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1.
Neurobiol Stress ; 14: 100323, 2021 May.
Article in English | MEDLINE | ID: mdl-33912629

ABSTRACT

In highly stressful environments, individuals with diverging stress-reactivity can perform differently. Identification of blood markers of stress-reactivity is of major significance to help human performance during stress. Candidate transcripts were identified between stressed and non-stressed strains of rats' blood and brain, and overlapping significant differentially expressed genes were selected. Serum levels of human orthologues of these proteins, in lieu of blood RNA, in addition to classic stress and general clinical markers, were measured in 33 Battlefield Airmen undergoing a 52 day long preparatory training course before their course of initial entry (COIE). Blood samples and factors of affective state, negative valence "Threat" and positive valence "Challenge", were obtained five times across different days of training which included either routine physical exercise or prolonged and intense physical and mental training. During training, levels of chloride (Cl), dehydroepiandrosterone-sulfate (DHEA-S), creatinine kinase (CK), and total carbon dioxide (TCO2) differed between airmen who subsequently graduated from their COIE and those who did not. Time dependent changes of serum TCO2 and neuropeptide Y (NPY), as well as the affective factor Challenge differed by future graduation status throughout the training. Serum levels of parvin beta (PARVB) correlated with the affective factor Threat, while those of NPY, testosterone, coactosin like F-actin binding protein 1 (COTL1) and C-reactive protein (CRP) correlated with factor Challenge during the extended, intensive periods of training, consistently. These pilot data suggest that the identified panel of blood markers can measure stress responsiveness, which has the potential to advance individualized stress-management strategies.

2.
Proc Natl Acad Sci U S A ; 98(21): 12044-9, 2001 Oct 09.
Article in English | MEDLINE | ID: mdl-11593014

ABSTRACT

Chromatid catenation is actively monitored in human cells, with progression from G(2) to mitosis being inhibited when chromatids are insufficiently decatenated. Mitotic delay was quantified in normal and checkpoint-deficient human cells during treatment with ICRF-193, a topoisomerase II catalytic inhibitor that prevents chromatid decatenation without producing topoisomerase-associated DNA strand breaks. Ataxia telangiectasia (A-T) cells, defective in DNA damage checkpoints, showed normal mitotic delay when treated with ICRF-193. The mitotic delay in response to ICRF-193 was ablated in human fibroblasts expressing an ataxia telangiectasia mutated- and rad3-related (ATR) kinase-inactive ATR allele (ATR(ki)). BRCA1-mutant HCC1937 cells also displayed a defect in ICRF-193-induced mitotic delay, which was corrected by expression of wild-type BRCA1. Phosphorylations of hCds1 or Chk1 and inhibition of Cdk1 kinase activity, which are elements of checkpoints associated with DNA damage or replication, did not occur during ICRF-193-induced mitotic delay. Over-expression of cyclin B1 containing a dominant nuclear localization signal, and inhibition of Crm1-mediated nuclear export, reversed ICRF-193-induced mitotic delay. In combination, these results imply that ATR and BRCA1 enforce the decatenation G(2) checkpoint, which may act to exclude cyclin B1/Cdk1 complexes from the nucleus. Moreover, induction of ATR(ki) produced a 10-fold increase in chromosomal aberrations, further emphasizing the vital role for ATR in genetic stability.


Subject(s)
BRCA1 Protein/metabolism , CDC2 Protein Kinase/metabolism , Cell Cycle Proteins , Cyclin B/metabolism , Mitosis/physiology , Protein Serine-Threonine Kinases/metabolism , Signal Transduction , Topoisomerase II Inhibitors , Ataxia Telangiectasia , Ataxia Telangiectasia Mutated Proteins , Cell Line , Cell Nucleus/metabolism , Checkpoint Kinase 1 , Checkpoint Kinase 2 , Cyclin B1 , DNA-Binding Proteins , Diketopiperazines , G2 Phase , Humans , Mitosis/drug effects , Phosphorylation , Piperazines/pharmacology , Protein Kinases/metabolism , Tumor Suppressor Proteins
3.
Oncogene ; 20(15): 1839-51, 2001 Apr 05.
Article in English | MEDLINE | ID: mdl-11313932

