Acid phosphatase and phosphoamino acid phosphatases in murine erythroleukaemic cells

The activities of acid and alkaline phosphatases and phosphotyrosine, phosphoserine and phosphothreonine phosphatases were measured in Friend murine erythroleukaemic (MEL) cells. The effects of treating the cells with dimethyl sulphoxide (DMSO), an inducer of differentiation, were examined. In untreated cells alkaline phosphatase activity was undetectable, though there were significant amounts of acid phosphatase (76 +/- 15 mU/mg protein) and phosphotyrosine phosphatase (16 +/- 0.9mU/mg protein); phosphoserine and phosphothreonine phosphatase activities (9 +/- 0.4 and 7 +/- 0.6mU/mg protein, respectively) were lower than for phosphotyrosine phosphatase. Addition of 1 or 2% DMSO to the culture medium resulted in the expected cell death within 2 weeks. With 0.5% DMSO, cells remained viable for at least 8 weeks, but while some appeared to have smaller nuclei and retained their rounded appearance, others became fibroblastic within several days and adhered to the culture vessel. The treated cells which had kept their morphology showed no difference in acid phosphatase activities as compared with untreated controls; phosphotyrosine phosphatase was lower (9 +/- 0.8mU/mg protein) and phosphoserine and phosphothreonine phophatases higher (11 +/- 0.5 and 10 +/- 0.4mU/mg protein, respectively) than in the controls. The Km values for p-nitrophenyl phosphate were similar in untreated and treated cells (0.069 and 0.068mM, respectively); for phosphotyrosine the Km value was lower in the treated cells (0.97mM) than in the controls (1.9mM).     

Phosphoamino acid phosphatases in human normal and leukaemic lymphocytes

The activities of phosphoamino acid phosphatases were measured in lymphocytes from normal adults. For cells from white individuals the mean value (+/- SD) for phosphotyrosine phosphatase activity was 342 +/- 120 mU/mg protein; mean values for phosphothreonine and phosphoserine phosphatase activities were 35.6 +/- 17.1 and 21.6 +/- 10.2 mU/mg protein, respectively. The corresponding activities were similar to these for both the Indian and Bantu population groups. The activity of phosphotyrosine phosphatase was significantly lower in lymphocytes from white children with acute lymphocytic leukaemia than in cells from normal children or adults. For phosphothreonine and phosphoserine phosphatases, there was no significant difference between activities obtained in lymphocytes from normal and leukaemic children.     

Regulation of protein phosphatase activity by regucalcin localization in rat liver nuclei.

The regulatory role of regucalcin on protein phosphatase activity in isolated rat liver nuclei was investigated. Phosphatase activity toward phosphotyrosine and phosphoserine was significantly increased by the addition of CaCl(2) (10(-5) and 10(-4) M) in the enzyme reaction mixture. Trifluoperazine (25 and 50 microM), an antagonist of calmodulin, significantly inhibited protein phosphatase activity toward phosphoserine, while it had no effect on the enzyme activity toward phosphotysine and phosphothreonine. Cyclosporin A (10(-6)-10(-4) M), an inhibitor of Ca(2+)/calmodulin-dependent protein phosphatase activity toward phosphoserine, but not phosphotyrosine and phosphoserine. Thus, Ca(2+)/calmodulin-dependent phosphatases were present in liver nuclei. Regucalcin (0.25 and 0.5 microM) had an inhibitory effect on liver nuclear phosphatase activity toward phosphotyrosine, phosphoserine, and phosphothreonine. The presence of anti-regucalcin monoclonal antibody (25 and 50 ng/ml) in the enzyme reaction mixture caused a significant elevation of nuclear phosphatase activity toward three phosphoaminoacids. An analysis with sodium sulfate-polyacrylamide gel electrophoresis suggested a possibility of localization of regucalcin in liver nuclei. Moreover, regucalcin was determined in liver nuclei using enzyme-linked immunoadsorbent assay. The present study demonstrates that the endogenous regucalcin inhibits phosphatase activity in the liver nuclei.     

