共查询到20条相似文献,搜索用时 17 毫秒
1.
Distinct specificities of repressible acid phosphatase from yeast toward phosphoseryl and phosphotyrosyl phosphopeptides 总被引:1,自引:0,他引:1
A Donella-Deana K Lopandic S Barbaric L A Pinna 《Biochemical and biophysical research communications》1986,139(3):1202-1209
By using [32P]-labeled phosphoaminoacids it has been shown that, at mu molar range concentrations, Tyr-32P but neither Ser-32P nor Thr-32P can be significantly dephosphorylated by highly purified repressible acid phosphatase from Saccharomyces cerevisiae. The phosphopeptide Arg-Arg-Ala-Ser(32P)-Val-Ala however, reproducing the phosphorylation site of pyruvate kinase and previously phosphorylated by cAMP-dependent protein kinase, can be very readily dephosphorylated with favourable kinetic constants (Km 0.28 microM, Vmax = 62 units/micrograms) while its derivatives Ala-Ser(32P)-Val-Ala, Arg-Arg-Ala-Thr(32P)-Val-Ala, Arg-Arg-Pro-Ser(32P)-Pro-Ala as well as other peptides and protein substrates phosphorylated by either protein kinase-C or casein kinase-2 are either unaffected or very slowly dephosphorylated by the phosphatase. Conversely Tyr-32P containing angiotensin, poly (Glu, Tyr) 4:1 and the phosphopeptide Asp-Ala-Glu-Tyr(32P)-Ala-Ala-Arg-Arg-Arg-Gly are all dephosphorylated with kinetic constants comparable to those of free phosphotyrosine (Km 0.2-1 microM; Vmax = 4-10 units/micrograms). It is proposed that, while acid phosphatase exhibits a broad specificity toward phosphotyrosine and phosphotyrosyl polypeptides, it is highly selective toward phosphoseryl sites fulfilling definite structural requirements which are reminiscent of those determining phosphorylation by cAMP-dependent protein kinase. 相似文献
2.
This study determined whether all protein kinase A (PKA) and protein kinase C (PKC) phosphorylation sites on the alpha4 subunit of rat alpha4beta2 neuronal nicotinic receptors could be localized to the M3/M4 cytoplasmic domain of the protein, and investigated specific amino acid substrates for the kinases through two-dimensional phosphopeptide mapping and site-directed mutagenesis. Experiments were conducted using alpha4beta2 receptors expressed in Xenopus oocytes and a fusion protein corresponding to the M3/M4 cytoplasmic domain of alpha4 (alpha4(333-594) ). When oocytes expressing alpha4beta2 receptors were incubated with [(32) P]orthophosphate in order to label endogenous ATP stores, phosphorylation of alpha4 subunits was evident. Incubation of either immunoprecipitated receptors or the fusion protein with [(32) P]ATP and either PKA or PKC followed by trypsinization of the samples demonstrated that the kinases phosphorylated alpha4 subunits on multiple phosphopeptides, and that the phosphorylated full-length alpha4 protein and fusion protein produced identical phosphopeptide maps. Site-directed mutagenesis of Ser365, Ser472 and Ser491 to alanines in the fusion protein eliminated phosphopeptides phosphorylated by PKA, but not by PKC. Other mutations investigated, Ser470, Ser493, Ser517 and Ser590, did not alter the phosphopeptide maps. Results indicate that Ser365, Ser472 and Ser491 on neuronal nicotinic receptor alpha4 subunits are phosphorylated by PKA and are likely to represent post-translational regulatory sites on the receptor. 相似文献
3.