ABSTRACT

Entry into mitosis requires activation of the Cdc2 protein kinase by the Cdc25C protein phosphatase. The interactions between Cdc2 and Cdc25C are negatively regulated throughout interphase and in response to G2 checkpoint activation. This is accomplished in part by maintaining the Cdc25 phosphatase in a phosphorylated form that binds 14-3-3 proteins. Here we report that 14-3-3 binding regulates the intracellular trafficking of Cdc25C. Although primarily cytoplasmic, Cdc25C accumulated in the nuclei of leptomycin B (LMB)-treated cells, indicating that Cdc25C is actively exported out of the nucleus. A mutant of Cdc25C that is unable to bind 14-3-3 was partially nuclear in the absence of LMB and its nuclear accumulation was greatly enhanced by LMB-treatment. A nuclear export signal (NES) was identified within the amino terminus of Cdc25C. Although mutation of the NES did not effect 14-3-3 binding, it did cause nuclear accumulation of Cdc25C. These results demonstrate that 14-3-3 binding is dispensable for the nuclear export of Cdc25C. However, complete nuclear accumulation of Cdc25C required loss of both NES function and 14-3-3 binding and this was accomplished both pharmacologically and by mutation. These findings suggest that the nuclear export of Cdc25C is mediated by an intrinsic NES and that 14-3-3 binding negatively regulates nuclear import.


Subject(s)
Active Transport, Cell Nucleus , Cell Cycle Proteins/metabolism , Tyrosine 3-Monooxygenase/metabolism , cdc25 Phosphatases/metabolism , 14-3-3 Proteins , Alkaloids/pharmacology , Amino Acid Sequence , Cell Cycle Proteins/chemistry , Cytoplasm/metabolism , Fatty Acids, Unsaturated/pharmacology , HeLa Cells , Humans , Molecular Sequence Data , Protein Binding , Staurosporine/analogs & derivatives , cdc25 Phosphatases/chemistry
4.
J Biol Chem ; 275(8): 5600-5, 2000 Feb 25.
Article in English | MEDLINE | ID: mdl-10681541

ABSTRACT

A checkpoint operating in the G(2) phase of the cell cycle prevents entry into mitosis in the presence of DNA damage. UCN-01, a protein kinase inhibitor currently undergoing clinical trials for cancer treatment, abrogates G(2) checkpoint function and sensitizes p53-defective cancer cells to DNA-damaging agents. In most species, the G(2) checkpoint prevents the Cdc25 phosphatase from removing inhibitory phosphate groups from the mitosis-promoting kinase Cdc2. This is accomplished by maintaining Cdc25 in a phosphorylated form that binds 14-3-3 proteins. The checkpoint kinases, Chk1 and Cds1, are proposed to regulate the interactions between human Cdc25C and 14-3-3 proteins by phosphorylating Cdc25C on serine 216. 14-3-3 proteins, in turn, function to keep Cdc25C out of the nucleus. Here we report that UCN-01 caused loss of both serine 216 phosphorylation and 14-3-3 binding to Cdc25C in DNA-damaged cells. In addition, UCN-01 potently inhibited the ability of Chk1 to phosphorylate Cdc25C in vitro. In contrast, Cds1 was refractory to inhibition by UCN-01 in vitro, and Cds1 was still phosphorylated in irradiated cells treated with UCN-01. Thus, neither Cds1 nor kinases upstream of Cds1, such as ataxia telangiectasia-mutated, are targets of UCN-01 action in vivo. Taken together our results identify the Chk1 kinase and the Cdc25C pathway as potential targets of G(2) checkpoint abrogation by UCN-01.


