共查询到20条相似文献,搜索用时 35 毫秒
1.
P Heldin 《Archives of biochemistry and biophysics》1987,257(2):269-275
Human fibrinogen was phosphorylated by casein kinase TS. The [32P]phosphate incorporated varied between 0.5 and 1 mol of phosphate per mole of fibrinogen. The phosphate was localized to Ser523 and Ser590 and serine and threonine residues between amino acids 259 and 268 in the A alpha-chain. In addition, Thr416 and Ser420 were phosphorylated in the gamma'-chain, which is a variant of the gamma-chain, constituting 7-10% of the gamma-chain population. The functional significance of casein kinase TS-induced phosphorylation of fibrinogen remains unknown; however, a slight but consistent increase of the turbidity in a gelation assay was observed for phosphorylated compared to unphosphorylated fibrinogen. 相似文献
2.
Partially purified phospholipid- and Ca2+-dependent protein kinase C from human placenta catalyzes the Mg-ATP-dependent phosphorylation of serine residues of purified rabbit muscle actin. Two tryptic [32P]-phosphopeptides were found on HPLC separation. Confirming the previous report by Machicao and Wieland [(1985) Curr. Top. Cell. Regul. 27, 95-105], actin is phosphorylated at serine residues by human placental membranes, and this is stimulated by insulin. In the absence of insulin trypsin treatment yielded eight [32P]phosphopeptides, two of which coincided with the ones due to protein kinase C. Insulin led to the appearance of three new [32P]phosphopeptides. These results suggest that insulin stimulates (a) serine protein kinase(s) which, like protein kinase C, is present in placental membranes. 相似文献
3.
Glucokinase, purified from rat liver, was phosphorylated to an extent of 1 mol [32P]-phosphate/mol of enzyme when incubated with [32P]ATP and protein kinase A from pig or rabbit muscle. The phosphate was bound to serine residues. K0.5 increased and Vmax decreased upon phosphorylation. The phosphate group was removed during incubation of the phosphorylated glucokinase with alkaline phosphatase. Enzymatically inactive glucokinase was not phosphorylated by the protein kinase. 相似文献
4.
Photoreceptors of vertebrate retinas contain a 33,000-dalton phosphoprotein, phosducin, which complexes with the beta, gamma subunits of the photoreceptor G-protein (guanine nucleotide-binding protein), transducin. In situ, the retinal content of phosphorylated phosducin is modulated by light in conjunction with light-triggered changes in intracellular cyclic nucleotide concentration. In vitro, phosducin is phosphorylated by either exogenous or endogenous protein kinase A. 32P-Labeled rat retina phosducin was isolated by immunoprecipitation either after phosphorylation by protein kinase A in the presence of [gamma-32P]ATP or after incubation of retinas in darkness with 32Pi. In either case, phosphoamino acid analysis showed that greater than 98% of 32P was linked to serine, with less than 2% to threonine. Two-dimensional peptide mapping showed that [32P]phosphoserine was associated with the same characteristic set of tryptic peptides. Furthermore, Cleveland peptide analysis using four different proteases showed that either sample exhibited identical patterns of phosphopeptides which were characteristic of the protease used. Identical phosphopeptide maps were also obtained from 32P-labeled bovine retina phosducin, indicating that the serine phosphorylation site for protein kinase A is conserved between rat and bovine. Edman degradation of phosphopeptides derived from 32P-labeled bovine phosducin showed that radioactive phosphate was incorporated into serine residue 73 which is located within a consensus phosphorylation sequence for protein kinase A (-R-K-M-S73(P)-). These observations are uniformly in agreement with protein kinase A being the endogenous kinase that phosphorylates phosducin in vivo. 相似文献
5.
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
6.
