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     

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.     

Phosphorylation of triton X-100 soluble and insoluble protein substrates in a demembranated/reactivated human sperm model

Sperm motility is a process which involves a cascade of events mediated by cAMP and Ca²⁺, cAMP in the initiation of flagellar movement, and Ca²⁺ in the regulation of beat asymmetry, and it has been suggested that these two messengers act through phosphorylation/dephosphorylation of axonemal proteins. Only a few studies on human sperm protein phosphorylation have been reported and no relation of this process with motility or other function has been established. In the present study, phosphorylation of human sperm proteins was performed using detergent-demembranated spermatozoa, in which motility is reactivated by the addition of ATP. This system allows direct accessibility of intracellular kinases to [³²P]-γATP and allows some relation between protein phosphorylation and flagellar movements. After electrophoresis and autoradiography, numerous phosphoproteins were detected. Phosphorylation of 2 proteins (36 and 51 kDa) was stimulated by cAMP in a concentration-dependent manner, and this increase was prevented by inhibitors of cAMP-dependent protein kinase. In order to characterize phosphoproteins originating from the cytoskeleton or axoneme, detergent extracted spermatozoa were also subjected to phosphorylation. Three major phosphorylated proteins (14.8, 15.3, and 16.2 kDa) were detected, the first two expressing cAMP-dependency according to their cAMP concentration-dependent increase in phosphorylation and the reversal of this effect by inhibitors of cAMP-dependent protein kinase. Proteins phosphorylation during the reactivation of demembranated spermatozoa previously immobilized H₂O₂, xanthine + xanthine oxidase-generated reactive oxygen species, or the oxidative phosphorylation uncoupler rotenone, revealed increases in cAMP-independent phosphorylation of proteins of 16.2, 46, and 93 kDa. These results documenting human sperm phosphoproteins form a base for further studies on the role of protein phosphorylation in sperm functions. © 1996 Wiley-Liss, Inc.     

Protein kinases associated with the yeast phosphoproteome

Background  

Protein phosphorylation is an extremely important mechanism of cellular regulation. A large-scale study of phosphoproteins in a whole-cell lysate of Saccharomyces cerevisiae has previously identified 383 phosphorylation sites in 216 peptide sequences. However, the protein kinases responsible for the phosphorylation of the identified proteins have not previously been assigned.     

Protein Kinase Activities Associated with Actin Cytoskeleton in Xenopus laevis Oocytes and Eggs

Protein phosphorylation with specific protein kinases plays the key role in the regulation of meiotic maturation of oocytes. However, little is known about the contribution of kinases to the temporal and positional regulation of the cytoskeleton rearrangement in maturing oocytes, including the actin cytoskeleton. In order to study a relationship between the kinase activities and actin cytoskeleton rearrangement, we analyzed protein phosphorylation in the isolated actin cytoskeleton of Xenopus laevis oocytes. Analysis of the full grown oocytes and eggs injected with [-³²P]ATP has revealed phosphorylation of many proteins associated with the actin cytoskeleton and shown the appearance of three additional major phosphoproteins, 20, 43, and 69 kDa, during oocyte maturation. A significant number of these phosphoproteins were also found after incubation of the isolated cytoskeleton with [-³²P]ATP in vitro, thus confirming that the kinases modifying these substrates are also specifically associated with actin. The in vivo and in vitro kinase activities were also stimulated during maturation. Analysis of kinase self-phosphorylation in situ and protein phosphorylation in solutions and substrate containing gels revealed a set of actin-associated kinases, including cAMP- and Ca²⁺-dependent kinases, as well as MAP, p34^cdc2, and tyrosine kinase activities. Their level was the highest in the eggs. The involvement of kinases in the actin cytoskeleton rearrangement during oocyte maturation is discussed.     