The effects of N6,O2-dibutyryl-adenosine 3',5'-monophosphate (Bt2cAMP) and sodium fluoride on the phosphorylation of ribosomal proteins S6 and on protein synthesis were examined. Rabbit reticulocytes were incubated in a nutritional medium containing 32Pi in the presence and absence of Bt2cAMP (1mM) and 3-isobutyl-1-methyl-xanthine (1mM). In the control cells, four phosphorylated derivatives of S6 were observed, with most of the radioactivity in the monophosphorylated form. Upon addition of cyclic nucleotide, a twofold increase in the phosphorylation of ribosomal protein S6 was observed. This was accompanied by an increase of radioactive phosphate in the diphosphorylated derivative. No alteration in protein synthesis was observed upon addition of cAMP and analogues of cAMP in conjunction with 3-isobutyl-1-methyl-xanthine or theophylline. The effects of sodium fluoride on phosphorylation of S6 and on protein synthesis were examined also. At 5 mM sodium fluoride, protein synthesis was inhibited by 85%. A 2.5-fold increase in the phosphorylation of ribosomal protein S6 was observed with an accumulation of 32Pi in the diphosphorylated, triphosphorylated and tetraphosphorylated derivatives. Inhibition of protein synthesis coincided with an increase in the more highly phosphorylated derivatives, whereas an increase of radioactive phosphate in the diphosphorylated derivative could not be correlated with an alteration in globin synthesis. 相似文献
4.
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
5.
Phorbol esters stimulate phosphorylation of eukaryotic initiation factors 3, 4B, and 4F 总被引:11,自引:0,他引:11
Eukaryotic initiation factor (eIF) 4F, a multiprotein cap binding complex, was isolated by m7 GTP-Sepharose affinity chromatography from rabbit reticulocytes incubated with [32P]orthophosphate. Following treatment of reticulocytes with phorbol 12-myristate 13-acetate (PMA) for 30 min, stimulation of phosphorylation of both the p25 and p220 subunits was observed (2.5-5-fold). Two variants were observed for p25 in the absence and presence of PMA when analyzed by two-dimensional gel electrophoresis. Only the more acidic of these was phosphorylated, with the level of phosphorylation increased upon PMA treatment. One main variant was observed for p220; following PMA stimulation, in addition to increased labeling of this variant, two more acidic phosphorylated variants were observed. Low levels of eIF-3 and -4B were associated with purified eIF-4F, and PMA treatment stimulated phosphorylation of eIF-3 (p170) by 2-4-fold and eIF-4B by 1.5-2.5 fold. Two-dimensional phosphopeptide mapping of p25 phosphorylated in the absence or presence of PMA generated a single tryptic phosphopeptide, suggesting a single phosphorylation site. A more complex phosphopeptide map was observed with p220 subunit. The maps for both subunits contained the same phosphopeptides as those obtained when eIF-4F was phosphorylated in vitro by the Ca2+/phospholipid-dependent protein kinase, indicating this protein kinase directly modulated eIF-4F in response to PMA. 相似文献
6.
Stimulation of protein phosphorylation during fertilization-induced maturation of Urechis caupo oocytes 总被引:2,自引:0,他引:2
Protein phosphorylation was studied during fertilization of Urechis caupo oocytes both in vivo, by measuring [32P]phosphate incorporation into 32P preloaded oocytes and in vitro, by measuring endogenous protein kinase and phosphatase activities in homogenates. During fertilization (and maturation) the rate of protein phosphorylation is dramatically increased. No change in the [32P]phosphate uptake, or the nucleotide levels was observed at fertilization, so the increase cannot be attributed to changes in substrate availability. In vitro enzyme assays showed changes in protein kinase activity which approximately mirrored the changes in the in vivo phosphorylation pattern. As there were no changes in protein phosphatase activity, these results suggest the phosphorylation change results from an increase in protein kinase activity. The pattern of change, investigated by SDS-polyacrylamide gel electrophoresis, shows that proteins that were phosphorylated in the unfertilized egg become phosphorylated to a greater degree after fertilization. One protein (48 kd) undergoes an increase followed by a decrease of its phosphorylation level. 相似文献
7.
