdisciplinary Studies, The University of Tokyo, Japan.

    Using 'oligo-capped' mRNA [Maruyama, K., Sugano, S., 1994. Oligo-capping: a simple method to replace the cap structure of eukaryotic mRNAs with oligoribonucleotides. Gene 138, 171-174], whose cap structure was replaced by a synthetic oligonucleotide, we constructed two types of cDNA library. One is a 'full length-enriched cDNA library' which has a high content of full-length cDNA clones and the other is a '5'-end-enriched cDNA library', which has a high content of cDNA clones with their mRNA start sites. The 5'-end-enriched library was constructed especially for isolating the mRNA start sites of long mRNAs. In order to characterize these libraries, we performed one-pass sequencing of randomly selected cDNA clones from both libraries (84 clones for the full length-enriched cDNA library and 159 clones for the 5'-end-enriched cDNA library). The cDNA clones of the polypeptide chain elongation factor 1 alpha were most frequently (nine clones) isolated, and more than 80% of them (eight clones) contained the mRNA start site of the gene. Furthermore, about 80% of the cDNA clones of both libraries whose sequence matched with known genes had the known 5' ends or sequences upstream of the known 5' ends (28 out of 35 for the full length-enriched library and 51 out of 62 for the 5'-end-enriched library). The longest full-length clone of the full length-enriched cDNA library was about 3300 bp (among 28 clones). In contrast, seven clones (out of the 51 clones with the mRNA start sites) from the 5'-end-enriched cDNA library came from mRNAs whose length is more than 3500 bp. These cDNA libraries may be useful for generating 5' ESTs with the information of the mRNA start sites that are now scarce in the EST database.

    Gene 1997;200;1-2;149-56

  • 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.

    Knippschild U, Milne DM, Campbell LE, DeMaggio AJ, Christenson E, Hoekstra MF and Meek DW

    Biomedical Research Centre, Ninewells Hospital and Medical School, University of Dundee, UK.

    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.

    Oncogene 1997;15;14;1727-36

  • Isolation and characterization of human casein kinase I epsilon (CKI), a novel member of the CKI gene family.

    Fish KJ, Cegielska A, Getman ME, Landes GM and Virshup DM

    Program in Human Molecular Biology, University of Utah, Salt Lake City 84112, USA.

    The casein kinase I (CKI) gene family is a rapidly enlarging group whose members have been implicated in the control of cytoplasmic and nuclear processes, including DNA replication and repair. We report here the cloning and characterization of a novel isoform of CKI from a human placental cDNA library. The cDNA for this isoform, hCKI epsilon, predicts a basic polypeptide of 416 amino acids and a molecular mass of 47.3 kDa. It encodes a core kinase domain of 285 amino acids and a carboxyl-terminal tail of 123 amino acids. The kinase domain is 53-98% identical to the kinase domains of other CKI family members and is most closely related to the delta isoform. Localization of the hCKI epsilon gene to chromosome 22q12-13 and the hCKI delta gene to chromosome 17q25 confirms that these are distinct genes in the CKI family. Northern blot analysis shows that hCKI epsilon is expressed in multiple human cell lines. Recombinant hCKI epsilon is an active enzyme that phosphorylates known CKI substrates including a CKI-specific peptide substrate and is inhibited by CKI-7, a CKI-specific inhibitor. A budding yeast isoform of CKI, HRR25, has been implicated in DNA repair responses. Expression of hCKI epsilon but not hCKI alpha rescued the slow-growth phenotype of a Saccharomyces cerevisiae strain with a deletion of HRR25. Human CKI epsilon is a novel CKI isoform with properties that overlap those of previously described CKI isoforms.

    Funded by: NCI NIH HHS: 5 P30 CA42014; NHGRI NIH HHS: 5P30 HG00199; NIAID NIH HHS: R01 AI31657

    The Journal of biological chemistry 1995;270;25;14875-83

  • Cell cycle regulation of the activity and subcellular localization of Plk1, a human protein kinase implicated in mitotic spindle function.

