Proteolytic degradation of nuclear matrix proteins in the rat liver, Zajdela's hepatoma and hepatoma 22A in the presence of ATP

An intense proteolytic degradation of both proteins and phosphoproteins has been observed in isolated nuclear matrices from rat liver, Zajdela Hepatoma and Hepatoma 22a, incubated with NP-40, DTT and gamma-[32P] ATP being most intense in Hepatoma 22a. Practically all phosphoproteins of Hepatoma 22a nuclear matrix degraded. This implies either an extremely high proteolytic activity in the preparation or the presence of a specific to phosphoproteins protease absent from rat liver and Zajdela Hepatoma nuclear matrices.     

The cytoplasmic phosphoproteome of the Gram-negative bacterium Campylobacter jejuni: evidence for modification by unidentified protein kinases

We have undertaken a comprehensive analysis of cytoplasmic protein phosphorylation in Campylobacter jejuni by mass spectrometric identification of phosphoproteins and localization of the sites of modification by phosphopeptide analyses. Cell extracts, enriched for phosphoproteins using Fe(III) IMAC or commercial phosphoprotein purification kits, were analyzed by 1-D and 2-D SDS-PAGE and subjected to mass fingerprinting by in-gel tryptic digestion and MALDI-TOF MS. Fifty-eight phosphopeptides were identified from 1-D gel bands by nano-LC-MS/MS and automated searching in a C. jejuni ORF database resulting in the unequivocal identification of 36 phosphoproteins of diverse function. In addition to elongation factors and chaperonins, which have been reported to be phosphorylated in other bacteria, the major phosphoproteins included bacterioferritin and superoxide dismutase. The sequences around the phosphorylated Ser and Thr residues are indicative of specific kinases being responsible for some of the modifications. However, many of the other identified proteins are enzymes that have phosphorylated substrates, including ATP, hence other modifications may arise from autophosphorylation. Comparative analyses of IMAC extracts from the Escherichia coli strain AD202 and Helicobacter pylori resulted in the identification of homologs of six of the C. jejuni phosphoproteins, though their overall phosphoproteome maps were distinctly different.     

Phosphoproteins and the phosphoenolpyruvate:sugar phosphotransferase system of Streptococcus salivarius. Detection of two different ATP-dependent phosphorylations of the phosphocarrier protein HPr

Phosphoproteins which arise from incubation of Streptococcus salivarius ATCC25975 crude extracts with [32P]phosphoenolpyruvate and [gamma-32P]ATP, were separated and detected by sodium dodecyl sulphate-polyacrylamide gel electrophoresis and autoradiography. These procedures were carried out using the methodology that has been developed to allow for the detection of phosphoproteins containing 1-P-histidinyl and 3-P-histidinyl residues, and also to distinguish between these and phosphoproteins containing acid-stable phosphoamino acids such as phosphoserine, phosphothreonine, and phosphotyrosine. Extracts of cells which had been grown with various sugars as carbon sources were investigated to determine both constitutive and inducible phosphoproteins. No evidence was found for phosphoproteins specifically induced by a sugar, and in particular no evidence was found for any IIIsugar phosphocarrier protein of the phosphoenolpyruvate:sugar phosphotransferase system (PTS). Incubation with [gamma-32P]ATP showed that histidine-containing phosphocarrier protein (HPr) of the PTS could be phosphorylated to give both acid-stable and acid-labile phosphoamino acid residues. The acid-labile ATP-dependent phosphorylation activity was activated by glucose-6-P and appeared to produce a 3-P-histidinyl residue in HPr.     

Studies in pig heart tissue on various 60,000 Da phosphoproteins

Pig heart tissue have been shown to contain 3 different 60,000 Da phosphoproteins. Different purification procedures were used in order to separate them, suggesting that the 3 phosphoproteins differ in their environmental parameters. The 2 major ones appear essentially as peripheral phosphoproteins that are associated with cellular membranes through ionic forces, whereas the third minor phosphoprotein behaves as an integral plasma membrane protein. The three phosphoproteins also differ in their relative amount of phosphorylated serine, threonine and tyrosine residues after in vitro protein kinase assay. Evidence that the 3 phosphoproteins are related arises from the similarity between their respective phosphopeptide maps after partial hydrolysis with proteases, an experiment that also points out relatedness in primary structure between them and the transforming protein of Rous sarcoma virus, pp60v-src. The 3 phosphoproteins, however, do not appear to be immunologically related to pp60v-src since none of them is immunoprecipitated by sera that precipitate pp60v-src. The possibility that the three 60,000 Da phosphoproteins under study represent 3 differentially localized and phosphorylated products of c-src and/or c-src related genes is discussed.     