S Corvera P J Roach A A DePaoli-Roach M P Czech 《The Journal of biological chemistry》1988,263(7):3116-3122
Insulin caused a rapid, dose-dependent increase in the binding of 125I-insulin-like growth factor-II (IGF-II) to the surface of cultured H-35 hepatoma cells. The [32P]phosphate content of the IGF-II receptors, immunoprecipitated from extracts of H-35 cell monolayers previously incubated with [32P]phosphate for 24 h, was decreased after brief exposure of the cells to insulin. Analysis of tryptic digests of labeled IGF-II receptors by bidimensional peptide mapping revealed that the decrease in the content of [32P]phosphate occurred to varying degrees on three tryptic phosphopeptides. Thin layer electrophoresis of an acid hydrolysate of isolated IGF-II receptors revealed the presence of [32P] phosphoserine and [32P]phosphothreonine. Insulin treatment of cells caused a decrease in the labeled phosphoserine and phosphothreonine content of IGF-II receptors. The ability of a number of highly purified protein kinases (cAMP-dependent protein kinase, protein kinase C, phosphorylase kinase, and casein kinase II) to catalyze the phosphorylation of purified IGF-II receptors was examined. Casein kinase II was the only kinase capable of catalyzing the phosphorylation of the IGF-II receptor on serine and threonine residues under the conditions of our assay. Bidimensional peptide mapping revealed that the kinase catalyzed phosphorylation of the IGF-II receptor on a tryptic phosphopeptide which comigrated with the main tryptic phosphopeptide found in receptors obtained from cells labeled in vivo with [32P]phosphate. IGF-II receptors isolated by immunoadsorption from insulin-treated H-35 cells were phosphorylated in vitro by casein kinase II to a greater extent than the receptors isolated from control cells. Similarly, IGF-II receptors from plasma membranes obtained from insulin-treated adipocytes were phosphorylated by casein kinase II to a greater extent than the receptors from control adipocyte plasma membranes. Thus, the insulin-regulated phosphorylation sites on the IGF-II receptor appear to serve as substrates in vivo for casein kinase II or an enzyme with similar substrate specificity. 相似文献
7.
P O Forsberg S C Martin B Nilsson P Ekman U R Nilsson L Engstr?m 《The Journal of biological chemistry》1990,265(5):2941-2946
Complement factor C3, recently found to contain covalently bound phosphate, was phosphorylated in vitro by cyclic AMP-dependent protein kinase (protein kinase A) and Ca2(+)-activated, phospholipid-dependent protein kinase (protein kinase C). Both protein kinases phosphorylated the same serine residue(s) located in the C3a portion of the alpha-chain. In addition, protein kinase C phosphorylated the beta-chain to a lesser extent. Protein kinase A gave a maximal incorporation of 1 mol of phosphate/mol of C3 while that value with protein kinase C was 1.5 mol of phosphate/mol of C3. The velocity in pmol of [32P]phosphate/(min x unit kinase) was 20 times higher for protein kinase C than for protein kinase A although a 10 times lower ratio of protein kinase to C3 was used in the former case. The apparent Km for C3 was 2.6 microM when protein kinase C was used. The phosphorylated C3 was found to be more resistant to partial degradation by trypsin than unphosphorylated C3. It was also found that phosphorylation of C3 in the C3a portion of the alpha-chain inhibited both the classical and alternative complement activation pathways on an approximately stoichiometric basis. 相似文献
8.
