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1.
E Humble P Heldin P O Forsberg L Engstr?m 《Archives of biochemistry and biophysics》1985,241(1):225-231
Human, dog, and rabbit fibrinogen served as substrates for calcium-activated, phospholipid-dependent protein kinase, cAMP-dependent protein kinase, casein kinase TS, and casein kinase S. The chains of phosphorylated fibrinogen were separated by polyacrylamide gel electrophoresis and the phosphorylation patterns, obtained on autoradiography of the gels, were found to be characteristic for each of the four protein kinases. Dog, and even more so rabbit, fibrinogen was phosphorylated more rapidly than human fibrinogen by calcium-activated, phospholipid-dependent protein kinase and by casein kinase TS. Dog fibrinogen was not a good substrate for cAMP-dependent protein kinase. The rate of phosphorylation with casein kinase S did not differ very much between the fibrinogens of the three species. In most cases the A alpha-chain was most rapidly phosphorylated. However, in dog fibrinogen incubated with casein kinase TS the B beta-chain was most rapidly phosphorylated. A substantial part of this phosphate seemed to be incorporated as phosphorylthreonine into fibrinopeptide B. In human fibrinogen incubated with the casein kinase TS preparation the gamma-chain as well as the A alpha-chain appeared to be phosphorylated. 相似文献
2.
Casein kinase 2 from rat liver cytosol phosphorylated human fibrinogen in a reaction that was not stimulated by Ca2+ or cyclic AMP, but was markedly inhibited by heparin, and proceeded at a similar rate when either ATP or GTP was used as phosphate donor. Analysis of casein kinase 2 by glycerol-density-gradient centrifugation showed that the activities towards fibrinogen, casein, phosvitin, high-mobility-group protein 14 and glycogen synthase coincided. Maximal incorporation into fibrinogen by casein kinase 2 averaged 1 mol of phosphate/mol of protein substrate, most of it in the alpha-chain, although some phosphorylation of the beta-chain was also detected. Analysis of phosphorylated alpha-chain revealed that most of the phosphate was incorporated on serine. Phosphorylation of human fibrinogen was also performed by casein kinase 2 from human polymorphonuclear leucocytes, lymphocytes and platelets. 相似文献
3.
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
4.
Phosphate groups as substrate determinants for casein kinase I action 总被引:22,自引:0,他引:22
H Flotow P R Graves A Q Wang C J Fiol R W Roeske P J Roach 《The Journal of biological chemistry》1990,265(24):14264-14269
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. 相似文献
5.
Phosphorylation of fibrinogen by casein kinase 1 总被引:3,自引:0,他引:3
E Itarte M Plana M D Guasch C Martos 《Biochemical and biophysical research communications》1983,117(2):631-636
Casein kinase 1 phosphorylated human fibrinogen, in a reaction that did not use GTP as phosphoryl donor and was neither stimulated by cyclic AMP or Ca2+, nor inhibited by the cyclic AMP-dependent protein kinase inhibitor protein. Maximal incorporation averaged 4 mol of phosphate per mol of fibrinogen, most of it in the largest CNBr-fragment of the alpha-chain. Phosphoamino acid analysis revealed that phosphorylation occurred only at seryl residues. The phosphorylation of fibrinogen by casein kinase 1 was reverted by alkaline phosphatase. 相似文献
6.
Phosphorylation by cell surface protein kinase of bovine and human fibrinogen and fibrin 总被引:1,自引:0,他引:1
Human and bovine fibrinogen as well as fibrin are shown to be phosphorylated by Co631 (monolayer, hamster) and RPL12 (suspension, chicken) cells by their surface protein kinase of the casein kinase II type. The phosphate label is introduced into the alpha-peptide. The kinase system phosphorylates serine residues and utilizes GTP equally well as ATP. The participation of intact cell surfaces indicates the possibility of phosphorylation of extracellular fibrinogen independently of the site of its biosynthesis. 相似文献
7.