Subject(s)
Alkaloids/pharmacology , Antineoplastic Agents/pharmacology , Cell Cycle Proteins/antagonists & inhibitors , Protein Kinase Inhibitors , cdc25 Phosphatases/antagonists & inhibitors , Checkpoint Kinase 1 , Checkpoint Kinase 2 , Cloning, Molecular , DNA Damage , Dose-Response Relationship, Radiation , G2 Phase/drug effects , HeLa Cells , Humans , Membrane Proteins/metabolism , Models, Biological , Phosphorylation/drug effects , Phosphorylation/radiation effects , Plasmids , Protein Kinases/metabolism , Protein Serine-Threonine Kinases/antagonists & inhibitors , Protein Serine-Threonine Kinases/metabolism , Protein-Tyrosine Kinases/antagonists & inhibitors , Protein-Tyrosine Kinases/metabolism , Serine/metabolism , Staurosporine/analogs & derivatives , Time Factors
5.
Mol Cell Biol ; 18(9): 5229-38, 1998 Sep.
Article in English | MEDLINE | ID: mdl-9710607

ABSTRACT

By binding to serine-phosphorylated proteins, 14-3-3 proteins function as effectors of serine phosphorylation. The exact mechanism of their action is, however, still largely unknown. Here we demonstrate a requirement for 14-3-3 for Raf-1 kinase activity and phosphorylation. Expression of dominant negative forms of 14-3-3 resulted in the loss of a critical Raf-1 phosphorylation, while overexpression of 14-3-3 resulted in enhanced phosphorylation of this site. 14-3-3 levels, therefore, regulate the stoichiometry of Raf-1 phosphorylation and its potential activity in the cell. Phosphorylation of Raf-1, however, was insufficient by itself for kinase activity. Removal of 14-3-3 from phosphorylated Raf abrogated kinase activity, whereas addition of 14-3-3 restored it. This supports a paradigm in which the effects of phosphorylation on serine as well as tyrosine residues are mediated by inducible protein-protein interactions.


Subject(s)
Protein Structure, Secondary , Proteins/chemistry , Proteins/metabolism , Proto-Oncogene Proteins c-raf/chemistry , Proto-Oncogene Proteins c-raf/metabolism , Tyrosine 3-Monooxygenase , 14-3-3 Proteins , Amino Acid Sequence , Animals , Binding Sites , Cell Line , Chloramphenicol O-Acetyltransferase/biosynthesis , Cloning, Molecular , Glutathione Transferase , Humans , Mice , Models, Molecular , Mutagenesis, Site-Directed , Phosphorylation , Phosphoserine , Phosphotyrosine , Polymerase Chain Reaction , Protein Biosynthesis , Recombinant Fusion Proteins/biosynthesis , Recombinant Fusion Proteins/metabolism , Spectrum Analysis , Transfection
6.
Cell Growth Differ ; 9(3): 197-208, 1998 Mar.
Article in English | MEDLINE | ID: mdl-9543386

ABSTRACT

Cdc25C is a dual-specificity protein kinase that controls entry into mitosis by dephosphorylating Cdc2 on both threonine 14 and tyrosine 15. Cdc25C is phosphorylated on serine 216 throughout interphase but not during mitosis. Serine 216 phosphorylation mediates the binding of 14-3-3 protein to Cdc25C, and Cdc25C/14-3-3 complexes are present throughout interphase but not during mitosis. Here we report the cloning of a human kinase denoted C-TAK1 (for Cdc twenty-five C associated protein kinase) that phosphorylates Cdc25C on serine 216 in vitro. C-TAK1 is ubiquitously expressed in human tissues and cell lines and is distinct from the DNA damage checkpoint kinase Chk1, shown previously to phosphorylate Cdc25C on serine 216. Cotransfection of Cdc25C with C-TAK1 resulted in enhanced phosphorylation of Cdc25C on serine 216. In addition, a physical interaction between C-TAK1 and Cdc25C was observed upon transient overexpression in COS-7 cells. Finally, coproduction of Cdc25C and C-TAK1 in bacteria resulted in the stoichiometric phosphorylation of Cdc25C on serine 216 and facilitated 14-3-3 protein binding in vitro. Taken together, these results suggest that one function of C-TAK1 may be to regulate the interactions between Cdc25C and 14-3-3 in vivo by phosphorylating Cdc25C on serine 216.