Protein phosphorylation and the respiratory burst

The exposure of 32P-loaded neutrophils to any of a variety of activating agents induces changes in the levels of phosphorylation of a large number of phosphoproteins. The uptake of phosphate by one set of phosphoproteins in particular, a family whose members migrate at Mr 48K with near neutral pI values, appears to be closely related to the activation of the respiratory burst oxidase, the O2--producing enzyme of phagocytes that is responsible for the generation of microbicidal oxidants by these cells. Evidence for the relationship between the phosphorylation of these proteins and the activation of the respiratory burst oxidase has been furnished by kinetic studies as well as by studies on protein phosphorylation in neutrophils from patients with chronic granulomatous disease, a group of inherited disorders affecting this oxidase. The details of this relationship are obscure, although the evidence suggests that these phosphoproteins act in substoichiometric amounts with respect to the oxidase.     

Properties of the 23,000-Da phosphoproteins in cardiac sarcolemma and sarcoplasmic reticulum

The calmodulin- and cAMP-dependent protein kinase-mediated phosphorylations of isolated sarcolemma and sarcoplasmic reticulum vesicles have been compared. Similarities in the calmodulin-mediated phosphorylation of the sarcolemma and sarcoplasmic reticulum 23,000-Da phosphoproteins included their Mg2+, Na+, Ca2+, and calmodulin sensitivities, as well as the size of their dissociated subunits. In contrast, a number of differences between these phosphoproteins were indicated in their sensitivity to detergents (Triton X-100 and sodium dodecyl sulfate) and calmodulin antagonists (R24571 and trifluoperazine). Furthermore, in contrast to the sarcoplasmic reticulum phosphoprotein, the sarcolemma phosphoprotein could not be affinity labeled with 125I-calmodulin. While these results indicate the probable chemical similarity of the sarcolemma and sarcoplasmic reticulum 23,000-Da phosphoproteins, they also indicate there are differences in the lipid/phosphoprotein interactions in these two membranes.     

Phosphoproteins and the phosphoenolpyruvate: sugar phosphotransferase system in Salmonella typhimurium and Escherichia coli: evidence for IIImannose, IIIfructose, IIIglucitol, and the phosphorylation of enzyme IImannitol and enzyme IIN-acetylglucosamine

Phosphoproteins produced by the incubation of crude extracts of Salmonella typhimurium and Escherichia coli with either [32P]phosphoenolpyruvate or [gamma 32P]ATP have been resolved and detected using sodium dodecyl sulphate polyacrylamide gel electrophoresis and autoradiography. Simple techniques were found such that distinctions could be made between phosphoproteins containing acid-labile or stable phosphoamino acids and between N1-P-histidine and N3-P-histidine. Phosphoproteins were found to be primarily formed from phosphoenolpyruvate, but because of an efficient phosphoexchange, ATP also led to the formation of the major phosphoenolpyruvate-dependent phosphoproteins. These proteins had the following apparent subunit molecular weights: 65,000, 65,000, 62,000, 48,000, 40,000, 33,000, 25,000, 20,000, 14,000, 13,000, 9,000, 8,000. Major ATP-dependent phosphoproteins were detected with apparent subunit molecular weights of 75,000, 46,000, 30,000, and 15,000. Other minor phosphoproteins were detected. The phosphorylation of the 48,000- and 25,000-MW proteins by phosphoenolpyruvate was independent of the phosphoenolpyruvate:sugar phosphotransferase system (PTS). The PTS phosphoproteins were identified as enzyme I (soluble; MW = 65,000); enzyme IIN-acetylglucosamine (membrane bound; MW = 65,000); enzyme IImannitol (membrane bound; MW = 62,000); IIIfructose (soluble; MW = 40,000); IIImannose (partially membrane associated; MW = 33,000); IIIglucose (soluble; MW = 20,000); IIIglucitol (soluble; MW = 13-14,000); HPr (soluble; MW = 9,000); FPr (fructose induced HPr-like protein (soluble; MW = 8,000). HPr and FPr are phosphorylated on the N-1 position of a histidyl residue while all the others appear to be phosphorylated on an N-3 position of a histidyl residue. These studies identify some previously unknown proteins of the PTS and show the phosphorylation of others, which although previously known, had not been shown to be phosphoproteins.     