Phosphorylation of human fibrinogen in vitro by incubation with [gamma-32P]ATP and protein kinase C purified from pig spleen, led to incorporation of [32P]phosphate at serine residues located in the A alpha-chain. In order to identify the residues that were phosphorylated, the A alpha-chain of fibrinogen was isolated and subjected to consecutive cleavage by cyanogen bromide, trypsin, and chymotrypsin. The resulting radioactive phosphopeptides were purified by gel chromatography and high-performance liquid chromatography using a reversed-phase column. Subsequent amino acid analysis and manual Edman degradation of the purified phosphopeptides revealed that Ser557, Ser558, Ser559, and Ser599 were phosphorylated. These serine residues are located in the carboxy-terminal part of the A alpha-chain. This region also contains lysine residues participating in the cross-linking of fibrin and, possibly, a site involved in the binding of fibrinogen to receptors on platelets. In addition, peptides derived from the middle section of the polypeptide chain were found to contain [32P]phosphate; in these cases, however, the exact localization of the phosphate could not be determined, due to the low yield of radioactivity. Two glutamine residues, Gln328 and Gln366, in this portion of the A alpha-chain take part in the cross-linking of fibrin. 相似文献
8.
Phosphorylation of avian gizzard caldesmon by casein kinase II was investigated. The enzyme incorporates about 1 mol of phosphate per mol of caldesmon. All sites of phosphorylation are located in short chymotryptic peptides with Mr 25-27 kDa or in the short N-terminal peptide formed after cleavage of chicken gizzard caldesmon at Cys153. The primary structure of the tryptic peptide containing the main site of duck gizzard caldesmon phosphorylation is S-E-V-N-A-Q-N-X-V-A-E-D-E-T-K, where X is an unidentified residue, presumed to be phosphoserine. Thus, Ser73 is the main site phosphorylated by casein kinase II in avian gizzard caldesmon. 相似文献
9.
Multisite phosphorylation of glycogen synthase from rabbit skeletal muscle. Identification of the sites phosphorylated by casein kinase-I 总被引:3,自引:0,他引:3
A casein kinase was highly purified from rabbit skeletal muscle whose substrate specificity and enzymatic properties were virtually identical to those of casein kinase-I from rabbit reticulocytes. Prolonged incubation of glycogen synthase with high concentrations of skeletal muscle casein kinase-I and Mg-ATP resulted in the incorporation of greater than 6 mol phosphate/mol subunit and decreased the activity ratio (+/- glucose-6P) from 0.8 to less than 0.02. The sites phosphorylated by casein kinase-I were all located in the N and C-terminal cyanogen bromide peptides, termed CB-1 and CB-2. At an incorporation of 6 mol phosphate/mol subunit, approximately equal to 2 mol/mol was present in CB-1 and approximately equal to 4 mol/mol in CB-2. Within CB-1, casein kinase-I phosphorylated the serines that were 3, 7 and 10 residues from the N-terminus of glycogen synthase, with minor phosphorylation at threonine-5. Within CB-2, approximately equal to 90% of the phosphate incorporated was located between residues 28 and 53, and at least five of the seven serine residues in this region were phosphorylated. The remaining 10% of phosphate incorporated into CB-2 was located between residues 98 and 123, mainly at a serine residue(s). Two of the major sites labelled by casein kinase-I (serine-3 and serine-10 of CB-1) are not phosphorylated by any other protein kinase. This will enable the role of casein kinase-I as a glycogen synthase kinase in vivo to be evaluated. 相似文献
10.
