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Query: UNIPROT:P06889 (Mol)
 630,302 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Protein phosphorylation-dephosphorylation appears to be an essential component in the regulation of many cellular processes by hormones and drugs. This concept has developed primarily from in vitro biochemical studies in which various purified proteins have been phosphorylated and dephosphorylated by distinct protein kinases and phosphoprotein phosphatases. However, the more difficult, but essential, task of demonstrating the physiological occurrence of these reactions in intact tissue or cell preparations in many cases has not been undertaken in a quantitative manner. There are 4 basic approaches for assessing the extent of protein phosphorylation in vivo and in intact cell systems, each having particular advantages and disadvantages. These are summarized in Table 2. The applicability of any one procedure will be highly dependent upon the protein under investigation. For instance, chemical measurements of total protein-bound phosphate may provide only limited information for proteins which are phosphorylated at multiple sites but could be highly useful for those proteins such as glycogen phosphorylase which are phosphorylated at single sites. The relative ease and the high sensitivity of measuring 32P incorporation into proteins will tempt many investigators to rely heavily on this approach. It is a very powerful procedure, particularly for the initial identification of phosphoproteins, but ultimately quantitative conclusions regarding 32P incorporation must be corroborated by one or more of the other procedures. There is no simple, single experimental approach that may be used under all circumstances, but by integrating these procedures firm conclusions may be drawn regarding the physiological importance of phorphorylation of specific proteins.
Mol Cell Endocrinol 1980 Jul
PMID:Protein phosphorylation: quantitative analysis in vivo and in intact cell systems. 624

Protein phosphatase, active on non-histone phosphoprotein substrate, was partially purified from rat liver cell nuclei by means of salt extraction, ammoniumsulfate precipitation, DEAE cellulose chromatography, gel filtration and preparative isoelectrofocusing. Rat liver nuclei contain a heterogenous population of different protein phosphatases. All the enzyme fractions eluted from DEAE cellulose are of low molecular weight between 12,000--31,000. The pH 5.5 peak fraction of preparative isoelectrofocusing was characterized in detail. It has a pH optimum of 6.8 using nuclear phosphoprotein substrate. It is inhibited by Na+ at 80 mM, and to a lesser extent by K+, activated by Mg2+ (5 mM) and Mn2+ (1 mM). However, the latter is inhibitory at 6 mM. The nuclear protein phosphatase is also active on labelled F1 and F2b histones and casein, however, its V is lower on histones and it contains component(s) active specifically on nuclear phosphoprotein substrate but not on casein.
Mol Cell Biochem 1980 Aug 29
PMID:Protein phosphatase from rat liver nuclei. 625 11

We have examined the phosphorylation of a 50,000-dalton cellular polypeptide associated with the Rous sarcoma virus (FSV) transforming protein pp60-src. It has been shown that pp60src forms a complex with two cellular polypeptides, an 89,000-dalton heat-shock protein (89K) and a 50,000-dalton phosphoprotein (50K). The pp60src-associated protein kinase activity phosphorylates at tyrosine residues, and the 50K polypeptide present in the complex contains phosphotyrosine and phosphoserine. These observations suggest that the 50K polypeptide may be a substrate for the protein kinase activity of pp60src. To examine this possibility, we isolated the 50K polypeptide by two-dimensional polyacrylamide gel electrophoresis from lysates of uninfected or virally infected cells. Tryptic phosphopeptide analysis indicated that the 50K polypeptide isolated by this method was the same polypeptide as that complexed to pp60src. In uninfected cells or cells infected by a transformation-defective mutant, the 50K polypeptide contained phosphoserine but little or no phosphotyrosine. In cells infected by Schmidt-Ruppin or Prague RSV, there was a 40- to 50-fold increase in the quantity of phosphotyrosine in the 50K protein. Thus, the phosphorylation of the 50K polypeptide at tyrosine is dependent on the presence of pp60src. However, the 50K polypeptide isolated from cells infected by temperature-sensitive mutants of RSV was found to be phosphorylated at tyrosine at both permissive and nonpermissive temperatures; this behavior is different from that of other substrates or putative substrates of the pp60src kinase activity. It is possible that the 50K polypeptide is a high-affinity substrate of pp60src.
Mol Cell Biol 1982 Feb
PMID:Tyrosine phosphorylation of a 50K cellular polypeptide associated with the Rous sarcoma virus transforming protein pp60src. 628 29