Two-dimensional polyacrylamide gel analysis of changes in protein phosphorylation during maturation of Xenopus oocytes

A three- to five-fold increase in non-cAMP-dependent protein phosphorylation has previously been found to occur in progesterone-treated oocytes shortly before germinal vesicle breakdown (GVBD) or immediately following maturation-promoting factor (MPF) injection. Analysis of phosphoprotein from 32Pi-labeled oocytes by both equilibrium and nonequilibrium two-dimensional gel electrophoresis revealed a large number of qualitative changes in phosphoproteins at GVBD, including both phosphorylation and dephosphorylation events. Time-course studies demonstrated that some of the new phosphoproteins appeared as early as 0.36 GVBD50, and all changes were stable at least through GVBD. The pattern of new phosphoproteins at GVBD was similar in oocytes microinjected with a partially purified preparation of MPF. A number of the new phosphoproteins were heat stable, which may facilitate their purification and characterization. These results support the hypothesis that key regulatory events during oocyte maturation are controlled by protein phosphorylation.     

The primary structure of avian phosvitins. Contributions through the Edman degradation of methylmercaptovitins prepared from the constituent phosphoproteins

Methylmercaptovitins were prepared from the constituent phosvitin phosphoproteins as well as from the CNBr cleavage peptides derived from the methionine-containing phosphoproteins. Edman degradation of these methylmercaptovitins has afforded partial sequence information and identifies the serine phosphorylation sites in the phosphoprotein. Primary structure, determined for approximately seventy-seven residues from the amino terminus of the hen methionine-containing phosphoprotein, agrees fully with that deduced from recently published nucleotide sequence data, and provides corrections to results of earlier work on enzymatically dephosphorylated samples. Partial sequence data, together with corrections to earlier results, are also provided for phosphoproteins from duck and turkey phosvitins, as well as for the methionine-free phosphoprotein from hen phosvitin. All phosphoproteins have N-terminal leader sequences of low serine content. Sequences of the methionine-containing group are homologous, as are the sequences of the methionine-free group, but the groups differ significantly from one another. Unphosphorylated sites appear to have a fractional distribution over all available serine residues.     

Phosphoproteins of the chicken eggshell calcified layer

The chicken eggshell matrix is a complex mixture of proteins and proteoglycans. It also contains phosphoproteins that are thought to affect mineralization of the matrix. Several of the matrix phosphoproteins, such as the major component osteopontin, have already been identified as phosphoproteins in other tissues, but the phosphorylation status of the eggshell matrix forms was unknown. The phosphopeptides, obtained after cleavage of the matrix proteins with several different cleavage methods, were enriched by anion-exchange chromatography and reversible binding to titanium oxide and identified by LC-MS(n) or pseudo-MS(n) analysis following neutral loss scanning. Altogether we identified 39 phosphorylated matrix proteins, 22 of which were not known to be phosphorylated before. Eight of the proteins were identified as eggshell matrix components for the first time. Together these proteins contained more than 150 different phosphorylation sites, 103 of which were determined with high confidence. Among the major phosphorylated proteins of the chicken eggshell matrix were osteopontin and the eggshell-specific proteins ovocleidin-17, ovocleidin-116, and ovocalyxin-32.     