Threonine 1336 of the human insulin receptor is a major target for phosphorylation by protein kinase C 总被引:6,自引:0,他引:6
The ability of tumor-promoting phorbol diesters to inhibit both insulin receptor tyrosine kinase activity and its intracellular signaling correlates with the phosphorylation of the insulin receptor beta subunit on serine and threonine residues. In the present studies, mouse 3T3 fibroblasts transfected with a human insulin receptor cDNA and expressing greater than one million of these receptors per cell were labeled with [32P]phosphate and treated with or without 100 nM 4 beta-phorbol 12 beta-myristate 13 alpha-acetate (PMA). Phosphorylated insulin receptors were immunoprecipitated and digested with trypsin. Alternatively, insulin receptors affinity purified from human term placenta were phosphorylated by protein kinase C prior to trypsin digestion of the 32P-labeled beta subunit. Analysis of the tryptic phosphopeptides from both the in vivo and in vitro labeled receptors by reversed-phase HPLC and two-dimensional thin-layer separation revealed that PMA and protein kinase C enhanced the phosphorylation of a peptide with identical chromatographic properties. Partial hydrolysis and radiosequence analysis of the phosphopeptide derived from insulin receptor phosphorylated by protein kinase C indicated that the phosphorylation of this tryptic peptide occurred specifically on a threonine, three amino acids from the amino terminus of the tryptic fragment. Comparison of these data with the known, deduced receptor sequence suggested that the receptor-derived tryptic phosphopeptide might be Ile-Leu-Thr(P)-Leu-Pro-Arg. Comigration of a phosphorylated synthetic peptide containing this sequence with the receptor-derived phosphopeptide confirmed the identity of the tryptic fragment. The phosphorylation site corresponds to threonine 1336 in the human insulin receptor beta subunit.(ABSTRACT TRUNCATED AT 250 WORDS) 相似文献
8.
The effect of insulin on intracellular ph and ribosomal protein S6 phosphorylation in oocytes of Xenopus laevis 总被引:14,自引:0,他引:14
Both insulin and progesterone are capable of stimulating germinal vesicle breakdown (GVBD) of large, Stage VI oocytes of Xenopus laevis. Numerous studies have shown an increase in intracellular pH (pHi) and ribosomal protein S6 phosphorylation prior to GVBD in oocytes treated with progesterone. In this study the effect of insulin and progesterone on pHi and S6 phosphorylation was compared. Both hormones increased pHi and S6 phosphorylation to similar levels and the time course of pHi change was the same for both hormones. Half-maximal effects of insulin were observed at 7 X 10(-8) M concentrations. In the presence of 1 nM cholera toxin, the ability of progesterone to induce these two responses was inhibited while the action of insulin was unaffected. However, GVBD induced by either hormone was blocked by cholera toxin. In small, Stage IV oocytes that do not undergo GVBD in response to either progesterone or insulin, a partial increase in pHi without S6 phosphorylation occurred in response to progesterone but both events occurred in response to insulin. These results suggest that the inability of Stage IV oocytes to undergo GVBD in response to hormone is not due to a failure to increase pHi or phosphorylate S6. The results in this paper also indicate that these events are regulated differently by insulin and progesterone in Xenopus oocytes. 相似文献
9.
Haug LS Jensen V Hvalby O Walaas SI Ostvold AC 《The Journal of biological chemistry》1999,274(11):7467-7473
We have examined cyclic nucleotide-regulated phosphorylation of the neuronal type I inositol 1,4,5-trisphosphate (IP3) receptor immunopurified from rat cerebellar membranes in vitro and in rat cerebellar slices in situ. The isolated IP3 receptor protein was phosphorylated by both cAMP- and cGMP-dependent protein kinases on two distinct sites as determined by thermolytic phosphopeptide mapping, phosphopeptide 1, representing Ser-1589, and phosphopeptide 2, representing Ser-1756 in the rat protein (Ferris, C. D., Cameron, A. M., Bredt, D. S., Huganir, R. L., and Snyder, S. H. (1991) Biochem. Biophys. Res. Commun. 175, 192-198). Phosphopeptide maps show that cAMP-dependent protein kinase (PKA) labeled both sites with the same time course and same stoichiometry, whereas cGMP-dependent protein kinase (PKG) phosphorylated Ser-1756 with a higher velocity and a higher stoichiometry than Ser-1589. Synthetic decapeptides corresponding to the two phosphorylation sites (peptide 1, AARRDSVLAA (Ser-1589), and peptide 2, SGRRESLTSF (Ser-1756)) were used to determine kinetic constants for the phosphorylation by PKG and PKA, and the catalytic efficiencies were in agreement with the results obtained by in vitro phosphorylation of the intact protein. In cerebellar slices prelabeled with [32P]orthophosphate, activation of endogenous kinases by incubation in the presence of cAMP/cGMP analogues and specific inhibitors of PKG and PKA induced in both cases a 3-fold increase in phosphorylation of the IP3 receptor. Thermolytic phosphopeptide mapping of in situ labeled IP3 receptor by PKA showed labeling on the same sites (Ser-1589 and Ser-1756) as in vitro. In contrast to the findings in vitro, PKG preferentially phosphorylated Ser-1589 in situ. Because both PKG and the IP3 receptor are specifically enriched in cerebellar Purkinje cells, PKG may be an important IP3 receptor regulator in vivo. 相似文献
10.