Subject(s)
Cell Cycle Proteins/metabolism , Protein Serine-Threonine Kinases/metabolism , Proteins/metabolism , Serine/metabolism , Tyrosine 3-Monooxygenase , cdc25 Phosphatases , 14-3-3 Proteins , Amino Acid Sequence , Cloning, Molecular , DNA, Complementary/genetics , Gene Expression Regulation , Humans , Molecular Sequence Data , Organ Specificity , Phosphorylation , Protein Binding , Protein Serine-Threonine Kinases/genetics , RNA, Messenger/analysis , Sequence Analysis, DNA , Sequence Homology, Amino Acid
7.
J Biol Chem ; 272(43): 27281-7, 1997 Oct 24.
Article in English | MEDLINE | ID: mdl-9341175

ABSTRACT

PTPH1 is a human protein-tyrosine phosphatase with homology to the band 4.1 superfamily of cytoskeletal-associated proteins. PTPH1 was found to associate with 14-3-3beta using a yeast two-hybrid screen, and its interaction could be reconstituted in vitro using recombinant proteins. Examination of the interaction between 14-3-3beta and various deletion mutants of PTPH1 by two-hybrid tests suggested that the integrity of the PTP is important for this binding. Although both PTPH1 and Raf-1 form complexes with 14-3-3beta, they appear to do so independently. Binding of 14-3-3beta to PTPH1 in vitro was abolished by pretreating PTPH1 with potato acid phosphatase and was greatly enhanced by pretreating with Cdc25C-associated protein kinase. Thus the association between PTPH1 and 14-3-3beta is phosphorylation-dependent. Two novel motifs RSLS359VE and RVDS853EP in PTPH1 were identified as major 14-3-3beta-binding sites, both of which are distinct from the consensus binding motif RSXSXP recently found in Raf-1. Mutation of Ser359 and Ser853 to alanine significantly reduced the association between 14-3-3beta and PTPH1. Furthermore, association of PTPH1 and 14-3-3beta was detected in several cell lines and was regulated in response to extracellular signals. These results raise the possibility that 14-3-3beta may function as an adaptor molecule in the regulation of PTPH1 and may provide a link between serine/threonine and tyrosine phosphorylation-dependent signaling pathways.


Subject(s)
Cytoskeletal Proteins , Neuropeptides , Phosphoserine/metabolism , Protein Tyrosine Phosphatases/metabolism , Proteins/metabolism , Tyrosine 3-Monooxygenase , 14-3-3 Proteins , Amino Acid Sequence , Binding Sites , Cell Line , Cloning, Organism , Enzyme Inhibitors/chemistry , Enzyme Inhibitors/metabolism , Humans , Membrane Proteins/chemistry , Peptide Fragments/chemistry , Phosphorylation , Protein Tyrosine Phosphatase, Non-Receptor Type 3 , Protein Tyrosine Phosphatases/chemistry , Protein Tyrosine Phosphatases/isolation & purification , Proteins/chemistry , Proteins/isolation & purification , Recombinant Fusion Proteins/chemistry , Recombinant Fusion Proteins/metabolism , Saccharomyces cerevisiae , Sequence Alignment , Transfection , Tumor Cells, Cultured
8.
Science ; 277(5331): 1501-5, 1997 Sep 05.
Article in English | MEDLINE | ID: mdl-9278512

ABSTRACT

Human Cdc25C is a dual-specificity protein phosphatase that controls entry into mitosis by dephosphorylating the protein kinase Cdc2. Throughout interphase, but not in mitosis, Cdc25C was phosphorylated on serine-216 and bound to members of the highly conserved and ubiquitously expressed family of 14-3-3 proteins. A mutation preventing phosphorylation of serine-216 abrogated 14-3-3 binding. Conditional overexpression of this mutant perturbed mitotic timing and allowed cells to escape the G2 checkpoint arrest induced by either unreplicated DNA or radiation-induced damage. Chk1, a fission yeast kinase involved in the DNA damage checkpoint response, phosphorylated Cdc25C in vitro on serine-216. These results indicate that serine-216 phosphorylation and 14-3-3 binding negatively regulate Cdc25C and identify Cdc25C as a potential target of checkpoint control in human cells.