Similar effects of platelet-derived growth factor and epidermal growth factor on the phosphorylation of tyrosine in cellular proteins

Platelet-derived growth factor (PDGF) stimulates the phosphorylation of proteins at tyrosine when added to quiescent 3T3 cells, as evidenced by the increase in the amount of phosphotyrosine, relative to phosphoserine and phosphothreonine, in cellular proteins. The increase was detected within 1 min of adding PDGF and was maximal by 5 min. This effect showed the same dependence on PDGF concentration as did association of 125I-PDGF with the cells. In different 3T3 cell lines the magnitude of the increase was approximately proportional to the number of PDGF receptors per cell. A number of proteins phosphorylated at tyrosine in response to PDGF have been detected by two-dimensional gel electrophoresis. They include a pair of related 45 kilodalton phosphoproteins, a pair of related 43 kilodalton phosphoproteins and a 42 kilodalton phosphoprotein. Similar changes were noted when quiescent 3T3 cells were incubated with epidermal growth factor. Possibly, these phosphoproteins are primary substrates of the tyrosine protein kinases activated by the receptors for PDGF and epidermal growth factor, and are involved in physiological effects common to the two growth factors.     

Phosphoproteome analysis of the human Chang liver cells using SCX and a complementary mass spectrometric strategy

The liver is the largest organ in the body, with many complex, essential functions, such as metabolism, deintoxication, and secretion, often regulated via post-translational modifications, especially phosphorylation. Thus, the detection of phosphoproteins and phosphorylation sites is important to comprehensively explore human liver biological function. The human Chang liver cell line is among the first derived from non-malignant tissue, and its phosphoproteome profile has never been globally analyzed. To develop the complete phosphoproteome and probe the roles of protein phosphorylation in normal human liver, we adopted a shotgun strategy based on strong cation exchange chromatograph, titanium dioxide and LC-MS/MS to isolate and identify phosphorylated proteins. Two types of MS approach, Q-TOF and IT, were used and compared to identify phosphosites from complex protein mixtures of these cells. A total of 1035 phosphorylation sites and 686 phosphorylated peptides were identified from 607 phosphoproteins. A search using the public database of PhosphoSite showed that approximately 344 phosphoproteins and 760 phosphorylation sites appeared to be novel. In addition, N-terminal phosphorylated peptides were a greater fraction of all identified phosphopeptides. With GOfact analysis, we found that most of the identified phosphoproteins are involved in regulating metabolism, consistent with the liver's role as a key metabolic organ.     

Phosphorylation of Plasmodium berghei derived phosphoproteins associated with the host erythrocyte membrane by the spectrin kinase

Summary Plasmodium berghei derived phosphoproteins are associated with the host erythrocyte membrane. Effectors of the phosphorylation reaction regulate the phosphorylation of the P. berghei derived proteins and spectrin in a similar manner. The spectrin kinase also phosphorylates the P. berghei phosphoproteins in a reconstituted reaction at the same site(s) as the endogenously phosphorylated proteins. These results indicate that a host protein kinase may regulate parasite phosphoproteins during malaria.Abbreviations 2,3-DPG 2,3-diphosphoglyceric acid - SDS Sodium Dodecyl Sulfate     

Phosphoproteins in dictyostelium discoideum

The phosphoproteins of Dictyostelium discoideum were compared at different stages of development by polyacrylamide gel electrophoresis. Certain phosphoproteins of vegetative amoebae were conserved while others appeared and disappeared during development. Four major phosphoproteins with apparent subunit molecular weights of 50,000, 47,000, 38,000, and 34,000 disappeared precociously in response to exogenous cAMP. Two membranal phosphoproteins, with apparent subunit molecular weights of 80,000 and 81,000, appeared precociously in response to added cAMP. One of these phosphoproteins, molecular weight of 80,000, has been identified tentatively as the “contact site A” glycoprotein. Another membranal protein, with apparent subunit molecular weight of 42,000, unaffected in its appearance by cAMP, has been identified tentatively as phosphoactin.     

Two-dimensional analysis of proteins associated with heterogenous nuclear RNA in various animal cell lines.

