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1.
High-mobility-group proteins P1, I and Y as substrates of the M-phase-specific p34cdc2/cyclincdc13 kinase 总被引:4,自引:0,他引:4
L Meijer A C Ostvold S I Walass T Lund S G Laland 《European journal of biochemistry》1991,196(3):557-567
All dividing cells entering the M phase of the cell cycle undergo the transient activation of an M-phase-specific histone H1 kinase which was recently shown to be constituted of at least two subunits, p34cdc2 and cyclincdc13. The DNA-binding high-mobility-group (HMG) proteins 1, 2, 14, 17, I, Y and an HMG-like protein, P1, were investigated as potential substrates of H1 kinase. Among these HMG proteins, P1 and HMG I and Y are excellent substrates of the M-phase-specific kinase obtained from both meiotic starfish oocytes and mitotic sea urchin eggs. Anticyclin immunoprecipitates, extracts purified on specific p34cdc2-binding p13suc1-Sepharose and affinity-purified H1 kinase display strong HMG I, Y and P1 phosphorylating activities, demonstrating that the p34cdc2/cyclincdc13 complex is the active kinase phosphorylating these HMG proteins. HMG I and P1 phosphorylation is competitively inhibited by a peptide mimicking the consensus phosphorylation sequence of H1 kinase. HMG I, Y and P1 all possess the consensus sequence for phosphorylation by the p34cdc2/cyclincdc13 kinase (Ser/Thr-Pro-Xaa-Lys/Arg). HMG I is phosphorylated in vivo at M phase on the same sites phosphorylated in vitro by H1 kinase. P1 is phosphorylated by H1 kinase on sites different from the sites of phosphorylation by casein kinase II. The three thermolytic phosphopeptides of P1 phosphorylated in vitro by purified H1 kinase are all present in thermolytic peptide maps of P1 phosphorylated in vivo in proliferating HeLa cells. These phosphopeptides are absent in nonproliferating cells. These results demonstrate that the DNA-binding proteins HMG I, Y and P1 are natural substrates for the M-phase-specific protein kinase. The phosphorylation of these proteins by p34cdc2/cyclincdc13 may represent a crucial event in the intense chromatin condensation occurring as cells transit from the G2 to the M phase of the cell cycle. 相似文献
2.
Schwanbeck R Gymnopoulos M Petry I Piekiełko A Szewczuk Z Heyduk T Zechel K Wiśniewski JR 《The Journal of biological chemistry》2001,276(28):26012-26021
The high mobility group (HMG) proteins of the AT-hook family (HMGA) lie downstream in regulatory networks with protein kinase C, Cdc2 kinase, MAP kinase, and casein kinase 2 (CK2) as final effectors. In the cells of the midge Chironomus, almost all of the HMGA protein (cHMGA) is phosphorylated by CK2 at two adjacent sites. 40% of the protein population is additionally modified by MAP kinase. Using spectroscopic and protein footprinting techniques, we analyzed how individual and consecutive steps of phosphorylation change the conformation of an HMGA protein and affect its contacts with poly(dA-dT).poly(dA-dT) and a fragment of the interferon-beta promoter. We demonstrate that phosphorylation of cHMGA by CK2 alters its conformation and modulates its DNA binding properties such that a subsequent phosphorylation by Cdc2 kinase changes the organization of the protein-DNA complex. In contrast, consecutive phosphorylation by MAP kinase, which results in a dramatic change in cHMGA conformation, has no direct effect on the complex. Because the phosphorylation of the HMGA proteins attenuates binding affinity and reduces the extent of contacts between the DNA and protein, it is likely that this process mirrors the dynamics and diversity of regulatory processes in chromatin. 相似文献
3.