Expression of c-myc gene in human ovary carcinoma cells treated with vanadate

The widely accepted hypothesis of vanadate action on cells postulates that this ion inhibits protein phosphatase(s) that dephosphorylates protein phosphotyrosine residues. This inhibition causes tyrosine hyperphosphorylation of cell proteins followed by changes in physiological action of phosphoproteins resulting in stimulation of cell proliferation, expression of protooncogenes, and transient cell transformation. We have found that treatment of human ovary carcinoma (CaOv) cells with vanadate causes the increase in total protein phosphorylation from 1.5- to 2.0-fold whereas the ratio between phosphoserine, phosphothreonine, and phosphotyrosine content remains unchanged. At the same time, enhancement of c-myc gene expression (not c-fos) was observed. Hence, the increase in the ratio of phosphotyrosine to phosphoserine and phosphothreonine is not an obligatory intermediate stage before vanadate-dependent activation of c-myc expression.     

Inhibition of membrane phosphotyrosyl-protein phosphatase activity by vanadate

We have investigated the effect of vanadate on the phosphoserine- and phosphotyrosine-specific phosphoprotein phosphatase activities of A-431 cell membranes and have found that micromolar concentrations of vanadate strongly inhibit the membrane-dependent dephosphorylation of histones containing phosphotyrosine but that they do not inhibit the dephosphorylation of histones containing phosphoserine and phosphothreonine. In addition, the dephosphorylation of endogeneous membrane proteins of A-431 cells (which are known to be phosphorylated at tyrosine residues) was inhibited by vanadate. These results show that vanadate is a potent and selective inhibitor of phosphotyrosyl-protein phosphatase.     

Effects of epidermal growth factor and 12-O-tetradecanoylphorbol-13-acetate on metabolism of the epidermal growth factor receptor in normal human fibroblasts.

The biosynthesis, phosphorylation, and degradation of the epidermal growth factor (EGF) receptor were examined in normal human fibroblasts. The receptor was initially synthesized as an Mr = 160,000 immature form which matured to an Mr = 170,000 form in a monensin-sensitive manner. Tunicamycin treatment led to the accumulation of an Mr = 130,000 protein. The receptor was phosphorylated on serine and threonine residues in normally growing and quiescent cells, and treatment with EGF or the tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA) resulted in a two- to threefold increase in receptor-bound phosphate. EGF increased the amount of phosphoserine and phosphothreonine and caused the appearance of a minor amount of phosphotyrosine. TPA increased the levels of phosphoserine and phosphothreonine exclusively. Prior treatment with TPA inhibited the EGF-dependent appearance of phosphotyrosine in the receptor. Analysis of tryptic phosphopeptides revealed that six of the seven major peptides were common to the receptor from cells treated with EGF or TPA. EGF strongly stimulated [3H]thymidine incorporation in confluent cells, increased final saturation density three to fourfold, and increased whole-cell levels of phosphotyrosine about threefold. Treatment of cells with TPA before addition of EGF inhibited all three of these EGF-dependent responses. EGF also decreased the receptor half-life from 15 h to 1 h, but this was not inhibited by TPA. TPA alone had no detectable effect on the receptor half-life.     

Enhancement of neutral phosphatase activity in the cytosol and nuclei of regenerating rat liver: role of endogenous regucalcin.

The role of endogenous regucalcin (RC) in the regulation of neutral phosphatase activity in regenerating rat liver was investigated. The liver weight reduced by a partial hepatectomy (about 70%) was completely restored at 72 h after surgery. Phosphotyrosine, phosphoserine, and phosphothreonine were used as the substrate for the assay of phosphatase activity. Phosphatase activity toward phosphotyrosine in the hepatic cytosol and nuclei was significantly increased at 24-72 h after hepatectomy. Such an increase was not seen in the case of phosphoserine and phosphothreonine. However, the presence of anti-RC monoclonal antibody (200 ng/ml) in the enzyme reaction mixture caused a remarkable elevation of phosphatase activity toward three phosphoaminoacids in the hepatic cytosol at 24 and 48 h after hepatectomy. In the liver nuclei after sham operation or hepatectomy, phosphatase activity toward three phosphoaminoacids was significantly raised by the addition of anti-RC antibody (150 ng/ml). The nuclear phosphatase activity toward phosphothreonine in regenerating liver was significantly enhanced in the presence of anti-RC antibody (100 and 150 ng/ml). The effect of anti-RC antibody to increase phosphatase activity toward three phosphoaminoacids in the cytosol and nuclei of regenerating liver was completely blocked by the addition of exogenous RC (1.0 microM). The present study demonstrates that protein phosphatase activity in the cytoplasm and nuclei is enhanced in regenerating rat liver. This enhancement may be suppressed by endogenous RC.     