Subject(s)
Cell Cycle Proteins/metabolism , G2 Phase , Mitosis , Proteins/metabolism , Tyrosine 3-Monooxygenase , cdc25 Phosphatases , 14-3-3 Proteins , Amino Acid Sequence , Checkpoint Kinase 1 , DNA Damage , DNA Replication , Gamma Rays , HeLa Cells , Humans , Jurkat Cells , Molecular Sequence Data , Mutation , Phosphorylation , Phosphoserine/metabolism , Protein Kinases/metabolism , Recombinant Fusion Proteins/metabolism , S Phase
9.
J Biol Chem ; 270(37): 21689-94, 1995 Sep 15.
Article in English | MEDLINE | ID: mdl-7665585

ABSTRACT

Casein kinase I delta is a member of the casein kinase I (CKI) family, a group of second messenger independent protein kinases. We present evidence that the COOH-terminal domain of CKI delta has regulatory properties. CKI delta expressed in Escherichia coli was activated by heparin, as found previously, and by treatment with the catalytic subunit of type-1 protein phosphatase (CS1). Concomitant with activation by CS1, there was a reduction in the apparent molecular weight of CKI delta from 55,000 to 49,000 as judged by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. Truncation of CKI delta by removal of the COOH-terminal 110 amino acids eliminated the ability of CS1 to activate or to increase electrophoretic mobility. Casein kinase I alpha, a 37-kDa isoform that lacks an extended COOH-terminal domain, was not activated by CS1 or the presence of heparin. However, a chimeric enzyme consisting of CKI alpha fused to the COOH-terminal domain of CKI delta was activated by both heparin and CS1. Analysis of the effects of CS1 on a series of CKI delta COOH-terminal truncation mutants identified an inhibitory region between His317 and Pro342, which contained six potential phosphorylation sites. From analysis of the specific activites of these truncation mutants, removal of the same region resulted in enzyme with a specific activity nearly 10-fold greater than wild-type. Thus, CKI delta activity can be regulated by phosphorylation of its COOH terminus, which may serve to create an autoinhibitory domain. This mechanism of regulation could have important consequences in vivo.


Subject(s)
Protein Kinases/chemistry , Protein Kinases/metabolism , Amino Acid Sequence , Base Sequence , Casein Kinases , Cloning, Molecular , Enzyme Activation , Escherichia coli , Heparin/pharmacology , Histidine , Isoenzymes/chemistry , Isoenzymes/metabolism , Kinetics , Macromolecular Substances , Molecular Sequence Data , Molecular Weight , Mutagenesis, Site-Directed , Oligodeoxyribonucleotides , Phosphorylation , Proline , Protein Kinases/isolation & purification , Protein Tyrosine Phosphatase, Non-Receptor Type 1 , Protein Tyrosine Phosphatases , Recombinant Fusion Proteins/chemistry , Recombinant Fusion Proteins/metabolism , Recombinant Proteins/chemistry , Recombinant Proteins/isolation & purification , Recombinant Proteins/metabolism , Second Messenger Systems
10.
J Biol Chem ; 270(21): 12717-24, 1995 May 26.
Article in English | MEDLINE | ID: mdl-7759525