Annikka Linnala-Kankkunen Jorma Palvimo Pekka H. Mäenpää 《Biochimica et Biophysica Acta (BBA)/General Subjects》1984,799(2):122-127
Phosphorylation of acidic substrates such as casein and phosvitin by nuclear protein kinase II is stimulated by polyamines and inhibited by heparin, which mimics an endogenous proteoglycan inhibitor. The phosphorylation in vitro of the chromatin proteins HMG 14 and HMG 17 by nuclear protein kinase II were examined in this study focusing on the modifying effects of polyamines and heparin. Both HMG proteins were phosphorylated by the enzyme, but polyamines did not appreciably influence the extent of their phosphorylation. In addition, heparin did not inhibit the kinase reaction with the HMG proteins as substrates. These results indicate that the nuclear protein kinase II does actively phosphorylate HMG 14 and HMG 17 in vitro but that in contrast to some model substrates, polyamines and heparin do not appreciably affect their phosphorylation. 相似文献
4.
Phosphorylation of DARPP-32, a dopamine- and cAMP-regulated phosphoprotein, by casein kinase II 总被引:14,自引:0,他引:14
J A Girault H C Hemmings K R Williams A C Nairn P Greengard 《The Journal of biological chemistry》1989,264(36):21748-21759
DARPP-32 (dopamine- and cAMP-regulated phosphorprotein, Mr = 32,000 as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis) is an inhibitor of protein phosphatase-1 and is enriched in dopaminoceptive neurons possessing the D1 dopamine receptor. Purified bovine DARPP-32 was phosphorylated in vitro by casein kinase II to a stoichiometry greater than 2 mol of phosphate/mol of protein whereas two structurally and functionally related proteins, protein phosphatase inhibitor-1 and G-substrate, were poor substrates for this enzyme. Sequencing of chymotryptic and thermolytic phosphopeptides from bovine DARPP-32 phosphorylated by casein kinase II suggested that the main phosphorylated residues were Ser45 and Ser102. In the case of rat DARPP-32, the identification of these phosphorylation sites was confirmed by manual Edman degradation. The phosphorylated residues are located NH2-terminal to acidic amino acid residues, a characteristic of casein kinase II phosphorylation sites. Casein kinase II phosphorylated DARPP-32 with an apparent Km value of 3.4 microM and a kcat value of 0.32 s-1. The kcat value for phosphorylation of Ser102 was 5-6 times greater than that for Ser45. Studies employing synthetic peptides encompassing each phosphorylation site confirmed this difference between the kcat values for phosphorylation of the two sites. In slices of rat caudate-putamen prelabeled with [32P]phosphate, DARPP-32 was phosphorylated on seryl residues under basal conditions. Comparison of thermolytic phosphopeptide maps and determination of the phosphorylated residue by manual Edman degradation identified the main phosphorylation site in intact cells as Ser102. In vitro, DARPP-32 phosphorylated by casein kinase II was dephosphorylated by protein phosphatases-1 and -2A. Phosphorylation by casein kinase II did not affect the potency of DARPP-32 as an inhibitor of protein phosphatase-1, which depended only on phosphorylation of Thr34 by cAMP-dependent protein kinase. However, phosphorylation of DARPP-32 by casein kinase II facilitated phosphorylation of Thr34 by cAMP-dependent protein kinase with a 2.2-fold increase in the Vmax and a 1.4-fold increase in the apparent Km. Phosphorylation of DARPP-32 by casein kinase II in intact cells may therefore modulate its phosphorylation in response to increased levels of cAMP. 相似文献
5.
Phosphorylation of chromosomal high mobility group (HMG) protein 14 by casein kinase II has been characterized. Two mol of 32P are incorporated per mol of bovine HMG 14. Kinetic analysis provided evidence for two distinct sites with apparent Km values of 14.5 and 134 microM and respective Vmax values of 0.17 and 0.68 mumol/min/mg casein kinase II. Tryptic peptide mapping identified two phosphorylated products, each with phosphoserine. Amino acid composition and sequence analysis demonstrate that the major high affinity phosphorylation site for casein kinase II is serine 89. This sequence located at the carboxyl-terminal of HMG 14 contains the primary sequence determinants for casein kinase II. On the basis of reverse-phase high performance liquid chromatography and amino acid analysis, HMG 14, serine 99 represents the low affinity phosphorylation site. 相似文献
6.