Large-scale analysis of phosphorylated proteins in maize leaf

Phosphorylation is an ubiquitous regulatory mechanism governing the activity, subcellular localization, and intermolecular interactions of proteins. To identify a broad range of phosphoproteins from Zea mays, we enriched phosphopeptides from Zea mays leaves using titanium dioxide microcolumns and then extensively fractionated and identified the phosphopeptides by mass spectrometry. A total of 165 unique phosphorylation sites with a putative role in biological processes were identified in 125 phosphoproteins. Most of these proteins are involved in metabolism, including carbohydrate and protein metabolism. We identified novel phosphorylation sites on translation initiation factors, splicing factors, nucleolar RNA helicases, and chromatin-remodeling proteins such as histone deacetylases. Intriguingly, we also identified phosphorylation sites on several proteins associated with photosynthesis, and we speculate that these sites may be involved in carbohydrate metabolism or electron transport. Among these phosphoproteins, phosphoenolpyruvate carboxylase and NADH: nitrate reductase (NR) which catalyzes the rate-limiting and regulated step in the pathway of inorganic nitrogen assimilation were identified. A conserved phosphorylation site was found in the cytochrome b5 heme-binding domain of NADH: nitrate reductase, suggesting that NADH: nitrate reductase is phosphorylated by the same protein kinase or highly related kinases. These data demonstrate that the pathways that regulate diverse processes in plants are major targets of phosphorylation.     

Heat Shock Effects on the Circadian Rhythm of Protein Synthesis and Phosphorylation of Ribosomal Proteins in Gonyaulax polyedra

Circadian changes in protein synthesis and phosphorylation of ribosomal and cytoplasmic proteins in the marine dinoflagellate Gonyaulax polyedra were analyzed by radioactive labeling and polyacrylamide gel electrophoresis. Maximal rates of protein synthesis were found during the subjective night and minimal rates during the subjective day. Protein synthesis was inhibited by heat shock to a different extent at different circadian phases—maximally during the subjective night. Heat shock proteins (HSPs) having molecular weights of approximately 105, 89, 83, 66, 35, and 18 kDa were induced by these treatments. Induction of HSP89 and HSP35 showed circadian differences with maximal synthesis rates at CT 15, whereas most HSPs maintained a constant constitutive and induced synthesis. Recovery of normal protein synthesis after heat shock occurred faster during the subjective night than during the subjective day. Ribosomal proteins with molecular weights of 16 and 18 kDa were highly phosphorylated by [³⁵S] thio gamma adenosine triphosphate during day phase in a light-dark cycle or at CT 6 in constant dim light and labeled only to a minor degree during night phase or at CT 18. A ribosome-associated protein (35 kDa) was labeled during the day and not during the night, but after heat shock during both day and night. In the 200,000 g cytosolic fraction, a 35-kDa protein was found to be more intensely labeled at night than during the day phase after heat shock. The results of this study show a correlation between circadian changes in the overall protein synthesis and ribosomal protein phosphorylation. The rhythm of protein synthesis and phosphorylation of a ribosome-associated protein are drastically altered by heat shock and dependent on the circadian phase.     

A survey of the Arabidopsis thaliana mitochondrial phosphoproteome

Plant mitochondria play central roles in cellular energy production, metabolism and stress responses. Recent phosphoproteomic studies in mammalian and yeast mitochondria have presented evidence indicating that protein phosphorylation is a likely regulatory mechanism across a broad range of important mitochondrial processes. This study investigated protein phosphorylation in purified mitochondria from cell suspensions of the model plant Arabidopsis thaliana using affinity enrichment and proteomic tools. Eighteen putative phosphoproteins consisting of mitochondrial metabolic enzymes, HSPs, a protease and several proteins of unknown function were detected on 2‐DE separations of Arabidopsis mitochondrial proteins and affinity‐enriched phosphoproteins using the Pro‐Q Diamond phospho‐specific in‐gel dye. Comparisons with mitochondrial phosphoproteomes of yeast and mouse indicate that these three species share few validated phosphoproteins. Phosphorylation sites for seven of the eighteen mitochondrial proteins were characterized by titanium dioxide enrichment and MS/MS. In the process, 71 phosphopeptides from Arabidopsis proteins which are not present in mitochondria but found as contaminants in various types of mitochondrial preparations were also identified, indicating the low level of phosphorylation of mitochondrial components compared with other cellular components in Arabidopsis. Information gained from this study provides a better understanding of protein phosphorylation at both the subcellular and the cellular level in Arabidopsis.     