Gly-Pro-Arg-Pro modifies the glutamine residues in the alpha- and gamma-chains of fibrinogen: inhibition of transglutaminase cross-linking 总被引:1,自引:0,他引:1
During blood clotting Factor XIIIa, a transglutaminase, catalyzes the formation of covalent bonds between the epsilon-amino group of lysine and the gamma-carboxamide group of peptide-bound glutamine residues between fibrin molecules. We report that glycyl-L-prolyl-L-arginyl-L-proline (GPRP), a tetrapeptide that binds to the fibrin polymerization sites (D-domain) in fibrin(ogen), inhibits transglutaminase cross-linking by modifying the glutamine residues in the alpha- and gamma-chains of fibrinogen. Purified platelet Factor XIIIa, and tissue transglutaminase from adult bovine aortic endothelial cells were used for the cross-linking studies. Gly-Pro (GP) and Gly-Pro-Gly-Gly (GPGG), peptides which do not bind to fibrinogen, had no effect on transglutaminase cross-linking. GPRP inhibited platelet Factor XIIIa-catalyzed cross-linking between the gamma-chains of the following fibrin(ogen) derivatives: fibrin monomers, fibrinogen and polymerized fibrin fibers. GPRP functioned as a reversible, noncompetitive inhibitor of Factor XIIIa-catalyzed incorporation of [3H]putrescine and [14C]methylamine into fibrinogen and Fragment D1. GPRP did not inhibit 125I-Factor XIIIa binding to polymerized fibrin, demonstrating that the Factor XIIIa binding sites on fibrin were not modified. GPRP also had no effect on Factor XIIIa cross-linking of [3H]putrescine to casein. This demonstrates that GPRP specifically modified the glutamine cross-linking sites in fibrinogen, and had no effect on either Factor XIIIa or the lysine residues in fibrinogen. GPRP also inhibited [14C]putrescine incorporation into the alpha- and gamma-chains of fibrinogen without inhibiting beta-chain incorporation, suggesting that the intermolecular cross-linking sites were selectively affected. Furthermore, GPRP inhibited tissue transglutaminase-catalyzed incorporation of [3H]putrescine into both fibrinogen and Fragment D1, without modifying [3H]putrescine incorporation into casein. GPRP also inhibited intermolecular alpha-alpha-chain cross-linking catalyzed by tissue transglutaminase. This demonstrates that the glutamine residues in the alpha-chains involved in intermolecular cross-linking are modified by GPRP. This is the first demonstration that a molecule binding to the fibrin polymerization sites on the D-domain of fibrinogen modifies the glutamine cross-linking sites on the alpha- and gamma-chains of fibrinogen. 相似文献
9.
The incorporation of [32P]phosphate into the 20 kDa myosin light chain of phorbol dibutyrate-contracted artery was slightly increased as compared to that of resting muscle. Addition of K+ to the 1-h phorbol dibutyrate-contracted artery immediately doubled the force and greatly increased the light chain phosphorylation. Two-dimensional phosphopeptide mapping of light chain from phorbol dibutyrate-contracted muscle showed distinct peptides phosphorylated on serine residues by myosin light chain kinase and protein kinase C. In addition, the peptide phosphorylated on threonine residue by protein kinase C was revealed for the first time in intact muscle. Upon addition of K+, the distribution of phosphopeptides shifted toward the myosin light chain kinase catalyzed pattern. 相似文献
10.
Epidermal growth factor receptor threonine and serine residues phosphorylated in vivo 总被引:9,自引:0,他引:9
The epidermal growth factor (EGF) receptor is regulated by EGF-stimulated autophosphorylation and by phorbol ester-stimulated, protein kinase C (Ca2+/phospholipid-dependent enzyme) mediated phosphorylation at identified sites. The EGF receptor contains additional phosphorylation sites including a prominent phosphothreonine and several phosphoserines which account for the majority of phosphate covalently bound to the receptor in vivo. We have identified three of these sites in EGF receptor purified from 32P-labeled A431 cells. The major phosphothreonine was identified as threonine 669 in the EGF receptor sequence. Phosphoserine residues were identified as serines 671 and 1046/1047 of the EGF receptor. Two other phosphoserine residues were localized to tryptic peptides containing multiple serine residues located carboxyl-terminal to the conserved protein kinase domain. The amino acid sequences surrounding the three identified phosphorylation sites are highly conserved in the EGF receptor and the protein products of the v-erb B and neu oncogenes. Analysis of predicted secondary structure of the EGF receptor reveals that all of the phosphorylation sites are located near beta turns. In A431 cells phosphorylation of the serine residues was dependent upon serum. In mouse B82 L cells transfected with a wild type human EGF receptor. EGF increased the 32P content in all tryptic phosphopeptides. A mutant EGF receptor lacking protein tyrosine kinase activity was phosphorylated only at threonine 669. Regulated phosphorylation of the EGF receptor at these threonine and serine residues may influence aspects of receptor function. 相似文献
11.