ABSTRACT

Casein kinase I, one of the first protein kinases identified biochemically, is known to exist in multiple isoforms in mammals. Using a partial cDNA fragment corresponding to an isoform termed CK1 gamma, three full-length rat testis cDNAs were cloned that defined three separate members of this subfamily. The isoforms, designated CK1 gamma 1, CK1 gamma 2, and CK1 gamma 3, have predicted molecular masses of 43,000, 45,500, and 49,700. CK1 gamma 3 may also exist in an alternatively spliced form. The proteins are more than 90% identical to each other within the protein kinase domain but only 51-59% identical to other casein kinase I isoforms within this region. Messages for CK1 gamma 1 (2 kilobases (kb)), CK1 gamma 2 (1.5 and 2.4 kb), and CK1 gamma 3 (2.8 kb) were detected by Northern hybridization of testis RNA. Message for CK1 gamma 3 was also observed in brain, heart, kidney, lung, liver, and muscle whereas CK1 gamma 1 and CK1 gamma 2 messages were restricted to testis. All three CK1 gamma isoforms were expressed as active enzymes in Escherichia coli and partially purified. The enzymes phosphorylated typical in vitro casein kinase I substrates such as casein, phosvitin, and a synthetic peptide, D4. Phosphorylation of the D4 peptide was activated by heparin whereas phosphorylation of the protein substrates was inhibited. The known casein kinase I inhibitor CK1-7 also inhibited the CK1 gamma s although less effectively than the CK1 alpha or CK1 delta isoforms. All three CK1 gamma s underwent autophosphorylation when incubated with ATP and Mg2+. The YCK1 and YCK2 genes in Saccharomyces cerevisiae encode casein kinase I homologs, defects in which lead to aberrant morphology and growth arrest. Expression of mammalian CK1 gamma 1 or CK1 gamma 3 restored growth and normal morphology to a yeast mutant carrying a disruption of YCK1 and a temperature-sensitive allele of YCK2, suggesting overlap of function between the yeast Yck proteins and these CK1 isoforms.


Subject(s)
Casein Kinase I , Isoenzymes/genetics , Multigene Family/genetics , Protein Kinases/genetics , Saccharomyces cerevisiae Proteins , Amino Acid Sequence , Animals , Base Sequence , Casein Kinases , Cloning, Molecular , Genes, Fungal/genetics , Genetic Complementation Test , Heparin/pharmacology , Isoenzymes/classification , Male , Molecular Sequence Data , Phosphorylation , Protein Kinases/classification , Protein Kinases/drug effects , RNA, Messenger/analysis , Rats , Restriction Mapping , Saccharomyces cerevisiae/enzymology , Saccharomyces cerevisiae/genetics , Sequence Homology, Amino Acid , Testis/enzymology , Tissue Distribution
11.
J Biol Chem ; 268(9): 6394-401, 1993 Mar 25.
Article in English | MEDLINE | ID: mdl-8454611

ABSTRACT

We report the molecular cloning and characterization of a 49-kDa form of casein kinase I from rat testis. A cDNA clone encoding the enzyme, designated casein kinase I delta, contained an open reading frame of 1284 nucleotides that predicts a polypeptide of 428 amino acids with a M(r) of 49,121. The predicted amino acid sequence shares 76% identity with casein kinase I alpha, a 37-kDa form recently cloned from bovine brain (Rowles, J., Slaughter, C., Moomaw, C., Hsu, J., and Cobb, M. (1991) Proc. Natl. Acad. Sci. U. S. A. 88, 9548-9552), and 65% identity with HRR25, a 57-kDa form of casein kinase I from yeast shown to be involved in DNA repair (Hoekstra, M. F., Liskay, R. M., Ou, A. C., DeMaggio, A. J., Burbee, D. G., and Heffron, F. (1991) Science 253, 1031-1034). Northern analysis of rat or rabbit RNA revealed three hybridizing species of 3.5-4.1, 2.2, and 1.9 kilobase pairs (kb). The largest message was detected in all tissues examined, whereas the 1.9- and 2.2-kb species were found predominantly in testis. A probe corresponding to the 3'-untranslated region of the casein kinase I delta cDNA hybridized only to the 1.9-kb transcript. Expression of the casein kinase I delta cDNA in Escherichia coli resulted in active enzyme that phosphorylated casein, phosvitin, and the peptide substrate DDDDVASLPGLRRR. Enzyme activity was associated with a predominant polypeptide of 55-kDa, although COOH-terminal degradation products of 50 and 42 kDa were also present in partially purified enzyme. Recombinant casein kinase I delta was inhibited by the specific casein kinase I inhibitor, CKI-7, half-maximally at 12 microM. Heparin inhibited recombinant casein kinase I delta when phosvitin was the substrate, with half-maximal inhibition at 11.5 micrograms/ml. However, if the peptide substrate was used, heparin activated recombinant casein kinase I delta 4-5-fold, with half-maximal activation at 9.5 micrograms/ml. A truncated form of casein kinase I delta, lacking the COOH-terminal 111 amino acids, was no longer activated by heparin. Casein kinase I delta therefore represents a separate member of the casein kinase I family distinguished by its larger size and unique kinetic behavior with respect to heparin.