After phosphorylation of electric eel Na,K-ATPase by Na+, Mg2+, and ATP was terminated by removing the unbound Mg2+, the phosphoenzyme was able to bind ouabain upon the addition of 2 mM ouabain under certain conditions. This binding was demonstrated by a 50% inhibition of ATPase after the removal of unbound ouabain by a Sephadex G-50 column (ouabain trapping method). At 4 degrees, this ouabain binding was observed on the K+- sensitive phosphoprotein (E2P) formed in the presence of 10 mM Na+ but was not observed on the ADP-sensitive phosphoprotein (E1P) formed in the presence of 1000 mM Na+. The increase in the dephosphorylation rate of E2P with various concentrations of K+ paralleled the decrease in inhibition by the addition of 2 mM ouabain after the termination of phosphorylation. In 50-200 mM Na+, the eel enzyme used here formed the E1P-rich phosphoprotein, but this phosphoprotein could bind with ouabain, even though the presence of ADP or oligomycin, which prevents the conversion of E1P to E2P, partially interfered with this ouabain binding. At 25 degrees, ouabain binding with E1P-rich phosphoprotein was observed in higher yield (up to 71%), but in each of these cases ADP or oligomycin strongly inhibited ouabain binding. Moreover, ouabain binding with E2P-rich phosphoprotein did not significantly change with temperature, but ouabain binding with E1P-rich phosphoprotein increased more than 6 times at temperatures from 4 degrees to 25 degrees. From these results, it can be concluded that E2P can bind with ouabain in the absence of free Mg2+ whereas E1P cannot, and that the interconversion between E1P and E2P can be stimulated with ouabain binding and accelerated with elevation of temperature. ADP- and K+ -insensitive phosphoprotein probably is only a minor intermediate for ouabain binding.
Mol Pharmacol 1982 Nov
PMID:Interaction between ouabain and the phosphorylated intermediate of Na,K-ATPase. 629 61

Fibroblasts transformed by Abelson murine leukemia virus differ from normal fibroblasts in that they contain several cellular proteins, including one of 29 and one of 36 kilodaltons, which are phosphorylated at tyrosine residues. Since it has been shown before that these proteins also become phosphorylated at tyrosine after transformation of fibroblasts by a number of other retroviruses, their phosphorylation may play an important role in the transformation of these cells. In contrast, the 36-kilodalton phosphoprotein was not detectable in three of the four lines of Abelson virus-transformed B lymphoma cell lines studied here. These three cell lines, RAW307.1.1, 18-48, and 18-81, and a B lymphoma induced by mineral oil, WEHI 279, were all found to lack both the phosphorylated and unphosphorylated forms of the 36-kilodalton protein. It thus appears that expression of this major cell protein is not essential for the survival of B lymphoma cells in culture and that the phosphorylation of the 36-kilodalton protein at tyrosine is not essential for transformation of pre-B lymphocytes by Abelson virus.
Mol Cell Biol 1983 Jan
PMID:Some lymphoid cell lines transformed by Abelson murine leukemia virus lack a major 36,000-dalton tyrosine protein kinase substrate. 629 8