Identification of Ser(1275) and Ser(1309) as autophosphorylation sites of the human insulin receptor in intact cells 总被引:1,自引:0,他引:1
Tennagels N Telting D Parvaresch S Maassen JA Klein HW 《Biochemical and biophysical research communications》2001,282(2):387-393
In a previous report we described Ser(1275) and Ser(1309) as autophosphorylation sites of the human insulin receptor (IR) tyrosine kinase (TK) in vitro. The question remained whether the observed phosphorylation was exclusive for the in vitro activated receptor or a more general, mechanism of the activated receptor in situ. In this study, we determined the intrinsic activity of the IR to phosphorylate both serine residues in intact cells. For this purpose CHO-09 and NIH-3T3 derived cell-lines expressing the human IR were metabolically labelled with [(32)P]orthophosphate, followed by hormone stimulation of the receptor. The IR was isolated by immunoprecipitation and SDS-PAGE and subsequently analysed for serine phosphorylation by phosphopeptide mapping of HPLC-purified tryptic phosphopeptides. Activation of the IR in the intact cell appeared to result in phosphate incorporation into Ser(1275) and Ser(1309), providing strong evidence that both serine residues are phosphorylation sites of the activated receptor in intact cells. 相似文献
12.
Enhanced phosphorylation of a coated vesicle polypeptide in response to insulin stimulation of rat adipocytes 总被引:5,自引:0,他引:5
The effect of insulin to increase the cell surface concentration of various receptors is accompanied by an increase in the concentration of clathrin assembled on the plasma membrane (Corvera, S. (1990) J. Biol. Chem. 265, 2413-2416). In the present study, clathrin-coated membranes were purified from isolated adipocytes labeled isotopically with [32P]orthophosphate. Analysis of the coated vesicle preparation by polyacrylamide gel electrophoresis and autoradiography revealed the presence of a cluster of phosphopeptides of 90-100 kDa as well as other phosphorylated species of 125, 70, 58, 50, 43, and 32 kDa. Incubation of the coated vesicles in alkaline pH resulted in the elution of the majority of the phosphopeptides, suggesting that these components are part of the clathrin coat and not integral membrane proteins. A pronounced increase in the amount of phosphate incorporated into the 125-kDa species was observed in response to stimulation of labeled cells by low concentrations of insulin. Phosphoamino acid analysis of an acid hydrolysate of this band revealed that its phosphorylation occurred exclusively on serine residues. The increased serine phosphorylation of this protein was apparent after only 2 min of exposure of cells to insulin and persisted for at least 60 min. The effect of insulin to increase the cell surface concentration of receptors and the assembly of clathrin on the plasma membrane displays a similar time course. Phorbol esters or dibutyryl cyclic AMP did not mimic the effects of insulin to stimulate the incorporation of [32P]phosphate into the 125-kDa polypeptide. Phosphorylation of the 125-kDa polypeptide was not observed after incubation of purified adipocyte-coated vesicles with [gamma-32P]ATP, suggesting that the kinase responsible for this reaction may not be contained within the clathrin-coated vesicle itself. These results suggest that phosphorylation of this 125-kDa polypeptide in intact cells may play a role in the regulation of clathrin-coated membrane formation and receptor-mediated endocytosis in response to insulin. 相似文献
13.