Activation of ribosomal protein S6 phosphorylation during meiotic maturation of Xenopus laevis oocytes: in vitro ordered appearance of S6 phosphopeptides. 总被引:4,自引:1,他引:3
下载免费PDF全文
![点击此处可从《The EMBO journal》网站下载免费的PDF全文](/ch/ext_images/free.gif)
During meiotic maturation of Xenopus laevis stage 6 oocytes into unfertilized eggs, 40S ribosomal protein S6 undergoes multiple phosphorylation. Extracts prepared from unfertilized eggs are up to 10-fold more efficient in phosphorylating S6 than those prepared from immature oocytes. When analyzed by DEAE chromatography the S6 kinase activity elutes as a single peak. If extracts from unfertilized eggs are prepared in the absence of beta-glycerol phosphate, a putative phosphatase inhibitor, there is a severe reduction in recovered S6 kinase activity. Under optimal conditions, incubation of unfertilized egg extracts with 40S ribosomes in the presence of ATP leads to the average incorporation of 3.5 mol of phosphate/mol of S6. Prior incubation of these extracts with the cAMP-dependent protein kinase inhibitor does not inhibit S6 phosphorylation indicating that another kinase is responsible. Analysis of the in vitro phosphorylated peptides demonstrates that they migrate to the equivalent position of those observed previously in vivo and in vitro. More strikingly, if each of the increasingly phosphorylated derivatives of S6 is analyzed independently, it is found that the phosphopeptides appear in a specific order. 相似文献
11.
The [32P]phosphoproteins and [35S]thiophosphoproteins were analyzed by electrophoresis and autoradiography after microinjection of [gamma-32P]ATP or of [35S]ATP-gamma-S into living Xenopus oocytes. The level of 32P incorporation into a 20-kDA protein was decreased following progesterone treatment (between 1 and 2 hr). This 20-kDa protein was partially thiophosphorylated in vivo by [35S]ATP-gamma-S. Furthermore it was found that this phosphoprotein was partially purified by TCA (1%) extraction and heat treatment. Microinjection of the C-subunit of cAMP-dependent protein kinase (0.6 to 1.2 pmole) inhibited maturation and provoked an increase in the level of phosphorylation of the 20-kDa protein and of a 32-kDa protein, indicating that both proteins were in vivo substrates (directly or indirectly) for cAMP-dependent protein kinase. When inhibitor-1 of protein phosphatase-1 was microinjected (5 to 10 pmole per oocyte) meiotic maturation was inhibited and the level of phosphorylation of the 32-kDa protein was increased; the same result was obtained following ATP-gamma-S (1 mM) microinjection. Altogether these results suggest that a 20-kDa phosphoprotein, whose level of phosphorylation is decreased by progesterone, could be involved in the regulation of maturation by lowering the level phosphorylation of a 32-kDa phosphoprotein. An attractive hypothesis would be that the 20-kDa phosphoprotein is an inhibitor of protein phosphatase-1. 相似文献
12.