The replication band of Euplotes is enriched with phosphoproteins

Summary— Employing several antibodies to phosphorylated protein epitopes, we demonstrate by immunostaining that the macronuclear replication band (RB) of the ciliated protozoan Euplotes eurystomus contains a high concentration of phosphoproteins. Enrichment is principally within the rear zone of the RB, the region of DNA synthesis and chromatin assembly. By immunoblot analysis, the various antibodies reacted with a diversity of macronuclear phosphoproteins, one of which was phosphorylated histone Hl. This diversity of phosphoproteins was also supported by examination of the macronuclear matrix generated by high NaCl extraction. Available evidence clearly indicates that the ultrastructural wave of chromatin modulation accompanying DNA replication is spatially correlated with a wave of localized nuclear protein phosphorylation.     

Differential effects of polyamines on the phosphorylation of chromatin-associated proteins

Studies are presented on the nature of chromatin-associated phosphoproteins whose phosphorylation is influenced by polyamines. After labelling with 32P, chromatin-associated proteins were separated into four fractions. Fraction I comprised neutral and basic non-histone phosphoproteins, including high-mobility-group non-histones; fraction II consisted mostly of histones; fraction III consisted of a class of (salt-soluble) acidic non-histone phosphoproteins; and fraction IV consisted of residual (salt-insoluble) acidic non-histone phosphoproteins. The average relative distribution of protein in the four fractions (I-IV) was about 1:4:2:1 for both liver and prostate. However, tissue-dependent differences were observed in the incorporation of 32P in various protein fractions. In the presence of polyamines (e.g. 1 mM-spermine or 2 mM-spermidine) maximal stimulation of phosphorylation was observed in non-histone proteins of fraction I (160-180%), followed by that in non-histone proteins of fraction III (80-110%). The phosphorylation of residual non-histone proteins in fraction IV, and the small extent of phosphorylation of histones in fraction II, remained unaltered in the presence of polyamines. Thus polyamines do not stimulate the phosphorylation of all non-histone proteins; their stimulative effect is most prominent in the phosphorylation of neutral and basic non-histone proteins and a class of salt-soluble acidic non-histone proteins. In accord with our hypothesis, these differential effects of polyamines on phosphorylation of endogenous non-histone proteins may relate to the conformation of these substrates rather than to endogenous kinases.     

Ser/Thr/Tyr phosphoproteome analysis of pathogenic and non‐pathogenic Pseudomonas species

Protein phosphorylation on serine, threonine, and tyrosine is well established as a crucial regulatory posttranslational modification in eukaryotes. With the recent whole‐genome sequencing projects reporting the presence of serine/threonine kinases and two‐component proteins both in prokaryotes and eukaryotes, the importance of protein phosphorylation in archaea and bacteria is gaining acceptance. While conventional biochemical methods failed to obtain a snapshot of the bacterial phosphoproteomes, advances in MS methods have paved the way for in‐depth mapping of phosphorylation sites. Here, we present phosphoproteomes of two ecologically diverse non‐enteric Gram‐negative bacteria captured by a nanoLC‐MS‐based approach combined with a novel phosphoenrichment method. While the phosphoproteome data from the two species are not very similar, the results reflect high similarity to the previously published dataset in terms of the pathways the phosphoproteins belong to. This study additionally provides evidence to prior observations that protein phosphorylation is common in bacteria. Notably, phosphoproteins identified in Pseudomonas aeruginosa belong to motility, transport, and pathogenicity pathways that are critical for survival and virulence. We report, for the first time, that motility regulator A, probably acting via the novel secondary messenger cyclic diguanylate monophosphate, significantly affects protein phosphorylation in Pseudomonas putida.     