Phosphorylation of high-mobility-group protein 14 by two specific kinases modifies its interaction with histone oligomers in free solution 总被引:1,自引:0,他引:1
E Espel J Bernués M D Guasch E Querol M Plana E Itarte 《Biochimica et biophysica acta》1987,909(3):190-200
Chromosomal protein HMG14 can be specifically phosphorylated by the cyclic AMP-dependent protein kinase at the N-terminus and by casein kinase 2 at the acidic C-terminus. Under the same conditions used for HMG14, HMG17 is not significantly phosphorylated by either of the two kinases. Further, we have studied the effect of phosphorylation by these kinases on the interaction of HMG14 with histone oligomers, using chemical cross-linking. Our results indicate that the phosphorylation of HMG14 by casein kinase 2 enhances its interaction with histone oligomers in free solution, whereas a minor effect was observed by phosphorylation with cyclic AMP-dependent protein kinase. In contrast, HMG17 does not interact at all with any histone oligomer in free solution under the conditions used. To gain insight into the possible effect that phosphorylation may play in vivo, the pattern of distribution among different chromatin fractions was analysed. It was found that, although phosphorylation of HMG14 by both kinases allowed reconstitution of HMG14 to chromatin, the patterns obtained showed some slight differences. 相似文献
7.
DNA minicircles, where the length of DNA is below the persistence length, are highly effective, preferred, ligands for HMG-box proteins. The proteins bind to them "structure-specifically" with affinities in the nanomolar range, presumably to an exposed widened minor groove. To understand better the basis of this preference, we have studied the binding of HMG1 (which has two tandem HMG boxes linked by a basic extension to a long acidic tail) and Drosophila HMG-D (one HMG box linked by a basic region to a short and less acidic tail), and their HMG-box domains, to 88 bp and 75 bp DNA minicircles. In some cases we see cooperative binding of two molecules to the circles. The requirements for strong cooperativity are two HMG boxes and the basic extension; the latter also appears to stabilize and constrain the complex, preventing binding of further protein molecules. HMG-D, with a single HMG box, does not bind cooperatively. In the case of HMG1, the acidic tail is not required for cooperativity and does not affect binding significantly, in contrast to a much greater effect with linear DNA, or even four-way junctions (another distorted DNA substrate). Such effects could be relevant in the hierarchy of binding of HMG-box proteins to DNA distortions in vivo, where both single-box and two-box proteins might co-exist, with or without basic extensions and acidic tails. 相似文献
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9.
T Hagiwara K Nakaya Y Nakamura H Nakajima S Nishimura Y Taya 《European journal of biochemistry》1992,209(3):945-950
The central region of the N-myc protein has a characteristic amino acid sequence EDTLSDSDDEDD, which is very similar to those of particular domains of adenovirus E1A, human papilloma virus E7, Simian virus 40 large T, c-myc and L-myc proteins. Domains of these three viral oncoproteins have recently been shown to be specific binding sites for the tumor-suppressor gene retinoblastoma protein. We have noted that the sequence of serine followed by a cluster of acidic amino acids is exactly the same as that of a typical substrate of casein kinase II (CKII). Therefore, we investigated whether these nuclear oncoproteins are phosphorylated by CKII. For this purpose, we fused the beta-galactosidase and N-myc genes including this domain and expressed it in Escherichia coli cells. Several mutant N-myc genes, containing single amino acid substitutions in this domain, were also used to produce fused proteins. Strong phosphorylation by CKII was detected with the fused protein of wild-type N-myc. However, no phosphorylation of beta-galactosidase itself was observed and the phosphorylations of fused mutant proteins were low. Another fused N-myc protein containing most of the C-terminal region downstream of this acidic region was not phosphorylated by CKII. Analysis of phosphorylation sites in synthetic peptides of this acidic region identified the major sites phosphorylated by CKII as Ser261 and Ser263. On two-dimensional tryptic mapping of phosphorylated N-myc proteins, major spots of in vitro-labeled and in-vivo-labeled N-myc proteins were detected in the same positions. These results suggest that two serine residues of the acidic central region of the N-myc protein are phosphorylated by CKII in vivo as well as in vitro. The functional significance of this acidic domain is discussed. 相似文献
10.