Site-specific phosphorylation of tyrosine hydroxylase after KCl depolarization and nerve growth factor treatment of PC12 cells 总被引:3,自引:0,他引:3
The phosphorylation and activation of tyrosine hydroxylase was examined in PC12 cells following depolarization with KCl or treatment with nerve growth factor. Both treatments activate tyrosine hydroxylase (TH) and increase enzyme phosphorylation. Site-specific analysis of the tryptic phosphopeptides of TH isolated from [32P]phosphate-labeled PC12 cells demonstrated that the major phosphorylated peptide (termed "H25") did not contain any of the previously reported phosphorylation sites. Phosphoamino acid analysis of this peptide demonstrated that the phosphorylated residue was a serine. Synthetic tryptic peptides containing putative phosphorylation sites were prepared, and subjected to high performance liquid chromatography analysis and isoelectric focusing. The tryptic phosphopeptide containing serine 31 comigrated with the H25 peptide during both of these analytical techniques. The tryptic phosphopeptide produced by the phosphorylation of tyrosine hydroxylase by the recently discovered proline-directed protein kinase and the phosphorylated synthetic phosphopeptide TH2-12 are clearly separated from H25 by this analysis. We conclude that serine 31 is phosphorylated during KCl depolarization and nerve growth factor treatment of PC12 cells and that this phosphorylation is responsible for the activation of tyrosine hydroxylase. Since this site is not located in a sequence selective for any of the "classical" protein kinases, we suggest that a novel protein kinase may be responsible for the phosphorylation of this site. Since serine 31 has a proline residue on the carboxyl-terminal side, the possibility that this kinase may be related to the recently reported proline-directed protein kinase is discussed. Other sites that are also phosphorylated on TH during KCl depolarization include serine 19, which is known to be phosphorylated by calmodulin-dependent protein kinase II. A schematic model for the regulation of tyrosine hydroxylase activity by phosphorylation of the NH2-terminal regulatory domain is presented. 相似文献
13.
The ribosomal protein S6 in exocrine cells is phosphorylated during stimulation of exocytosis by cAMP-dependent or calcium-dependent agonists. Under both conditions the same tryptic S6 phosphopeptides (termed A, B, and C) were found [Padel, Kruppa, Jahn & S?ling (1983) FEBS Lett. 159, 112-118]. Studies have now been made of the phosphorylation pattern of protein S6 from purified guinea pig parotid ribosomes following in vitro phosphorylation with calmodulin-dependent, phospholipid-dependent, and cAMP-dependent protein kinases. Only the phospholipid-dependent enzyme led to the phosphorylation of peptides A, B, and C, while the cAMP-dependent enzyme phosphorylated only peptides A and C, and the calmodulin-dependent enzyme did not phosphorylate any of the phosphopeptides found in S6 from unstimulated or stimulated intact cells. Guinea pig parotid microsomes contain substantial phospholipid-dependent protein kinase activity. Stimulation of intact parotid glands with tetradecanoylphorbol acetate led to a significant phosphorylation of S6 and a similar tryptic S6 phosphopeptide pattern as seen with carbamoylcholine. It is concluded that activation of phospholipid-dependent protein kinase is responsible for the phosphorylation of protein S6 during stimulation with calcium-dependent and cAMP-dependent secretagogues. 相似文献
14.