Phosphorylation of the insulin receptor by casein kinase I 总被引:1,自引:0,他引:1
Insulin receptor was examined as a substrate for the multipotential protein kinase casein kinase I. Casein kinase I phosphorylated partially purified insulin receptor from human placenta as shown by immunoprecipitation of the complex with antiserum to the insulin receptor. Analysis of the phosphorylated complex by polyacrylamide gel electrophoresis under nonreducing conditions showed a major phosphorylated band at the position of the alpha 2 beta 2 complex. When the phosphorylated receptor was analyzed on polyacrylamide gels under reducing conditions, two phosphorylated bands, Mr 95,000 and Mr 135,000, were observed which corresponded to the alpha and beta subunits. The majority of the phosphate was associated with the beta subunit with minor phosphorylation of the alpha subunit. Phosphoamino acid analysis revealed that casein kinase I phosphorylated only seryl residues. The autophosphorylated alpha 2 beta 2 receptor purified by affinity chromatography on immobilized O-phosphotyrosyl binding antibody was also a substrate for casein kinase I. Reduction of the phosphorylated alpha 2 beta 2 receptor indicated that casein kinase I incorporated phosphate into seryl residues only in the beta subunit. 相似文献
11.
The 204-kDa smooth muscle myosin heavy chain is phosphorylated in intact cells by casein kinase II on a serine near the carboxyl terminus 总被引:2,自引:0,他引:2
The heavy chain of smooth muscle myosin was found to be phosphorylated following immunoprecipitation from cultured bovine aortic smooth muscle cells. Of a variety of serine/threonine kinases assayed, only casein kinase II and calcium/calmodulin-dependent protein kinase II phosphorylated the smooth muscle myosin heavy chain to a significant extent in vitro. Two-dimensional maps of tryptic peptides derived from heavy chains phosphorylated in cultured cells revealed one major and one minor phosphopeptide. Identical tryptic peptide maps were obtained from heavy chains phosphorylated in vitro with casein kinase II but not with calcium/calmodulin-dependent protein kinase II. Of note, the 204-kDa smooth muscle myosin heavy chain but not the 200-kDa heavy chain isoform was phosphorylated by casein kinase II. Partial sequence of the tryptic phosphopeptides generated following phosphorylation by casein kinase II yielded Val-Ile-Glu-Asn-Ala-Asp-Gly-Ser*-Glu-Glu-Glu-Val. The Ser* represents the Ser(PO4) which is in an acidic environment, as is typical for casein kinase II phosphorylation sites. By comparison with the deduced amino acid sequence for rabbit uterine smooth muscle myosin (Nagai, R., Kuro-o, M., Babij, P., and Periasamy, M. (1989) J. Biol. Chem. 264, 9734-9737), we have localized the phosphorylated serine residue to the non-helical tail of the 204-kDa isoform of the smooth muscle myosin heavy chain. The ability of the 204-kDa isoform, but not the 200-kDa isoform, to serve as a substrate for casein kinase II suggests that these two isoforms can be regulated differentially. 相似文献
12.
Tyrosine hydroxylase was maximally phosphorylated by protein kinase C, with a stoichiometry of 0.43 mol of phosphate/mol of tyrosine hydroxylase subunit at Ser40, and by calmodulin-dependent protein kinase II, with stoichiometries of 0.43 mol/mol at Ser40 and 0.76 mol/mol at Ser19, respectively, without undergoing any significant direct activation. In contrast, the enzyme was maximally phosphorylated with a stoichiometry of 0.78 mol of phosphate/mol of subunit at Ser40 by cAMP-dependent protein kinase, which resulted in a large activation of the enzyme (about 3-fold activation under the assay conditions). Incubation of the enzyme, which had previously been maximally phosphorylated by calmodulin-dependent protein kinase II, with protein kinase C under phosphorylating conditions resulted in no additional incorporation of phosphate into the enzyme, suggesting that both protein kinases phosphorylated Ser40 of the same subunits of the enzyme. Since tyrosine hydroxylase is thought to be composed of four identical subunits, the results may indicate that calmodulin-dependent protein kinase II or protein kinase C phosphorylates only two of the four subunits of the enzyme at Ser40 without affecting the enzyme activity and that cAMP-dependent protein kinase phosphorylates Ser40 of all four subunits of the enzyme molecule, causing a marked activation. Based on a linear relationship between phosphorylation and the resulting activation of the enzyme by cAMP-dependent protein kinase, possible mechanisms for the activation of the enzyme by the protein kinase are discussed. 相似文献
13.