Subject(s)
Protein Kinases/genetics , Testis/enzymology , Amino Acid Sequence , Animals , Base Sequence , Blotting, Northern , Casein Kinases , Cloning, Molecular , DNA/isolation & purification , Escherichia coli , Male , Molecular Sequence Data , Mutagenesis, Site-Directed , Organ Specificity , Protein Kinases/metabolism , Rabbits , Rats , Recombinant Proteins/metabolism , Restriction Mapping , Sequence Homology, Amino Acid
12.
Biochem Biophys Res Commun ; 189(2): 944-9, 1992 Dec 15.
Article in English | MEDLINE | ID: mdl-1472067

ABSTRACT

The casein kinase I (CKI) family consists of widely distributed monomeric Ser/Thr protein kinases that have a preference for acidic substrates. Four mammalian isoforms are known. A full length cDNA encoding the CKI alpha isoform was cloned from a rabbit skeletal muscle cDNA library and was utilized to construct a bacterial expression vector. Active CKI alpha was expressed in Escherichia coli as a polypeptide of Mr 36,000. The protein kinase phosphorylated casein, phosvitin and a specific peptide substrate (D4). The enzyme was inhibited by the isoquinolinesulfonamide CKI-7, half-maximally at 70 microM. Heparin inhibited phosphorylation of the D4 peptide or phosvitin by CKI alpha. Polylysine activated when the D4 peptide was the substrate but had no effect on phosvitin phosphorylation. It is becoming clear that the individual CKI isoforms have different kinetic properties and hence could have quite distinct cellular functions.


Subject(s)
Heparin/pharmacology , Muscles/enzymology , Polylysine/pharmacology , Protein Kinases/metabolism , Animals , Casein Kinases , Cloning, Molecular , Codon , Enzyme Activation , Escherichia coli/genetics , Kinetics , Protein Kinase Inhibitors , Protein Kinases/isolation & purification , Rabbits , Recombinant Proteins/antagonists & inhibitors , Recombinant Proteins/isolation & purification , Recombinant Proteins/metabolism , Restriction Mapping
13.
Proc Natl Acad Sci U S A ; 89(1): 28-32, 1992 Jan 01.
Article in English | MEDLINE | ID: mdl-1729698

ABSTRACT

We report the isolation of an essential pair of Saccharomyces cerevisiae genes that encode protein kinase homologues. The two genes were independently isolated as dosage-dependent suppressors. Increased dosage of YCK1 suppressed defects caused by reduced SNF1 protein kinase activity, and increased dosage of YCK2 relieved sensitivity of wild-type cells to salt stress. The two genes function identically in the two growth assays, and loss of function of either gene alone has no discernible effect on growth. However, loss of function of both genes results in inviability. The two predicted protein products share 77% overall amino acid identity and contain sequence elements conserved among protein kinases. Partial sequence obtained for rabbit casein kinase I shares 64% identity with the two yeast gene products. Moreover, an increase in casein kinase I activity is observed in extracts from cells overexpressing YCK2. Thus YCK1 and YCK2 appear to encode casein kinase I homologues.