Nucleoside diphosphate (NDP) kinase from Escherichia coli was purified to homogeneity and was crystallized. Gel filtration analysis of the purified enzyme indicated that it forms a tetramer. The enzyme was phosphorylated with [gamma-32P]ATP, and the pH stability profile of the phosphoenzyme indicated that two different amino acid residues were phosphorylated. Both a histidine residue and serine residues, including Ser-119 and Ser-121, appear to be phosphorylated. A Ser119Ala/Ser121Ala double mutant (i.e., with a Ser-to-Ala double mutation at positions 119 and 121), as well as Ser119Ala and Ser121Ala mutants, was isolated. All of these retained NDP kinase activity; also, both the Ser119Ala and Ser121Ala mutants could still be autophosphorylated. In the case of the double mutant, a slight autophosphorylation activity, which was resistant to acid treatment, was still detected, indicating that an additional minor autophosphorylation site besides His-117 exists. These results are discussed in light of the recent report of N. J. MacDonald et al. on the autophosphorylation of human NDP kinase (J. Biol. Chem. 268:25780-25789, 1993). 相似文献
14.
Studies on the incorporation of [32P]phosphate into pyruvate dehydrogenase in intact rat fat-cells. Effects of insulin. 总被引:9,自引:8,他引:1
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1. Intact rat epididymal fat-cells were incubated with 32Pi, and the intracellular proteins were separated by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. One of the separated bands of phosphorylated proteins had an apparent subunit mol.wt. of 42 000, which is the same as that of the alpha-subunit of the pyruvate dehydrogenase complex. By using a combination of subcellular fractionation, immunoprecipitation with antiserum raised against pyruvate dehydrogenase complex and two-dimensional electrophoresis it was apparent that the incorporation into alpha-subunits accounted for 35--45% of the total incorporation into this band of phosphoproteins. 2. The increase in the initial activity of pyruvate dehydrogenase that follows brief exposure of fat-cells to insulin was shown to be associated with a decrease in the steady-state incorporation of 32P into the alpha-subunits of pyruvate dehydrogenase. 3. Tryptic peptide analysis of pyruvate dehydrogenase [32P]phosphate, labelled in intact fat-cells, indicated that three serine residues on the alpha-subunit were phosphorylated, corresponding to the three sites phosphorylated when purified pig heart pyruvate dehydrogenase was incubated with [gamma-32P]ATP. The relative phosphorylation of all three serine residues appeared to be similar in 32P-labelled alpha-subunits in both control and insulin-treated fat-cells. 相似文献
15.
Dephosphorylation of cardiac myofibril C-protein by protein phosphatase 1 and protein phosphatase 2A 总被引:1,自引:0,他引:1
C-protein purified from chicken cardiac myofibrils was phosphorylated with the catalytic subunit of cAMP-dependent protein kinase to nearly 3 mol [32P]phosphate/mol C protein. Digestion of 32P-labeled C-protein with trypsin revealed that the radioactivity was nearly equally distributed in three tryptic peptides which were separated by reversed-phase HPLC. Fragmentation of 32P-labeled C-protein with CNBr showed that the isotope was incorporated at different ratios in three CNBr fragments which were separated on polyacrylamide gels in the presence of sodium dodecyl sulfate. Phosphorylation was present in both serine and threonine residues. Incubation of 32P-labeled C-protein with the catalytic subunit of protein phosphatase 1 or 2A rapidly removed 30-40% of the [32P]phosphate. The major site(s) dephosphorylated by either one of the phosphatases was a phosphothreonine residue(s) apparently located on the same tryptic peptide and on the same CNBr fragment. CNBr fragmentation also revealed a minor phosphorylation site which was dephosphorylated by either of the phosphatases. Increasing the incubation period or the phosphatase concentration did not result in any further dephosphorylation of C-protein by phosphatase 1, but phosphatase 2A at high concentrations could completely dephosphorylate C-protein. These results demonstrate that C-protein phosphorylated with cAMP-dependent protein kinase can be dephosphorylated by protein phosphatases 1 and 2A. It is suggested that the enzyme responsible for dephosphorylation of C-protein in vivo is phosphatase 2A. 相似文献
16.