Phosphoproteomic analysis of the human pathogen Trypanosoma cruzi at the epimastigote stage

Trypanosoma cruzi is the etiologic agent of Chagas disease, which affects millions of people in Latin America and has become a public health concern in the United States and areas of Europe. The possibility that kinase inhibitors represent novel anti‐parasitic agents is currently being explored. However, fundamental understanding of the cell‐signaling networks requires the detailed analysis of the involved phosphorylated proteins. Here, we have performed a comprehensive MS‐based phosphorylation mapping of phosphoproteins from T. cruzi epimastigote forms. Our LC‐MS/MS, dual‐stage fragmentation, and multistage activation analysis has identified 237 phosphopeptides from 119 distinct proteins. Furthermore, 220 phosphorylation sites were unambiguously mapped: 148 on serine, 57 on threonine, and 8 on tyrosine. In addition, immunoprecipitation and Western blotting analysis confirmed the presence of at least seven tyrosine‐phosphorylated proteins in T. cruzi. The identified phosphoproteins were subjected to Gene Ontology, InterPro, and BLAST analysis, and categorized based on their role in cell structure, motility, transportation, metabolism, pathogenesis, DNA/RNA/protein turnover, and signaling. Taken together, our phosphoproteomic data provide new insights into the molecular mechanisms governed by protein kinases and phosphatases in T. cruzi. We discuss the potential roles of the identified phosphoproteins in parasite physiology and drug development.     

Inhibitor analysis of protein phosphorylation/dephosphorylation in maize mitochondria in relation to redox conditions

The role of protein phosphorylation/ dephosphorylation in the redox regulation of mitochondrial functioning was investigated. Incubation of isolated mitochondria of maize (Zea mays L.) in the presence of γ-³²P-ATP revealed phosphorylation of polypeptides with mol wt of 66, 60, 55, 48/50 doublet, 45, 29, 22, and 19 kD. The presence in the incubation medium of oxidized glutathione significantly reduced the level of protein phosphorylation. The addition of reduced glutathione diminished phosphorylation of proteins with mol wt of 60 and 48/50 kD and slightly increased phosphorylation of proteins with mol wt of 66, 55, and 45 kD. The reducing agent, sodium dithionite decreased phosphorylation of proteins with mol wt of 60, 45, 29, 22, and 19 kD but increased phosphorylation of 55 kD protein. The inhibitors of protein kinases and protein phosphatases significantly modified the effects of redox agents. For example, simultaneous action of an oxidant K₃[Fe(CN)₆] and NaF enhanced phosphorylation level compared to separate treatments with these agents. The combined application of sodium dithionite and NaF elevated phosphorylation level of 55 kD protein. Phosphoprotein with mol wt of about 66 kD was identified immunochemically as a heat shock protein (HSP 60). The results indicate the presence in mitochondria of redox-sensitive protein kinases and protein phosphatases. Differential changes in the pattern of mitochondrial phosphoproteins under the action of various redox agents suggest that phosphorylation is probably involved in the transduction of redox signal in plant mitochondria.     

Effects of Corticotropin-(1–24)-Tetracosapeptide on Polyphosphoinositide Metabolism and Protein Phosphorylation in Rabbit Iris Subcellular Fractions