The Drosophila homolog of cAMP-response element-binding protein (CREB), dCREB2, exists with serine 231, equivalent to mammalian serine 133, in a predominantly phosphorylated state. Thus, unlike the mammalian protein, the primary regulation of dCREB2 may occur at a different step from serine 231 phosphorylation. Although bacterially expressed dCREB2 bound cAMP-response element sites, protein from Drosophila extracts was unable to do so unless treated with phosphatase. Phosphorylation of recombinant protein by casein kinase (CK) I or II, but not calcium-calmodulin kinase II or protein kinase A, inhibited DNA binding. Up to four conserved CK sites likely to be phosphorylated in vivo were responsible for this effect, and these sites were phosphorylated by a kinase present in Drosophila cell extracts that biochemically resembles CKII. We propose that the relative importance of different signaling pathways in regulating CREB activity may differ between Drosophila and mammals. In Drosophila, the dephosphorylation of CK sites appears to be the major regulatory step, while phosphorylation of serine 231 is necessary but secondary. 相似文献
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Phosphorylation of neurofilament H subunit at the tail domain by CDC2 kinase dissociates the association to microtubules 总被引:14,自引:0,他引:14
S Hisanaga M Kusubata E Okumura T Kishimoto 《The Journal of biological chemistry》1991,266(32):21798-21803
We sought the mammalian neurofilament tail domain-specific kinase. Several well known kinases including cAMP-dependent protein kinase, protein kinase C, Ca(2+)-calmodulin-dependent protein kinase II, casein kinase I, and casein kinase II phosphorylated the high (NF-H) and middle molecular mass subunit (NF-M) of bovine neurofilaments, but they did not reduced the electrophoretic mobility of the dephosphorylated form of NF-M and NF-H by phosphorylation nor was the amount of phosphorylation increased by dephosphorylation of NF proteins, indicating that the phosphorylation sites by these kinases are not major in vivo phosphorylation sites at the tail domain. In contrast, cdc2 kinase phosphorylated specifically the dephosphorylated form of NF-H. 4 mol of phosphates were incorporated per mol of NF-H and this phosphorylation returned the electrophoretic mobility of the dephosphorylated form of NF-H to the position of the isolated, fully phosphorylated form of NF-H. Furthermore, the phosphorylation by cdc2 kinase dissociated the binding of dephosphorylated NF-H to microtubules. Phosphorylation sites were located at the carboxyl-terminal tail domain. The KSPXK motif, but not KSPXX, in the repetitive sequence was suggested to be the phosphorylation site by using synthetic peptides. 相似文献
14.
HMG-D is an architecture-specific protein that preferentially binds to DNA containing the dinucleotide TG. 总被引:6,自引:3,他引:3
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M E Churchill D N Jones T Glaser H Hefner M A Searles A A Travers 《The EMBO journal》1995,14(6):1264-1275
The high mobility group (HMG) protein HMG-D from Drosophila melanogaster is a highly abundant chromosomal protein that is closely related to the vertebrate HMG domain proteins HMG1 and HMG2. In general, chromosomal HMG domain proteins lack sequence specificity. However, using both NMR spectroscopy and standard biochemical techniques we show that binding of HMG-D to a single DNA site is sequence selective. The preferred duplex DNA binding site comprises at least 5 bp and contains the deformable dinucleotide TG embedded in A/T-rich sequences. The TG motif constitutes a common core element in the binding sites of the well-characterized sequence-specific HMG domain proteins. We show that a conserved aromatic residue in helix 1 of the HMG domain may be involved in recognition of this core sequence. In common with other HMG domain proteins HMG-D binds preferentially to DNA sites that are stably bent and underwound, therefore HMG-D can be considered an architecture-specific protein. Finally, we show that HMG-D bends DNA and may confer a superhelical DNA conformation at a natural DNA binding site in the Drosophila fushi tarazu scaffold-associated region. 相似文献
15.