Masaru Suzuki Yasushi Sakamoto Kunio Kitamura Kohji Fukunaga† Hideyuki Yamamoto† Eishichi Miyamoto† Keiichi Uyemura‡ 《Journal of neurochemistry》1990,55(6):1966-1971
The P0 protein in mammalian PNS myelin is known to undergo several posttranslational modifications, such as glycosylation, acylation, sulfation, and phosphorylation. Phosphorylation of purified P0 protein in vitro was studied comparatively using three enzymes, i.e., calcium/phospholipid-dependent protein kinase (protein kinase C), calcium/calmodulin-dependent protein kinase II (CaM kinase II), and the catalytic subunit of cyclic AMP-dependent protein kinase (A kinase). The phosphorylation of P0 protein by CaM kinase II was the greatest, followed by that by protein kinase C; phosphorylation by A kinase, however, was much lower. In order to identify phosphorylation sites, P0 protein was phosphorylated with [32P]ATP and each kinase and then digested with lysylendopeptidase. The resulting phosphopeptides were isolated by HPLC. Subsequent amino acid sequence analysis and comparison with the known sequence of P0 protein revealed that Ser181 and Ser204 were strongly phosphorylated by both protein kinase C and CaM kinase II. In addition, Ser214 was also phosphorylated by protein kinase C, but not by CaM kinase II. Because all of these sites are located in the cytoplasmic domain of P0 protein, phosphorylation may be important for maintenance of the major dense line of PNS myelin. 相似文献
17.
cAMP-dependent protein kinase stimulates epidermal growth factor-dependent phosphorylation of epidermal growth factor receptors 总被引:6,自引:0,他引:6
Epidermal growth factor (EGF)-dependent transfer of radiolabeled phosphate from [gamma-32P]ATP to 160-kDa EGF receptor solubilized from human epidermoid carcinoma A431 cell surface membranes was stimulated up to 3-fold by addition of 3',5'-cAMP and purified cAMP-dependent protein kinase. Phosphorylation of EGF receptors was stimulated to the same extent when cAMP-dependent protein kinase catalytic subunit was substituted for 3',5'-cAMP and cAMP-dependent protein kinase. Phosphoamino acid analysis revealed that the extent of phosphorylation of EGF receptor at tyrosine residues was the same regardless of whether cAMP-dependent protein kinase catalytic subunit was present in or omitted from the system. Increased EGF receptor phosphorylation occurring in response to cAMP-dependent protein kinase catalytic subunit was accounted for by phosphorylation at serine or threonine residues. In samples phosphorylated in the presence of cAMP-dependent protein kinase catalytic subunit, phosphate was present in tyrosine, serine, and threonine in a ratio of 32:60:8. Two-dimensional mapping of radiolabeled phosphopeptides produced from EGF receptors by digestion with trypsin revealed the generation of one additional major phosphoserine-containing peptide when cAMP-dependent protein kinase was present with EGF in the EGF receptor kinase system. Degradation of 160-kDa EGF receptors to a 145-kDa form by purified Ca2+-activated neutral protease produced a 145-kDa fragment with phosphoserine content increased over that present initially in the 160-kDa precursor. 相似文献
18.