Amino acid sequence of the phosphorylation site of rat liver 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase 总被引:5,自引:0,他引:5
K J Murray M R El-Maghrabi P D Kountz T J Lukas T R Soderling S J Pilkis 《The Journal of biological chemistry》1984,259(12):7673-7681
6-Phosphofructo-2-kinase/fructose-2,6-bisphosphatase from rat liver was phosphorylated by cyclic AMP-dependent protein kinase and [gamma-32P]ATP. Treatment of the 32P-labeled enzyme with thermolysin removed all of the radioactivity from the enzyme core and produced a single labeled peptide. The phosphopeptide was purified by ion exchange chromatography, gel filtration, and reverse phase high pressure liquid chromatography. The sequence of the 12-amino acid peptide was found to be Val-Leu-Gln-Arg-Arg-Arg-Gly-Ser(P)-Ser-Ile-Pro-Gln. Correlation of the extent of phosphorylation with activity showed that a 50% decrease in the ratio of kinase activity to bisphosphate activity occurred when only 0.25 mol of phosphate was incorporated per mol of enzyme subunit, and maximal changes occurred with 0.7 mol incorporated. The kinetics of cyclic AMP-dependent protein kinase-catalyzed phosphorylation of the native bifunctional enzyme was compared with that of other rat liver protein substrates. The Km for 6-phosphofructo-2-kinase/fructose-2, 6-bisphosphatase (10 microM) was less than that for rat liver pyruvate kinase (39 microM), fructose-1,6-bisphosphatase (222 microM), and 6- phosphofructose -1-kinase (230 microM). Comparison of the initial rate of phosphorylation of a number of protein substrates of the cyclic AMP-dependent protein kinase revealed that only skeletal muscle phosphorylase kinase was phosphorylated more rapidly than the bifunctional enzyme. Skeletal muscle glycogen synthase, heart regulatory subunit of cyclic AMP-dependent protein kinase, and liver pyruvate kinase were phosphorylated at rates nearly equal to that of 6-phosphofructo-2-kinase/fructose-2, 6-bisphosphatase, while phosphorylation of fructose-1,6-bisphosphatase and 6-phosphofructo-1-kinase was barely detectable. Phosphorylation of 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase was not catalyzed by any other protein kinase tested. These results are consistent with a primary role of the cyclic AMP-dependent protein kinase in regulation of the enzyme in intact liver. 相似文献
15.
The 70-kDa neurofilament protein subunit (NF-L) is phosphorylated in vivo on at least three sites (L1 to L3) (Sihag, R. K. and Nixon, R. A. (1989) J. Biol. Chem. 264, 457-464). The turnover of phosphate groups on NF-L during axonal transport was determined after the neurofilaments in retinal ganglion cells were phosphorylated in vivo by injecting mice intravitreally with [32P]orthophosphate. Two-dimensional phosphopeptide maps of NF-L from optic axons of mice 10 to 90 h after injection showed that radiolabel decreased faster from peptides L2 and L3 than from L1 as neurofilaments were transported. To identify phosphorylation sites on peptide L2, axonal cytoskeletons were phosphorylated by protein kinase A in the presence of heparin. After the isolated NF-L subunits were digested with alpha-chymotrypsin, 32P-peptides were separated by high performance liquid chromatography on a reverse-phase C8 column. Two-dimensional peptide mapping showed that the alpha-chymotrypsin 32P-peptide accepting most of the phosphates from protein kinase A migrated identically with the in vivo-labeled phosphopeptide L2. The sequence of this peptide (S-V-R-R-S-Y) analyzed by automated Edman degradation corresponded to amino acid residues 51-56 of the NF-L sequence. A synthetic 13-mer (S-L-S-V-R-R-S-Y-S-S-S-S-G) corresponding to amino acid residues 49-61 of NF-L was also phosphorylated by protein kinase A. alpha-Chymotryptic digestion of the 13-mer generated a peptide which contained most of the phosphates and co-migrated with the phosphopeptide L2 on two-dimensional phosphopeptide maps. Edman degradation of the phosphorylated 13-mer identified serine residue 55 which is located within a consensus phosphorylation sequence for protein kinase A as the major site of phosphorylation. Since protein kinase A-mediated phosphorylation influences intermediate filament assembly/disassembly in vitro, we propose that the phosphopeptide L2 region is a neurofilament-assembly domain and that the cycle of phosphorylation and dephosphorylation of Ser-55 on NF-L, which occurs relatively early after subunit synthesis in vivo, regulaaes a step in neurofilament assembly or initial interactions during axonal transport. 相似文献
16.