Alkaline phosphatase and phosphoamino acid phosphatases in normal and cancerous tissues of the human larynx

The activities of alkaline phosphatase and phosphoamino acid phosphatases were measured in normal and cancerous regions of the human larynx. For each larynx, alkaline phosphatase and phosphotyrosine phosphatase activities were higher in the tumor than in the corresponding normal tissue. Phosphothreonine and phosphoserine phosphatase activities were relatively low and there were no consistent trends. The increased alkaline phosphatase activity in the tumors supports histological observations that ossification of cartilage seems to occur at the site of invasion; the phosphatase acting on phosphotyrosine could serve as a regulator of cell differentiation during tumorigenesis.     

Inhibitory effect of regucalcin on protein phosphatase activity in the nuclei of rat kidney cortex

The role of regucalcin, which is a regulatory protein of calcium signaling, in the regulation of protein phosphatase activity in the nuclei of rat kidney cortex was investigated. Protein phosphatase activity towards phosphotyrosine, phosphoserine, and phosphothreonine was found in the nuclei. The enzyme activity towards three phosphoamino acids was significantly increased by the addition of calcium chloride (10-50 microM) in the enzyme reaction mixture. This increase was significantly inhibited by trifluoperazine (25 or 50 microM), an antagonist of calmodulin. The presence of regucalcin (50 or 100 nM) in the enzyme reaction mixture caused a significant decrease in protein phosphatase activity towards three phosphoamino acids. This effect was also seen in the presence of calcium (25 microM) and/or calmodulin (5 microg/ml). Protein phosphatase activity towards three phosphoamino acids was significantly increased in the presence of anti-regucalcin monoclonal antibody (25 or 50 ng/ml) in the enzyme reaction mixture. This effect was completely blocked by the addition of regucalcin (100 nM). The effect of antibody (25 ng/ml) in increasing protein phosphatase activity towards phosphotyrosine was significantly inhibited by vanadate (10(-4) M). Also, the antibody's effect towards phosphoserine and phosphothreonine was significantly inhibited by cyclosporin A (10(-5) M). Endogenous regucalcin was found in the nuclei of rat kidney cortex using Western blot analysis. Nuclear regucalcin level was significantly reduced by the administration of saline (0.9% NaCl) for seven days in rats. Protein phosphatase activity towards three phosphoamino acids was significantly decreased by saline administration. The effect of anti-regucalcin monoclonal antibody (25 ng/ml) in increasing protein phosphatase activity towards three phosphoamino acids was weakened in the renal cortex nuclei of saline-administrated rats. The present study demonstrates that endogenous regucalcin plays a suppressive role in the regulation of protein phosphatase activity in the nuclei of rat kidney cortex cells.     

Localisation of High Acid Phosphotyrosine Phosphatase Activity in Afferent Arterioles and Glomeruli of Human Kidney