Phosphorylation of neuromodulin (GAP-43) by casein kinase II. Identification of phosphorylation sites and regulation by calmodulin. 总被引:7,自引:0,他引:7
E D Apel D W Litchfield R H Clark E G Krebs D R Storm 《The Journal of biological chemistry》1991,266(16):10544-10551
Neuromodulin (P-57, GAP-43, B-50, F-1) is a neurospecific calmodulin-binding protein believed to play a role in regulation of neurite outgrowth and neuroplasticity. Neuromodulin is phosphorylated by protein kinase C, and this phosphorylation prevents calmodulin from binding to neuromodulin (Alexander, K. A., Cimler, B. M., Meier, K. E. & Storm, D. R. (1987) J. Biol. Chem. 262, 6108-6113). The only other protein kinase known to phosphorylate neuromodulin is casein kinase II (Pisano, M. R., Hegazy, M. G., Reimann, E. M. & Dokas, L. A. (1988) Biochem. Biophys. Res. Commun. 155, 1207-1212). Phosphoamino acid analyses revealed that casein kinase II modified serine and threonine residues in both native bovine and recombinant mouse neuromodulin. Two serines located in the C-terminal end of neuromodulin, Ser-192 and Ser-193, were identified as the major casein kinase II phosphorylation sites. Thr-88, Thr-89, or Thr-95 were identified as minor casein kinase II phosphorylation sites. Phosphorylation by casein kinase II did not affect the ability of neuromodulin to bind to calmodulin-Sepharose. However, calmodulin did inhibit the phosphorylation of neuromodulin by casein kinase II with a Ki of 1-2 microM. Calmodulin inhibition of casein kinase II phosphorylation was due to calmodulin binding to neuromodulin rather than to the protein kinase. These data suggest that the minimal secondary and tertiary structure exhibited by neuromodulin may be sufficient to juxtapose its calmodulin-binding domain, located at the N-terminal end, with the neuromodulin casein kinase II phosphorylation sites at the C-terminal end of the protein. We propose that calmodulin regulates casein kinase II phosphorylation of neuromodulin by binding to neuromodulin and sterically hindering the interaction of casein kinase II with its phosphorylation sites on neuromodulin. 相似文献
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Ishihara K Yasuda K Hatayama T 《Biochemical and biophysical research communications》2000,270(3):927-931
The 105-kDa heat shock protein alpha (HSP105alpha) and HSP105beta are mammalian heat shock proteins that belong to the HSP105/HSP110 family. Both HSP105alpha and HSP105beta consist of acidic and basic isoforms. Here we report that the acidic isoforms are serine phosphorylated HSP105alpha or HSP105beta. Furthermore, using an in-gel kinase assay with HSP105alpha or HSP105beta as the substrate, the protein kinase that phosphorylates HSP105alpha and HSP105beta was identified as casein kinase II. Since phosphorylated HSP105alpha is especially prominent in the brain compared to other tissues of mice and rats, the phosphorylation of HSP105alpha by casein kinase II may be biologically significant. 相似文献
18.