An increase in intracellular pH (pHi) and ribosomal protein S6 phosphorylation during Xenopus oocyte maturation has been reported by several laboratories. In this paper, the question of whether the pHi increase is necessary to induce S6 phosphorylation, an increase in protein synthesis, or germinal vesicle breakdown (GVBD) was assessed using sodium-free medium and the putative Na/H exchange blocker amiloride. Sodium-free medium decreased basal pHi by 0.3 unit and prevented increases in pHi in response to both insulin and progesterone, but S6 phosphorylation occurred normally with both hormones. GVBD occurred normally in sodium-free medium in response to progesterone, but the effect of insulin was reduced by 60%. In sodium-containing medium, amiloride inhibited GVBD and prevented insulin or progesterone-induced increases in pHi but the hormone-induced increase in S6 phosphorylation was unaffected. In the absence of sodium, amiloride inhibited GVBD but did not affect pHi, indicating that amiloride inhibits GVBD by a pHi-independent mechanism. Both progesterone and insulin increased protein synthesis in oocytes by 35%, and amiloride inhibited basal protein synthesis but not the increase with hormone. In the presence of cholera toxin, protein synthesis increases with insulin were inhibited but increased S6 phosphorylation was unaffected. Priming of animals with pregnant mare's serum gonadotropin prior to oocyte isolation reduced the time required for progesterone-induced GVBD, and increased the synchrony of GVBD of the population. Priming also increased oocyte basal pHi and basal protein synthesis as well as the magnitude of the increase in protein synthesis with progesterone but had no effect on S6 phosphorylation. The results indicate that in Xenopus oocytes increased pHi is not necessary for increased S6 phosphorylation, increased protein synthesis, or GVBD in response to insulin or progesterone nor is increased S6 phosphorylation sufficient for GVBD or increased protein synthesis. 相似文献
17.
H Tokumitsu A Mizutani M Watanabe H Hidaka 《Archives of biochemistry and biophysics》1991,286(1):94-98
We found a novel 81-kDa acidic protein (ACAMP-81) in the bovine brain membrane fraction, which bound to calmodulin in a Ca(2+)-dependent manner. The present study reveals physicochemical properties and phosphorylation of this protein with various protein kinases in vitro. The Stokes radius and sedimentation coefficient were calculated to be 52 A and 2.05 S, respectively, suggesting that the structure of ACAMP-81 is highly elongated. Purified Ca2+/phospholipid-dependent protein kinase (protein kinase C), cAMP-dependent protein kinase, and Ca2+/calmodulin-dependent protein kinase II (Ca2+/CaM kinase II) catalyzed the incorporation of 1.46, 0.72, and 0.44 mol of phosphate/mol of ACAMP-81, respectively. The amino acid residues of ACAMP-81 phosphorylated by either protein kinase C or cAMP-dependent protein kinase were almost exclusively on serine. Sequential phosphorylation of ACAMP-81 by cAMP-dependent protein kinase and protein kinase C resulted in the additional incorporation of 1.15 mol of [32P]phosphate into ACAMP-81. Comparison of phosphopeptide maps of ACAMP-81 phosphorylated by each kinase revealed that there are two classes of phosphorylatable polypeptide, one is phosphorylatable by both protein kinases which contained two polypeptides and the others are specific sites for protein kinase C. 相似文献
18.