Abstract: Effects of the neuropeptide corticotropin-(1–24) -tetracosapeptide (ACTH) on the endogenous and exogenous phosphorylation of lipids and endogenous phosphorylation of proteins were investigated in microsomes and a 110,000 ×g supernatant fraction [30–50% (NH₄)₂SO₄ precipitate; ASP_30–50] obtained from rabbit iris smooth muscle. Subcellular distribution studies revealed that both of these fractions are enriched in diphosphoinositide (DPI) kinase. The ³²P labeling of lipids and proteins was measured by incubation of the subcellular fractions with [γ-³²P]ATP. The labeled lipids, which consisted of triphosphoinositide (TPI), DPI, and phosphatidic acid (PA) were isolated by TLC. The microsomal and ASP_30–50 fractions were resolved into six and nine labeled phosphoprotein bands, respectively, by sodium dodecyl sulfate polyacrylamide gel electrophoresis. The basal labeling of both lipids and proteins was rapid (30–60 s), and it was dependent on the presence of Mg²⁺ in the incubation medium; in general it was inhibited by high concentrations (>0.2 mM) of Ca²⁺. ACTH stimulated the labeling of TPI and inhibited that of PA in a dose-dependent manner, with maximal effect observed at 50–100 μ of the peptide. ACTH appears to increase TPI labeling by stimulating the DPI kinase. Under the same experimental conditions ACTH (100 μM) inhibited significantly the endogenous phosphorylation of six microsomal phosphoproteins (100K, 84K, 65K, 53K, 48K, and 17K). In the ASP_30–50 fraction, ACTH inhibited the phosphorylation of three phosphoproteins (53K, 48K, and 17K) and stimulated the labeling of six phosphoprotein bands (117K, 100K, 84K, 65K, 42K, and 35K). The effects of ACTH on lipid and protein phosphorylation are probably Ca²⁺-independent; thus the neuropeptide effects were not influenced by either 1 μM EGTA or low concentrations of Ca²⁺ (50 μ.M). We conclude that a relationship may exist between polyphosphoinositide metabolism and protein phosphorylation in the rabbit iris smooth muscle.     

Characteristics of Ca2+/calmodulin- and Ca2+/phosphatidylserine-stimulated phosphoproteins in rat striatum

The characteristics of endogenous Ca²⁺/calmodulin (CaM)- and Ca²⁺/phosphatidylserine (PS)-stimulated phosphorylated proteins in the striatum of rat were partially determined and compared in this study. The Ca²⁺/CaM-dependent phosphoproteins were associated with serine and threonine residues. The sensitivity of these proteins for phosphorylation by Ca²⁺/CaM was not affected by pretreatment of tissue with Ca²⁺ chelating agent, EGTA or with non-ionic detergent, Triton X-114. Triton X-114 phase separation experiments revealed that these Ca²⁺/CaM-dependent phosphoproteins were partitioned in the detergent rich phase suggesting that they are integral proteins of the striatal membrane. On the other hand, the Ca²⁺/PS-dependent phosphorylated proteins were primarily associated with the serine residue. Phosphorylation of these proteins by Ca²⁺/PS were abolished after the treatment with EGTA or Triton X-114. These results suggest that Ca²⁺/PS-dependent striatal phosphoproteins are biochemically unstable in maintaining their state of phosphorylation.     

Specific protein phosphorylation induced in Xanthomonas campestris pv. oryzae by bacteriophage Xp12

We have investigated the endogenous phosphorylation patterns of phosphorylated proteins of Xanthomonas campestris pv. oryzae induced by its bacteriophages. For bacteriophage Xp12-infected cells, at least three phosphoproteins with apparent molecular weights of 28, 28.5 and 45kDa were detected by in vitro labeling with [-³²P]-ATP. These Xp12-specific phosphoproteins only occurred with Xp12 infection, and were not shown in uninfected or Xp10-infected cells. The protein kinase(s) responsible could use either ATP or GTP as the nucleotide substrate with nearly the same efficiency. Magnesium was proved to be an essential factor for the phosphorylation. EGTA treatment excluding the possibility that the presumed protein kinase was calcium-dependent. Under our reaction conditions, the optimal phosphorylation occurred at pH 7 to 8, for 30 to 40 min at 25 to 37°C. The Xp12-specific protein phosphorylation hint the existence of a physiological regulation mechanism involved in the life cycle of bacteriophage Xp12. Furthermore, the presumed protein kinase was shown to be encoded by the genome of Xp12 rather than indirectly induced by Xp12 infection.     