H Jahn W Nastainczyk A R?hrkasten T Schneider F Hofmann 《European journal of biochemistry》1988,178(2):535-542
Five protein kinases were used to study the phosphorylation pattern of the purified skeletal muscle receptor for calcium-channel blockers (CaCB). cAMP kinase, cGMP kinase, protein kinase C, calmodulin kinase II and casein kinase II phosphorylated the 165-kDa and the 55-kDa proteins of the purified CaCB receptor. The 130/28-kDa and the 32-kDa protein of the receptor are not phosphorylated by these protein kinases. Among these protein kinases only cAMP kinase phosphorylated the 165-kDa subunit with 2-3-fold higher initial rate than the 55-kDa subunit. Casein kinase II phosphorylated the 165-kDa and the 55-kDa protein of the receptor with comparable rates. cGMP kinase, protein kinase C and calmodulin kinase II phosphorylated preferentially the 55-kDa protein. The 55-kDa protein is phosphorylated 50 times faster by cGMP kinase and protein kinase C than by calmodulin kinase II or casein kinase II and about 10 times faster by these enzymes than by cAMP kinase. Two-dimensional peptide maps of the 165-kDa subunit yielded a total of 11 phosphopeptides. Four or five peptides are phosphorylated specifically by cAMP kinase, cGMP kinase, casein kinase II and protein kinase C, whereas the other peptides are modified by several kinases. The same kinases phosphorylate 11 peptides in the 55-kDa subunit. Again, some of these peptides are modified specifically by each kinase. These results suggest that the 165-kDa and the 55-kDa subunit contain specific phosphorylation sites for cAMP kinase, cGMP kinase, casein kinase II and protein kinase C. Phosphorylation of these sites may be relevant for the in vivo function of the CaCB receptor. 相似文献
19.
HP1 is an essential heterochromatin-associated protein in Drosophila. HP1 has dosage-dependent effects on the silencing of euchromatic genes that are mislocalized to heterochromatin and is required for the normal expression of at least two heterochromatic genes. HP1 is multiply phosphorylated in vivo, and HP1 hyperphosphorylation is correlated with heterochromatin assembly during development. The purpose of this study was to test whether HP1 phosphorylation modifies biological activity and biochemical properties of HP1. To determine sites of HP1 phosphorylation in vivo and whether phosphorylation affects any biochemical properties of HP1, we expressed Drosophila HP1 in lepidopteran cultured cells using a recombinant baculovirus vector. Phosphopeptides were identified by matrix-assisted laser desorption ionization/time of flight mass spectroscopy; these peptides contain target sites for casein kinase II, protein tyrosine kinase, and PIM-1 kinase. Purified HP1 from bacterial (unphosphorylated) and lepidopteran (phosphorylated) cells has similar secondary structure. Phosphorylation has no effect on HP1 self-association but alters the DNA binding properties of HP1, suggesting that phosphorylation could differentially regulate HP1-dependent interactions. Serine-to-alanine and serine-to-glutamate substitutions at consensus protein kinase motifs resulted in reduction or loss of silencing activity of mutant HP1 in transgenic flies. These results suggest that dynamic phosphorylation/dephosphorylation regulates HP1 activity in heterochromatic silencing. 相似文献
20.
Christian Stemmer Andrea Schwander Guy Bauw Peter Fojan Klaus D Grasser 《The Journal of biological chemistry》2002,277(2):1092-1098
The high mobility group (HMG) proteins of the HMGB family are architectural factors in eukaryotic chromatin, which are involved in the regulation of various DNA-dependent processes. We have examined the post-translational modifications of five HMGB proteins from maize suspension cultured cells, revealing that HMGB1 and HMGB2/3, but not HMGB4 and HMGB5, are phosphorylated by protein kinase CK2. The phosphorylation sites have been mapped to the acidic C-terminal domains by analysis of tryptic peptides derived from HMGB1 and HMGB2/3 using nanospray ion trap mass spectrometry. In native HMGB1, Ser(149) is constitutively phosphorylated, whereas Ser(133) and Ser(136) are differentially phosphorylated. The functional significance of the CK2-mediated phosphorylation of HMGB proteins was analyzed by circular dichroism measurements showing that the phosphorylation increases the thermal stability of the HMGB proteins. Electrophoretic mobility shift assays demonstrate that the phosphorylation reduces the affinity of the HMGB proteins for linear DNA. The specific recognition of DNA minicircles is not affected by the phosphorylation, but a different pattern of protein-DNA complexes is formed. Collectively, these findings show that phosphorylation of residues within the acidic C-terminal domain of the HMGB proteins can modulate protein stability and the DNA binding properties of the HMGB proteins. 相似文献