Summary Endothelial cells contain a variety of specific protein tyrosine phosphatases and an acid phosphatase differing from other known phosphatases. The highest activity of this acid phosphatase with artificial or unspecific substrates is present in the afferent arterioles and glomeruli of human kidney, and the activity is inhibited by nephrotoxic fluoride concentrations, suggesting that it plays a role in circulatory regulation. Here the activity was characterised with physiological substrates. An incubation mixture containing phosphotyrosine or phosphoserine was stable at pH 5 when phosphate-precipitating lead was chelated with tartrate. The activities were studied in frozen sections. Only phosphotyrosine was hydrolysed by some cells. High activity of tartrate-resistant phosphotyrosine phosphatase was present in lymphocytes, endothelial cells of afferent arterioles, and glomerular mesangial cells of kidney, decidual cells, and alveolar macrophages. In lymphocytes the activity was fluoride-resistant and vanadate-sensitive, in other cells fluoride- and vanadate-sensitive. In decidual cells and alveolar macrophages, the activity is due to specific osteoclastic/macrophagic tartrate-resistant acid phosphatase, in lymphocytes to specific protein tyrosine phosphatases, and in endothelial and mesangial cells to a protein tyrosine phosphatase-like acid phosphatase. The results suggest that in endothelial cells of the afferent arterioles, mesangial cells, and lymphocytes the cellular activities are regulated by high constitutive phosphotyrosine phosphatase activity and this may be related to the exceptional cyclosporin A sensitivity of these cells.     

Characteristics of ether-linked glycerophospholipids in Friend erythroleukaemia cells differentiated by dimethyl sulphoxide or hexamethylenebisacetamide and in non-inducible clones treated with the inducers.

The present study deals with changes in ether-linked glycerophospholids which accompany differentiation of Friend erythroleukaemia (FEL) cells by dimethyl sulphoxide (DMSO) and hexamethylenebisacetamide (HMBA). We also tested clones of FEL cells non-inducible by DMSO or HMBA for ether-linked lipid changes not related to the differentiation process. FEL cells contained appreciable proportions of alkenylacyl and alkylacyl subfractions in phosphatidylethanolamine (PE) and phosphatidylcholine (PC). Compared with FEL cells, clones non-inducible by DMSO or HMBA had a greater amount of alkenylacyl PE associated with a lack of alkenylacyl PC. The differentiation of FEL cells by DMSO or HMBA was accompanied by a reduction of alkylacyl PE and PC. DMSO- and HMBA-differentiated FEL cells showed changes in alkenyl- and alkyl-chain profiles, some of which were also observed in non-inducible FEL cells treated with DMSO or HMBA.     

Tyrosine phosphorylation of the insulin receptor beta subunit activates the receptor-associated tyrosine kinase activity

The regulation of kinase activity associated with insulin receptor by phosphorylation and dephosphorylation has been examined using partially purified receptor immobilized on insulin-agarose. The immobilized receptor preparation exhibits predominately tyrosine but also serine and threonine kinase activities toward insulin receptor beta subunit and exogenous histone. Phosphorylation of the insulin receptor preparation with increasing concentrations of unlabeled ATP, followed by washing to remove the unreacted ATP, results in a progressive activation of the receptor kinase activity when assayed in the presence of histone and [gamma-32P]ATP. A maximal 4-fold activation is achieved by prior incubation of receptor with concentrations of ATP approaching 1 mM. High pressure liquid chromatographic analysis of tryptic hydrolysates of the 32P-labeled insulin receptor beta subunit reveals three domains of phosphorylation (designated peaks 1, 2, and 3). Phosphotyrosine and phosphoserine residues are present in these three domains while peak 2 contains phosphothreonine as well. Thus, at least seven sites are available for phosphorylation on the beta subunit of the insulin receptor. Incubation of the phosphorylated insulin receptor with alkaline phosphatase at 15 degrees C results in the selective dephosphorylation of the phosphotyrosine residues on the beta subunit of the receptor while the phosphoserine and phosphothreonine contents are not affected. The dephosphorylation of the receptor is accompanied by a marked 65% inhibition of the receptor kinase activity. Almost 90% of the decrease in [32P]phosphate content of the receptor after alkaline phosphatase treatment is accounted for by a decrease in phosphotyrosine content in peak 2, while very small decreases are observed in peaks 1 and 3, respectively. These results demonstrate that the extent of phosphorylation of tyrosine residues in receptor domain 2 closely parallels the receptor kinase activity state, suggesting phosphorylation of this domain may play a key role in regulating the insulin receptor tyrosine kinase.     

Protein tyrosine phosphorylation in the cyanobacterium Anabaena sp. strain PCC 7120.