Subject(s)
Casein Kinase I , Genes, Fungal , Protein Kinases/genetics , Saccharomyces cerevisiae Proteins , Saccharomyces cerevisiae/genetics , Amino Acid Sequence , Casein Kinases , Cloning, Molecular , Gene Expression , Genes, Suppressor , Genetic Complementation Test , Molecular Sequence Data , Multigene Family , Mutation , Protein Kinases/physiology , Restriction Mapping , Sequence Alignment
14.
Adv Enzyme Regul ; 31: 101-20, 1991.
Article in English | MEDLINE | ID: mdl-1652188

ABSTRACT

Mammalian glycogen synthase, with its complex multisite phosphorylation mechanisms, continues to provide interesting and novel examples of the regulation of protein function. The mammalian enzyme is phosphorylated in a hierarchal manner such that modification of certain sites requires the prior phosphorylation of other sites. Yeast contains two glycogen synthases that have extensive similarities to their mammalian counterpart but the greatest divergence in amino acid sequence is seen precisely in the regions likely to be involved in covalent control. We hope that examination of the control of the yeast glycogen synthase will be as informative as study of the mammalian enzymes, whether by revealing important parallels with the mammalian system or by uncovering major differences in mechanism.


Subject(s)
Glycogen Synthase/metabolism , Glycogen/metabolism , Saccharomyces cerevisiae/enzymology , Signal Transduction , Amino Acid Sequence , Animals , Calcium-Calmodulin-Dependent Protein Kinases , Genes , Glucuronosyltransferase/genetics , Glycogen Synthase/genetics , Homeostasis , Mammals , Molecular Sequence Data , Protein Kinases/metabolism
15.
J Biol Chem ; 265(24): 14264-9, 1990 Aug 25.
Article in English | MEDLINE | ID: mdl-2117608

ABSTRACT

Phosphorylation of rabbit muscle glycogen synthase by cyclic AMP-dependent protein kinase has been shown to enhance subsequent phosphorylation by casein kinase I (Flotow, H., and Roach, P. J. (1989) J. Biol. Chem. 264, 9126-9128). In the present study, synthetic peptides based on the sequences of the four phosphorylated regions in muscle glycogen synthase were used to probe the role of substrate phosphorylation in casein kinase I action. With all four peptides, prior phosphorylation significantly stimulated phosphorylation by casein kinase I. A series of peptides was synthesized based on the NH2-terminal glycogen synthase sequence PLSRTLS7VSS10LPGL, in which phosphorylation at Ser7 is required for modification of Ser10 by casein kinase I. The spacing between the P-Ser and the acceptor Ser was varied to have 1, 2, or 3 intervening residues. The peptide with a 2-residue spacing (-S(P)-X-X-S-) was by far the best casein kinase I substrate. When the P-Ser residue at Ser7 was replaced with P-Thr, the resulting peptide was still a casein kinase I substrate. However, substitution of Asp or Glu residues at Ser7 led to peptides that were not phosphorylated by casein kinase I. Phosphorylation of one of the other peptides showed that Thr could also be the phosphate acceptor. From these results, we propose that there are substrates for casein kinase I for which prior phosphorylation is a critical determinant of protein kinase action. In these instances, an important recognition motif for casein kinase I appears to be -S(P)/T(P)-Xn-S/T- with n = 2 much more effective than n = 1 or n = 3. Thus, casein kinase I may be involved in hierarchal substrate phosphorylation schemes in which its activity is controlled by the phosphorylation state of its substrates.


Subject(s)
Phosphopeptides/chemical synthesis , Protein Kinases/metabolism , Amino Acid Sequence , Animals , Casein Kinases , Caseins/metabolism , Glycogen Synthase/metabolism , Kinetics , Molecular Sequence Data , Muscles/enzymology , Peptides/chemical synthesis , Phosphopeptides/isolation & purification , Phosphorylation , Protein Kinases/isolation & purification , Rabbits , Substrate Specificity
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