Proteins encoded by adenovirus type 2 and type 5 early region 2A isolated from infected HeLa cells were compared to translation products of E2A-specific messenger RNA in a reticulocyte cell-free system and in Xenopus oocytes. The main cell-free translation product is a 72,000 Mr polypeptide which in HeLa cells as well as in Xenopus oocytes is converted into a 75,000 Mr phosphoprotein capable of binding to single-stranded DNA. Some minor proteins are proteolytic cleavage products of the major protein. In the cell-free system, three E2A polypeptides, 32,000, 37,000 and 44,000 Mr, are translated from minor polyadenylated mRNA species that can be separated from the major mRNA. Synthesis of all E2A polypeptides in vitro is inhibited by cap-analogs. The 44,000 Mr protein is also synthesized in Xenopus oocytes. Tryptic peptide maps of [35S]methionine-labeled E2A proteins were constructed using high pressure liquid chromatography and the position of the methionyl residues within each peptide was determined by amino acid sequencing procedures. This information and the DNA sequence of the adenovirus 5 E2A gene published by Kruijer et al. (1981) were used to align the peptides and to construct a map of the E2A proteins. Our data demonstrate that the major 75,000 Mr protein is coded for by a leftward reading frame of 529 amino acid residues located between 62 and 66 map units. The data also map six sites as targets for proteolytic enzymes. The minor E2A translation products have the same carboxy terminus as the major protein. The initiation codons of the 44,000, 37,000 and 32,000 Mr polypeptides probably correspond to amino acids 170, 243 or 244 and 290 of the major protein. Some functional properties of the major E2A protein are shared by the minor proteins and thus could be mapped. Major sites of phosphorylation, the region involved in binding to single-stranded DNA and the antigenic regions recognized by immune sera are located between amino acid residues 50 to 120, 170 to 470 and 170 to 240, respectively.
J Mol Biol 1983 Jan 15
PMID:Structural characterization of the proteins encoded by adenovirus early region 2A. 630 66

Injection of whole adenovirus DNA into Xenopus oocytes results in the synthesis of large amounts of the early region 2A DNA-binding protein (E2A-DBP) and smaller amounts of polypeptide IX. The lack of synthesis of any functional messenger RNAs transcribed from the major late promotor at 16.3 map units is remarkable. Cleavage of the adenovirus DNA outside the E2A gene proper by restriction enzymes decreases synthesis of the DBP to about 10% of the amount produced after injection of intact DNA. On the other hand, presence of the terminal (Bellett) protein on the injected template enhances DBP synthesis considerably. Experiments with injected DNA restriction fragments, as well as reconstructed genes cloned into pBR322, indicate that efficient synthesis of DBP in oocytes requires the presence of either or both of the two main promoters from which the E2A gene is transcribed plus an intact 3' end of the gene. In the absence of any known promotor, 100-fold lower amounts of otherwise normal DBP are produced. Unlike in a regular infection, synthesis of DBP in oocytes does not require the product of the E1A gene. The same series of experiments also demonstrates that the DBP, a phosphoprotein, is the substrate of a cellular rather than a virus-encoded protein kinase. Two minor E2A proteins, although colinear with the major DBP, are synthesized independently. Synthesis of a 44,000 Mr protein, probably corresponding to the carboxy-terminal 360 amino acid residues of the DBP, is not decreased after injection of "promotorless" E2A genes. Unlike the 44,000 Mr protein, production of a 67,000 Mr protein (carboxy-terminal 483 amino acid residues) by one DNA-construct is probably directed by a T-A-T-A-A-A-T-A sequence in the vector DNA.
J Mol Biol 1983 Jan 15
PMID:Analysis of expression of adenovirus DNA (fragments) by microinjection in Xenopus oocytes. Independent synthesis of minor early region 2 proteins. 630 67

Phosphoprotein phosphatase was purified from swine kidney by chromatography on DEAE-Sephadex A-50, Sephacryl S-200 and Sepharose 4B columns containing covalently bound hexanediamine and polylysine. The enzyme was purified more than 20000-fold and the homogeneous preparation had a specific activity of 2.8 micromol per min per mg of protein with saturating concentrations of 32P-histone as the substrate. The phosphatase showed only a single protein band when examined by polyacrylamide gel electrophoresis and a single protein peak containing all of the enzymatic activity was observed during chromatography on Sephadex G-100 column. The molecular weight of the purified enzyme was determined to be 70000 +/- 5000 by exclusion chromatography on a calibrated Sephadex G-100 column. Similar values were obtained by sucrose density centrifugation, 70000 +/- 5000, and polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate, 70000 +/- 3000. The purified enzyme catalyzed the dephosphorylation of the phosphorylated forms of glycogen synthase, phosphorylase, histone, phosphofructokinase, Type II regulatory subunit of cyclic AMP-dependent protein kinase, casein and protamine. The apparent Km values for these substrates were 3.6 microM, 2.8 microM, 66 microM, 3.3 microM, 8.0 microM, 6.6 microM and 100 microM, respectively. The enzyme did not hydrolyze low molecular weight phosphate esters such as glucose 6-phosphate, glycerol phosphate, adenosine nucleotides and inorganic pyrophosphate. The activity of the enzyme towards a phosphorylated protein substrate was competitively inhibited by the addition of other substrates. These results suggest that swine kidney contains a phosphoprotein phosphatase with a rather broad substrate specificity for a number of endogenous and exogenous phosphoprotein substrates.
Mol Cell Biochem 1983
PMID:Purification and properties of swine kidney phosphoprotein phosphatase. 630 89