Components of a protein tyrosine phosphorylation/dephosphorylation network were identified in the cyanobacterium Anabaena sp. strain PCC 7120. Three phosphotyrosine (P-Tyr) proteins of 27, 36, and 52 kDa were identified through their conspicuous immunoreactions with RC20H monoclonal antibodies specific for P-Tyr. These immunoreactions were outcompeted completely by free P-Tyr (5 mM) but not by phosphoserine or phosphothreonine. The P-Tyr content of the three major P-Tyr proteins and several minor proteins increased with their time of incubation in the presence of Mg-ATP and the protein phosphatase inhibitors sodium orthovanadate and sodium fluoride. Incubation of the same extracts with [gamma-32P]ATP but not [alpha-32P]ATP led to the phosphorylation of five polypeptides with molecular masses of 20, 27, 52, 85, and 100 kDa. Human placental protein tyrosine phosphatase 1B, with absolute specificity for P-Tyr, liberated significant quantities of 32Pi from four of the polypeptides, confirming that a portion of the protein-bound phosphate was present as 32P-Tyr. Alkaline phosphatase and the dual-specificity protein phosphatase IphP from the cyanobacterium Nostoc commune UTEX 584 also dephosphorylated these proteins and did so with greater apparent efficiency. Two of the polypeptides were partially purified, and phosphoamino analysis identified 32P-Tyr, [32P]phosphoserine, and [32P]phosphothreonine. Anabaena sp. strain PCC 7120 cell extracts contained a protein tyrosine phosphatase activity that was abolished in the presence of sodium orthovanadate and inhibited significantly by the sulfhydryl-modifying agents p-hydroxymercuriphenylsulfonic acid and p-hydroxymercuribenzoate as well as by heparin. In Anabaena sp. strain PCC 7120 the presence and/or phosphorylation status of P-Tyr proteins was influenced by incident photon flux density.     

Ecto-phosphatase activities on the cell surface of the amastigote forms of Trypanosoma cruzi

Live Trypanosoma cruzi amastigotes hydrolyzed p-nitrophenylphosphate (PNPP), phospho-amino-acids and 32P-casein under physiologically appropriate conditions. PNPP was hydrolysed at a rate of 80 nmol.mg-1.h-1 in the presence of 5 mM MgCl2, pH 7.2 at 30 degrees C. In the absence of Mg2+ the activity was reduced 40% and we call this basal activity. At saturating concentration of PNPP, half-maximal PNPP hydrolysis was obtained with 0.22 mM MgCl2. Ca2+ had no effect on the basal activity, could not substitute Mg2+ as an activator and in contrast inhibited the PNPP hydrolysis stimulated by Mg2+ (I50 = 0.43 mM). In the absence of Mg2+ (basal activity) the stimulating half concentration (S0.5) for PNPP was 1.57 mM, while at saturating MgCl2 concentrations the corresponding S0.5 for PNPP for Mg(2+)-stimulated phosphatase activity (difference between total minus basal phosphatase activity) was 0.99 mM. The Mg-dependent PNPP hydrolysis was strongly inhibited by sodium fluoride (NaF), vanadate and Zn2+ but not by tartrate and levamizole. The Mg-independent basal phosphatase activity was insensitive to tartrate, levamizole as well NaF and less inhibited by vanadate and Zn2+. Intact amastigotes were also able to hydrolyse phosphoserine, phosphothreonine and phosphotyrosine but only the phosphotyrosine hydrolysis was stimulated by MgCl2 and inhibited by CaCl2 and phosphotyrosine was a competitive inhibitor of the PNPP hydrolysis stimulated by Mg2+. The cells were also able to hydrolyse 32P-casein phosphorylated on serine and threonine residues but only in the presence of MgCl2. These results indicate that in the amastigote form of T. cruzi there are at least two ectophosphatase activities, one of which is Mg2+ dependent and can dephosphorylate phospho-amino acids and phosphoproteins under physiological conditions.     