The protein complement of heterogenous nuclear RNA . protein particles from human HeLA, mouse L and Chinese hamster (CHO) cells has been analysed by two-dimensional gel electrophoresis using the two techniques described by O'Farrell [J. Biol. Chem. (1975) 250, 4007--4021 and Cell (1977) 12, 1133--1142]. Over a hundred individual spots habe been reproducibly detected both L-[35S]methionine. Large similarities, especially in the 25 000--40 000 Mr cluster of basic protein, were found among these three mammalian species. As far as phosphoproteins are concerned, it was observed that the bands already described by one-dimensional gels [Eur. J. Biochem. (1978) 86, 301--310] with Mr values of 28 000, 30 000, 37 000 and 52 000 are resolved into about 15 individual spots, suggesting a corresponding number of distinct states of phosphorylation. It was also clearly demonstrated that phosphoproteins are unrelated to the major basic protein species. Particles of different size classes were analysed with respect to their content of individual proteins, both non-phosphorylated and phosphorylated. The most salient feature observed was that phosphoproteins become progressively more abundant with particles of increasing size. This raises the possibility that at least some of these phosphoproteins might belong to a nuclear structure to which hnRNA is normally bound.     

Proteomic analysis of phosphoproteins regulated by abscisic acid in rice leaves

Abscisic acid (ABA) is a hormone that regulates plant development and adaptation to environmental stresses. Protein phosphorylation has been recognized as an important mechanism for ABA signaling. However, the target phosphoproteins regulated by ABA are still largely unknown. Here, we report the identification of ABA-regulated phosphoproteins in rice using proteomic approaches. Six ABA-regulated phosphoproteins were identified as G protein beta subunit-like protein, ascorbate peroxidase, manganese superoxide dismutase, triosephosphate isomerase, putative Ca²⁺/H⁺ antiporter regulator protein, and glyoxysomal malate dehydrogenase. These results provide new insight into the regulatory mechanism for some ABA signaling proteins and implicate several previously unrecognized proteins in ABA action.     

On the biochemical significance of phosphoserine: a working hypothesis

The significance of the presence of free phosphoserine in living cells represents an intriguing problem. Its utilization for the synthesis of phosphoproteins and phospholipids has been ruled out. It is produced extensively by hydrolysis of phosphoproteins or phosphatidylserine since no phosphorylating enzyme for serine is present. So far the only significance of phosphoserine has been related to its participation in the exchange reaction with serine, the meaning of which is quite unclear. Evidence is presented that phosphoserine could modulate the activity of phospholipase A2, thus regulating the permeability properties of cellular and intracellular membranes which depend largely on phospholipase pattern. Phosphoserine in fact inhibits in a competitive way phospholipase A2 from cobra venom.     

Isolation of the Arabidopsis phosphoproteome using a biotin-tagging approach

Protein phosphorylation plays a key role in signal transduction in cells. Since phosphoproteins are present in low abundance, enrichment methods are required for their purification and analysis. Chemical derivatization strategies have been devised for enriching phosphoproteins and phosphopeptides. In this report, we employed a strategy that replaces the phosphate moieties on serine and threonine residues with a biotin-containing tag via a series of chemical reactions. Ribulose 1,5-bis-phosphate carboxylase/oxygenase (RUBISCO)-depleted protein extracts prepared from Arabidopsis seedlings were chemically modified for 'biotin-tagging'. The biotinylated (previously phosphorylated) proteins were then selectively isolated by avidin-biotin affinity chromatography, followed by two-dimensional gel electrophoresis (2-DE) and matrix-assisted laser-desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS). This led to the identification of 31 protein spots, representing 18 different proteins, which are implicated in a variety of cellular processes. Despite its current technical limitations, with further improvements in tools and techniques this strategy may be developed into a useful approach.     

Several enzymes of the central metabolism are phosphorylated in Staphylococcus aureus

Staphylococcus aureus is an important human and animal pathogen that harbors protein kinases. The proteins phosphorylated in this bacterium grown on glucose minimal medium have been in vivo labeled with[(32)P]-orthophosphate and analyzed by two-dimensional gel electrophoresis followed by MS. A total of 11 glycolytic phosphoproteins have been identified and verified. In vitro analyses have revealed that phosphorylation of these glycolytic enzymes is catalysed primarily through the activity of an endogenous serine/threonine kinase and to a lesser extent by a tyrosine kinase. The identification of these phosphoproteins should prove helpful in understanding and unravelling of the role of phosphorylation with respect to pathogenesis and virulence in this organism.