To begin to understand the regulation and roles of neurofilament phosphorylation, we localized the phosphorylated domains on the 140-145-kDa neurofilament subunit (NF-M) and identified the protein kinases that may specifically phosphorylate the sites within these domains in vivo. Mouse retinal ganglion cells were labeled in vivo by injecting mice intravitreally with [32P]orthophosphate, and neurofilament-enriched fractions were obtained from the optic axons. Two-dimensional phosphopeptide map analysis of NF-M after digestion with alpha-chymotrypsin and trypsin revealed seven major (M8-M14) and at least eight minor (M1-M7 and M15) phosphopeptides. Two-dimensional phosphopeptide map analyses of NF-M phosphorylated in vitro by individual purified or endogenous axonal cytoskeleton-associated protein kinases showed that five peptides (M9-M13) were substrates for the heparin-sensitive second messenger-independent protein kinase(s). Protein kinase A and/or protein kinase C phosphorylated eight other peptides (M1-M8). Two alpha-chymotryptic peptides (C1 and C2) that were phosphorylated by protein kinase A but not by the endogenous independent kinase(s) were isolated by high performance liquid chromatography on a reverse-phase C8 column. Partial sequence analysis of peptides C1 (S R V S G P S ...) and C2 (S R G S P S T V S ...) showed that the peptides were localized on the head domain of NF-M at 25 and 41 residues from the amino terminus, respectively. Tryptic digest of peptide C1 (less than 12 kDa) generated the phosphopeptides M1-M6. Peptide C2 was a breakdown product of peptide C1. Since the polypeptide sites targeted by second messenger-independent kinase(s) associated with neurofilaments are localized on the carboxyl-terminal domain, separate aspects of NF-M function appear to be regulated by separate kinase systems that selectively phosphorylate head or tail domains of the polypeptide. 相似文献
19.
In the posterior silk gland of Bombyx mori, ribosomal protein S1, homologous to S6 in mammals, is partially phosphorylated in a normally fed animal. Before the first meal of the fifth larval instar, S1 is completely dephosphorylated. Likewise, starvation induces rapid dephosphorylation of the protein in both free and membrane-bound ribosomes. Upon refeeding after 48 h of starvation, S1 becomes phosphorylated again, first on membrane-bound ribosomes, then on free ribosomes, with a lag time of about 3 h. Following 48 h of refeeding, the most highly phosphorylated form of S1 predominates in both populations of ribosomes. These variations in phosphorylation are correlated with the level of protein synthesis in the posterior silk gland, 70% of the ribosomes occurring in polysomes upon feeding and only 30% upon starvation [Prudhomme, J.-C. & Couble, P. (1979) Biochimie (Paris) 61, 215-227]. After in vivo 32P labelling, the phosphopeptides of S1 from free and membrane-bound ribosomes were found to be identical and phosphoserine (only) was found in each S1. These results suggest the involvement of S1 phosphorylation in the regulation of protein synthesis at the translational level and the existence of at least two different pathways controlling this phosphorylation: one for the free ribosomes, the other for the membrane-bound ribosomes. 相似文献
20.
Protein phosphorylation during activation of surf clam oocytes. 总被引:3,自引:0,他引:3
We have investigated the increase of phosphorylated proteins upon activation of surf clam (Spisula solidissima) oocytes, by measuring the cumulative incorporation of 32P in proteins and by performing an SDS-PAGE and autoradiographic analysis of 32P-labeled proteins, from oocytes initially radiolabeled with 32P-orthophosphate. The phosphorylation inhibitor 6-dimethylaminopurine (6-DMAP) inhibits both germinal vesicle breakdown (GVBD) and the normal increase in phosphorylated proteins observed upon activation by KCl, in a reversible and dose-dependent manner. Using different artificial seawaters (normal, Ca(2+)-free, Na(+)-free), we observed that the increase of phosphorylated proteins, upon K+ stimulation, occurs only when GVBD is allowed to proceed along with an increased Ca2+ influx, in normal or Na(+)-free seawater. Stimulation of oocytes by ammonia, which directly raises intracellular pH (pHi) but does not trigger GVBD, is without effect on the level or pattern of phosphorylated proteins. The link between the Ca2+ influx and the level of phosphorylated proteins was further investigated using conditions altering the duration or the level of Ca2+ influx upon K+ stimulation. In all conditions tested, both GVBD and the level of phosphorylated proteins were similarly affected by alterations of the Ca2+ influx, indicating that these processes are tightly coupled one with another. Upon activation of oocytes, six major proteins of estimated molecular weights of 31, 41, 48, 56, 80 and 86 kDa undergo an increased phosphorylation that is reversibly sensitive to 6-DMAP. Our results suggest that increased protein phosphorylation, sensitive to 6-DMAP, is necessary for GVBD and that it is indirectly linked to the increased Ca2+ influx that stands as an upstream trigger for activation, while an elevated pHi alone has no effect on these processes. 相似文献