Tyrosine phosphorylation catalyzed by a unique class of protein kinases is an important process in both normal cell proliferation and oncogenic transformation. In this study, phosphoprotein phosphatases specific for the dephosphorylation of phosphotyrosine residues were partially purified from secondary chicken embryo fibroblasts, using 32P-labeled immunoglobulin G phosphorylated by pp60src as substrate. Crude cell extracts contained ca. 70% of the activity in the soluble form and ca. 30% associated with a crude membrane fraction. The soluble activity was purified by using DEAE-cellulose and carboxymethyl cellulose column chromatography and gel filtration, and at least three enzyme species of apparent Mr 55,000 (pTPI), 50,000 (pTPII), and 95,000 (pTPIII)--comprising ca. 20, 45, and 35%, respectively, of the total activity--were resolved. All three enzymes possessed somewhat similar properties. They had a pH optimum of about 7.4, they were inhibited by Zn2+, vanadate, ATP, and ADP, and they were unaffected by divalent metal cations, EDTA, and F- under standard assay conditions employing a physiological ionic strength. These properties suggest that they represent a class of enzymes distinct from well-known phosphoseryl-phosphothreonyl-protein phosphatases and that dephosphorylation of phosphotyrosine-containing proteins may be carried out by a unique family of phosphoprotein phosphatases. Transformation by Rous sarcoma virus resulted in a small increase in phosphotyrosyl-protein phosphatase activity.
Mol Cell Biol 1984 Jun
PMID:Identification, purification, and characterization of phosphotyrosine-specific protein phosphatases from cultured chicken embryo fibroblasts. 633 May 24

A basic ribosomal phosphoprotein of 30,000 molecular weight was rapidly dephosphorylated in cultured Drosophila melanogaster cells heat shocked at 37 degrees C. The protein was associated with the 40S ribosomal subunit and had an electrophoretic mobility similar to that of purified rat liver protein S6 on basic two-dimensional polyacrylamide gels as well as a similar partial proteolysis peptide map. In logarithmically growing cultures, this D. melanogaster S6 protein appeared to have a single phosphorylated species consisting of 30 to 40% of the total cellular S6. Thus, the nearly complete dephosphorylation of this protein observed in heat shock involves a large fraction of the cellular S6. The significance of this dephosphorylation in the expression of the heat shock response was investigated by examining the phosphorylation status of S6 in recovery from heat shock and in response to chemical inducers of the heat shock response. During recovery from a 30-min heat shock, the recovery of normal protein synthesis was almost complete in 2 to 4 hr, whereas there was no significant rephosphorylation of S6 for 8 h. Two chemical inducers of the heat shock response, canavanine and sodium arsenite, induced the synthesis of heat shock proteins in D. melanogaster cells. Sodium arsenite also caused an inhibition of normal protein synthesis similar to that observed in heat shock. Neither agent, however, caused significant dephosphorylation of S6. These results suggest that the dephosphorylation of S6, although invariably observed in heat-shocked cells, may in some cases be dissociated from both the induction of heat shock protein synthesis and the turnoff of normal protein synthesis which occur in a heat shock response.
Mol Cell Biol 1983 Nov
PMID:Dephosphorylation of S6 and expression of the heat shock response in Drosophila melanogaster. 641 58


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