Purification of substrate proteins of casein kinases from the cytosol fraction of AH-66 hepatoma cells 总被引:1,自引:0,他引:1
We have attempted to purify endogenous substrate proteins for casein kinases I and II from the cytosol of AH-66 hepatoma cells. Utilizing the fact that only a few substrates are concentrated in the fraction eluted from DEAE-cellulose between 0.3 and 0.6 M NaCl, two substrates were purified from this fraction by DEAE-cellulose chromatography, hydroxyapatite chromatography, and HPLC on a DEAE-5PW column. The purified substrate proteins had molecular masses of 30.5 kDa and 31 kDa. The 31-kDa protein substrate was markedly phosphorylated by casein kinase II, but only slightly by casein kinase I. The radioactive phosphate incorporated into 31-kDa substrate by casein kinase II was 0.2 mol/mol of the protein and phosphorylation occurred on both threonine and serine residues. The 30.5 kDa protein was only slightly phosphorylated by casein kinase II, but not at all by casein kinase I. 相似文献
14.
Katsumi Shinkawa Shigeo Nakajo Kazuyasu Nakaya Yasuharu Nakamura 《Biochimica et Biophysica Acta (BBA)/Molecular Cell Research》1987,930(3)
We have attempted to purify endogenous substrate proteins for casein kinases I and II from the cytosol of AH-66 hepatoma cells. Utilizing the fact that only a few substrates are concentrated in the fraction eluted from DEAE-cellulose between 0.3 and 0.6 M NaCl, two substrates were purified from this fraction by DEAE-cellulose chromatography, hydroxyapatite chromatography, and HPLC on a DEAE-5PW column. The purified substrate proteins had molecular masses of 30.5 kDa and 31 kDa. The 31-kDa protein substrate was markedly phosphorylated by casein kinase II, but only slightly by casein kinase I. The radioactive phosphate incorporated into 31-kDa substrate by casein kinase II was 0.2 mol/mol of the protein and phosphorylation occurred on both threonine and serine residues. The 30.5 kDa protein was only slightly phosphorylated by casein kinase II, but not at all by casein kinase I. 相似文献
15.
F A Gr?sser S G?ttel P Haiss B Boldyreff O G Issinger N Mueller-Lantzsch 《Biochemical and biophysical research communications》1992,186(3):1694-1701
A major in vivo phosphorylation site of the Epstein-Barr virus nuclear antigen 2 (EBNA-2) was found to be localized at the C-terminus of the protein. In vitro phosphorylation studies using casein kinase 1 (CK-1) and casein kinase 2 (CK-2) revealed that EBNA-2 is a substrate for CK-2, but not for CK-1. The CK-2 specific phosphorylation site was localized in the 140 C-terminal amino acids using a recombinant trpE-C-terminal fusion protein. In a similar experiment, the 58 N-terminal amino acids expressed as a recombinant trpE-fusion protein were not phosphorylated. Phosphorylation of a synthetic peptide corresponding to amino acids 464-476 of EBNA-2 as a substrate led to the incorporation of 0.69 mol phosphate/mol peptide indicating that only one of three potential phosphorylation sites within the peptide was modified. The most likely amino acid residues for phosphorylation by CK-2 are Ser469 and Ser470. 相似文献
16.