Marie-Christine Ralet Dominique Fouques Joëlle Leonil Daniel Molle Jean-Claude Meunier 《The protein journal》1999,18(3):315-323
Protein kinase CK2 purified from the yeast Yarrowia lipolytica was used to phosphorylate soybean β-conglycinin α subunit. CK2 is known to phosphorylate serines and threonines in the consensus sequence Ser/Thr-X-X-Glu/Asp/SerP/TyrP. β-Conglycinin α subunit (68 kDa) presents seven consensus sequences, but only 0.5–1 mol P/mol α subunit was incorporated by CK2. [32P]Phosphorylated β-conglycinin α subunit was cleaved either by cyanogen bromide or by trypsin. 32P was incorporated into the largest cyanogen bromide fragment only (50 kDa, N-terminal) and only two radiolabeled zones were detected after HPLC of the trypsic digest. The corresponding phosphorylated zones were collected and further analyzed by RP-HPLC coupled to electrospray ionization mass spectrometry (LC-ESMS). Two phosphorylated sites, Ser 75 and Ser 117, were determined after MS-MS analysis of three phosphopeptides identified as 70–89, 116–126, and 116–127 sequences. Over the seven consensus sequences of β-conglycinin α subunit, Ser 75 is the only one which was phosphorylated. Ser 117 was phosphorylated although it is not an expected phosphorylation site according to the canonical consensus sequence criteria as there is no acidic determinant at the +3 position. Both Ser 75 and Ser 117 are located inside very acidic sequences, by contrast with the other unphosphorylated potential sites. 相似文献
19.
Identification of serine 24 as the unique site on the transferrin receptor phosphorylated by protein kinase C 总被引:5,自引:0,他引:5
R J Davis G L Johnson D J Kelleher J K Anderson J E Mole M P Czech 《The Journal of biological chemistry》1986,261(19):9034-9041
Addition of tumor-promoting phorbol diesters to [32P]phosphate-labeled A431 human epidermoid carcinoma cells caused an increase in the phosphorylation state of the transferrin receptor. The A431 cell transferrin receptor was also found to be a substrate for protein kinase C in vitro. Tryptic phosphopeptide mapping of the transferrin receptor resolved the same two phosphopeptides (X and Y) after either protein kinase C phosphorylation in vitro or treatment of labeled A431 cells with phorbol diesters. [32P]Phosphoserine was the only labeled phosphoamino acid detected. Phosphopeptide X was shown to be an incomplete tryptic digestion product which could be further digested with trypsin to generate the limit tryptic phosphopeptide (Y). Radiosequence analysis of [32P]phosphopeptide Y demonstrated that the [32P]phosphoserine was the second residue from amino terminus of the peptide. This receptor phosphopeptide was found to co-migrate with the synthetic peptide Phe-Ser(P)-Leu-Ala-Arg (where Ser(P) is phosphoserine) during reverse-phase high pressure liquid chromatography and two-dimensional thin layer electrophoresis and chromatography. The peptide Phe-Ser(P)-Leu-Ala-Arg is an expected tryptic fragment of the cytoplasmic domain of the transferrin receptor corresponding to residues 23-27. We conclude that the major site of protein kinase C phosphorylation of the transferrin receptor in vivo and in vitro is serine 24. This phosphorylation site is located within the intracellular domain of the transferrin receptor, 38 residues away from the predicted transmembrane domain. 相似文献
20.
The phosphorylation sites of polyomavirus large T antigen from infected or transformed cells were investigated. Tryptic digestion of large T antigen from infected, 32Pi-labeled cells revealed seven major phosphopeptides. Five of these were phosphorylated only at serine residues, and two were phosphorylated at serine and threonine residues. The overall ratio of phosphoserine to phosphothreonine was 6:1. The transformed cell line B4 expressed two polyomavirus-specific phosphoproteins: large T antigen, which was only weakly phosphorylated, and a truncated form of large T antigen of 34,000 molecular weight which was heavily phosphorylated. Both showed phosphorylation patterns similar to that of large T antigen from infected cells. Peptide analyses of large T antigens encoded by the deletion mutants dl8 and dl23 or of specific fragments of wild-type large T antigen indicated that the phosphorylation sites are located in an amino-terminal region upstream of residue 194. The amino acid composition of the phosphopeptides as revealed by differential labeling with various amino acids indicated that several phosphopeptides contain overlapping sequences and that all phosphorylation sites are located in four tryptic peptides derived from a region between Met71 and Arg191. Two of the potential phosphorylation sites were identified as Ser81 and Thr187. The possible role of this modification of large T antigen is discussed. 相似文献