Phosphorylation of thylakoid proteins during chloroplast biogenesis in greening etiolated and light-grown wheat leaves

Phosphorylation of polypeptides in isolated thylakoids was examined during chloroplast biogenesis in greening etiolated wheat leaves and 4 day-old wheat leaves grown under a diurnal light regime. At early stages of plastid development standard thylakoid preparations were heavily contaminated with nuclear proteins, which distorted the polypeptide phosphorylation profiles. Removal of contamination from membranes by sucrose density centrifugation demonstrated that the major membrane phosphoprotein in etioplasts was at 35 kDa. During etioplast greening a number of phosphoproteins appeared, of which the 25–27 kDa apoproteins of the light-harvesting chlorophylla/b protein complex associated with photosystem II (LHCII) became the most dominant. At the early stages of thylakoid development found at the base of the 4-day-old light grown leaf the LHCII apoproteins were evident as phosphoproteins; however the major phosphoprotein was polypeptide atca. 9kDA. Phosphorylation of both the LHCII apoproteins and the 9 kDa polypeptide in these thylakoids was not light-dependent. In the older thylakoids isolated from the leaf tip the LHCII apoproteins were the major phosphoproteins and their phosphorylation had become light-regulated; however phosphorylation of the 9 kDa polypeptide remained insensitive to light.     

Thylakoid-protein phosphorylation during the life cycle of Scenedesmus obliquus in synchronous culture

The phosphorylation of thylakoid proteins, which comprise apoproteins of the light-harvesting chlorophyll a/b-protein complex (LHCP), was investigated in vivo and in vitro during the development of Scenedesmus obliquus in synchronous cultures. The in-vitro and in-vivo protein phosphorylation exhibited a maximum activity in cells with maximum photosynthetic capacity (8th hour) and miximum activity in cells with minimum photosynthetic capacity (16th hour). The major phosphorylated polypeptides in vivo were the 24/25-kDa and 28–30-kDa apoprotein of the LHCP, a protein of about 32 kDa, and some smaller polypeptides within the range 10 to 20 kDa. In vitro, the main phosphoproteins were the 28–30-kDa apoprotein and the protein characterized by an apparent molecular weight of 32 kDa. Pulse-chase experiments in vivo established that the latter had the fastest radioactivity turnover of the thylakoidal phosphoproteins.Abbreviations DCMU 3-(3,4-dichlorophenyl)-1,1-dimethylurea - LHCP light-harvesting chlorophyll a/b-protein complex - PSII photosystem II Dedicated to Prof. Erwin Bünning on the occasion of his 80^th birthday     

Evidence for selective regulation of the phosphorylation of myocyte proteins by isoproterenol and prostaglandin E1

Both isoproterenol and prostaglandin E₁ increased the activation state of cyclic AMP-dependent protein kinase in cultured myocytes; however, only isoproterenol enhanced phosphorylase activity and contractile state. Following the incubation of intact myocytes with ³²PO₄^3?, 32 phosphoproteins were resolved from total cellular proteins by electrophoresis in sodium dodecyl sulfate polyacrylamide gels followed by autoradiography. Isoproterenol stimulated ³²PO₄^3? incorporated into 16 proteins, including 2 phosphoproteins not observed under control conditions. By contrast, prostaglandin E₁ neither caused a measurable change in the protein phosphorylation pattern nor interfered with isoproterenol's capacity to do so. Isoproterenol stimulated myocyte protein phosphorylation in either the presence or absence of extracellular Ca²⁺. The results suggest that the regulation of protein phosphorylation following adenylate cyclase stimulation is: (1) an agonist-specific process and not due solely to a random accumulation of intracellular cycle AMP and activation of protein kinase; (2) the Ca²⁺ mobilization component of β-receptor activation does not account for the paradoxical effects of isoproterenol and prostaglandins E₁; (3) activation of cyclic AMP-dependent protein kinase does not always result in an enhancement of protein phosphorylation.