Alterations in C3 activation and binding caused by phosphorylation by a casein kinase released from activated human platelets. 总被引:5,自引:0,他引:5
A casein kinase released from activated human platelets phosphorylates a number of plasma proteins extracellularly, and that activation of platelets in systemic lupus erythematosus patients parallels an increase in the phosphate content of plasma proteins, including C3. The present study was undertaken to characterize this platelet protein kinase and to further elucidate the effect(s) on C3 function of phosphorylation by platelet casein kinase. The phosphate content of human plasma C3 was increased from 0.15 to 0.60 mol phosphate/mol of C3 after platelet activation in whole blood or platelet-rich plasma. The platelet casein kinase was distinct from other casein kinases in terms of its dependence on cations, inhibition by specific protein kinase inhibitors, and immunological reactivity. C3 that had been phosphorylated with platelet casein kinase was tested for its susceptibility to cleavage by trypsin or the classical and alternative pathway convertases and its binding to EAC and IgG. Phosphorylation did not affect the cleavage of C3 into C3a and C3b, but the binding of fragments from phosphorylated C3 to EAC14oxy2 cells and to IgG in purified systems and in serum was increased by 1.6-4.5 times over that of unphosphorylated C3. A covariation was seen between the enhanced binding of C3 fragments to IgG after phosphorylation and an increased ratio of glycerol/glycine binding, from 2.0 for unphosphorylated C3 to 4.9 for phosphorylated C3. The present study suggests that an overall effect of phosphorylation of C3 by platelet casein kinase is to enhance the opsonization of immune complexes. 相似文献
17.
The nonapeptide DTDSEEEIR, corresponding to amino acid residues 78-86 of calmodulin, was synthesized, and its kinetics of phosphorylation by casein kinase 2 was examined. In the presence of 4 microM polylysine, the phosphorylation rate by casein kinase 2 was 16 times greater than that of synthetic substrate peptide RRREEETEEE reported previously, and almost 1 mol of 32p was incorporated per mol of nonapeptide in 60 min at 37 degrees C. The peptide was not phosphorylated by any other protein kinase. The Thr residue was phosphorylated by casein kinase 2, but Ser was not. The Km value of casein kinase 2 for the nonapeptide was 60 microM, comparable to that of casein, and Vmax for the nonapeptide was 4 times greater than that for casein. Addition of polylysine did not affect the Km value but markedly increased Vmax. 相似文献
18.
Conformational changes in human fibrinogen after in vitro phosphorylation and their relation to fibrinogen behaviour 总被引:1,自引:0,他引:1
The far-ultraviolet circular dichroism spectra of fibrinogens phosphorylated by protein kinase C or casein kinase II indicated a conformational change corresponding to an increase in ordered secondary structure. The spectra of protein kinase A- or casein kinase I-phosphorylated fibrinogens did not differ substantially from the control. Fluorescence studies indicated changes in the tertiary structure around tryptophan residues for protein kinase A- or C-phosphorylated fibrinogens, but failed to show any such change for fibrinogen phosphorylated by either of the casein kinases. This latter result was also confirmed by circular dichroism measurements in the near-ultraviolet region. The apparent increase in ordered structure was proposed as an explanation for the slower rate of plasmin degradation seen in fibrinogens after phosphorylation by protein kinase C [6], and casein kinase II, especially as both spectral changes and plasmin degradation rate were unaffected by alkaline phosphatase. 相似文献
19.
20.
Eukaryotic initiation factor 2B: identification of multiple phosphorylation sites in the epsilon-subunit and their functions in vivo 总被引:2,自引:0,他引:2
Wang X Paulin FE Campbell LE Gomez E O'Brien K Morrice N Proud CG 《The EMBO journal》2001,20(16):4349-4359
Eukaryotic initiation factor (eIF) 2B is a heteromeric guanine nucleotide exchange factor that plays an important role in regulating mRNA translation. Here we identify multiple phosphorylation sites in the largest, catalytic, subunit (epsilon) of mammalian eIF2B. These sites are phosphorylated by four different protein kinases. Two conserved sites (Ser712/713) are phosphorylated by casein kinase 2. They lie at the extreme C-terminus and are required for the interaction of eIF2Bepsilon with its substrate, eIF2, in vivo and for eIF2B activity in vitro. Glycogen synthase kinase 3 (GSK3) is responsible for phosphorylating Ser535. This regulatory phosphorylation event requires both the fourth site (Ser539) and a distal region, which acts to recruit GSK3 to eIF2Bepsilon in vivo. The fifth site, which lies outside the catalytic domain of eIF2Bepsilon, can be phosphorylated by casein kinase 1. All five sites are phosphorylated in the eIF2B complex in vivo. 相似文献