    Golsteyn RM, Mundt KE, Fry AM and Nigg EA

    Swiss Institute for Experimental Cancer Research (ISREC), Epalinges.

    Correct assembly and function of the mitotic spindle during cell division is essential for the accurate partitioning of the duplicated genome to daughter cells. Protein phosphorylation has long been implicated in controlling spindle function and chromosome segregation, and genetic studies have identified several protein kinases and phosphatases that are likely to regulate these processes. In particular, mutations in the serine/threonine-specific Drosophila kinase polo, and the structurally related kinase Cdc5p of Saccharomyces cerevisae, result in abnormal mitotic and meiotic divisions. Here, we describe a detailed analysis of the cell cycle-dependent activity and subcellular localization of Plk1, a recently identified human protein kinase with extensive sequence similarity to both Drosophila polo and S. cerevisiae Cdc5p. With the aid of recombinant baculoviruses, we have established a reliable in vitro assay for Plk1 kinase activity. We show that the activity of human Plk1 is cell cycle regulated, Plk1 activity being low during interphase but high during mitosis. We further show, by immunofluorescent confocal laser scanning microscopy, that human Plk1 binds to components of the mitotic spindle at all stages of mitosis, but undergoes a striking redistribution as cells progress from metaphase to anaphase. Specifically, Plk1 associates with spindle poles up to metaphase, but relocalizes to the equatorial plane, where spindle microtubules overlap (the midzone), as cells go through anaphase. These results indicate that the association of Plk1 with the spindle is highly dynamic and that Plk1 may function at multiple stages of mitotic progression. Taken together, our data strengthen the notion that human Plk1 may represent a functional homolog of polo and Cdc5p, and they suggest that this kinase plays an important role in the dynamic function of the mitotic spindle during chromosome segregation.

    The Journal of cell biology 1995;129;6;1617-28

  • Oligo-capping: a simple method to replace the cap structure of eukaryotic mRNAs with oligoribonucleotides.

    Maruyama K and Sugano S

    Institute of Medical Science, University of Tokyo, Japan.

    We have devised a method to replace the cap structure of a mRNA with an oligoribonucleotide (r-oligo) to label the 5' end of eukaryotic mRNAs. The method consists of removing the cap with tobacco acid pyrophosphatase (TAP) and ligating r-oligos to decapped mRNAs with T4 RNA ligase. This reaction was made cap-specific by removing 5'-phosphates of non-capped RNAs with alkaline phosphatase prior to TAP treatment. Unlike the conventional methods that label the 5' end of cDNAs, this method specifically labels the capped end of the mRNAs with a synthetic r-oligo prior to first-strand cDNA synthesis. The 5' end of the mRNA was identified quite simply by reverse transcription-polymerase chain reaction (RT-PCR).

    Gene 1994;138;1-2;171-4

Gene lists (5)

Gene List Source Species Name Description Gene count
L00000009 G2C Homo sapiens Human PSD Human orthologues of mouse PSD adapted from Collins et al (2006) 1080
L00000016 G2C Homo sapiens Human PSP Human orthologues of mouse PSP adapted from Collins et al (2006) 1121
L00000061 G2C Homo sapiens BAYES-COLLINS-MOUSE-PSD-CONSENSUS Mouse cortex PSD consensus (ortho) 984
L00000069 G2C Homo sapiens BAYES-COLLINS-HUMAN-PSD-FULL Human cortex biopsy PSD full list 1461
L00000071 G2C Homo sapiens BAYES-COLLINS-MOUSE-PSD-FULL Mouse cortex PSD full list (ortho) 1556
© G2C 2014. The Genes to Cognition Programme received funding from The Wellcome Trust and the EU FP7 Framework Programmes:
EUROSPIN (FP7-HEALTH-241498), SynSys (FP7-HEALTH-242167) and GENCODYS (FP7-HEALTH-241995).

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