小鼠腹水型肝癌H22a细胞浆内磷蛋白磷酸酶活力调节的研究

小鼠腹水型肝癌细胞胞浆内磷蛋白磷酸酶对磷酸化的组蛋白、酪蛋白、鱼精蛋白具有脱磷酸化活力,而对小分子底物P-Ser、P-Thr、P-Tyr、PNPP等无活力。二价金属离子Mn~(2+)、Co~(2+)、Mg~(2+)对酶有明显激活作用,而Zn~(2+)、F~-、Pi对酶有明显抑制作用。代谢中间物G-6-P、G-1-P、F-6-P、F-1.6-2P、ATP、ADP、GTP对酶有抑制作用,而磷酸化氨基酸和环核苷酸对酶活影响很小。还试验了碱性蛋白质和酸性蛋白质对酶活力的影响,肝素和组蛋白均对酶活力有抑制作用,当两者混和后,其抑制作用会相互抵消。     

A rapid microdetermination of phosphoserine, phosphothreonine, and phosphotyrosine in proteins by automatic cation exchange on a conventional amino acid analyzer

A cation-exchange chromatographic method for the separation and determination of phosphoserine, phosphothreonine, and phosphotyrosine in proteins after partial acid hydrolysis is described. The short column (0.6 X 8 cm) of an automatic amino acid analyzer was used and elution was carried out isocratically with 10 mM trifluoroacetic acid. The method is highly sensitive and each of the three O-phosphoamino acids can be accurately determined down to the 50-pmol level. Higher sensitivity may be obtained by the use of [32P]phosphate-labeled proteins. A correction factor for the decomposition of phosphoserine or phosphothreonine during acid hydrolysis can be deduced from the amount of inorganic phosphate recovered at the column void volume. The method is sensitive enough to be used for 32P-labeled proteins isolated by two-dimensional gel electrophoresis.     

Vasopressin dependent tyrosine phosphorylation of a 38 kDa protein in human platelets.

Arginine vasopressin administration (10(-10)-10(-6) M) to isolated human platelets induces an increase in the specific immunoblotting of a 38 kDa protein revealed by a phosphotyrosine antibody. This signal is biphasic with maximal stimulation within one minute. Neither forskolin (10(-5) M) nor phorbol ester (10(-6) M) produces a similar 38 kDa signal. The specific immunoblotted signals are competitively abolished by 1 mM phosphotyrosine but not phosphoserine or phosphothreonine. Electrophoretic separation at pH 3.5 of the acid hydrolysates of the 38 kDa proteins reveals a vasopressin dependent increase in levels of phosphotyrosine as well as phosphoserine and phosphothreonine. The 38 kDa phosphorylation is also induced by the specific arginine vasopressin V1 receptor agonist (Phe2Orn8Vastocina) and blocked by the V1 receptor antagonist [desGly(NH2)d(CH2)5Tyr(Me) AVPb]. These observations suggest that arginine vasopressin signal transduction may be associated with the tyrosine phosphorylation of a 38 kDa protein.     

Interleukin 1 and tumour necrosis factor increase phosphorylation of the small heat shock protein. Effects in fibroblasts, Hep G2 and U937 cells

Interleukin 1 alpha and tumour necrosis factor-alpha stimulated phosphorylation of three 27 kDa phosphoproteins in MRC-5 fibroblasts which was sustained for up to 2 h after adding the cytokines. All three phosphoproteins were immunoprecipitated by a specific antiserum to the small mammalian heat shock protein, hsp 27. The three phosphoproteins from stimulated or control cells contained phosphoserine but not phosphothreonine or phosphotyrosine. Similar increases in phosphorylation of immunoprecipitable 27 kDa proteins were seen in U937 cells stimulated by TNF alpha and Hep G2 cells stimulated by IL1 alpha.     

Purification and characterization of a phosphotyrosyl-protein phosphatase from wheat seedlings

A neutral phosphatase which catalyzes the hydrolysis of p-nitrophenylphosphate has been purified to homogeneity from wheat seedlings. The enzyme is a monomeric glycoprotein exhibiting a molecular weight of 35,000, frictional ratio of 1.22, Stokes' radius of 260 nm, and sedimentation coefficient of 3.2 S. That the enzyme is a glycoprotein is surmised from its chromatographic property on Concanavalin A-Sepharose column. An examination of the substrate specificity indicates that the enzyme exhibits a preference for phosphotyrosine over a number of phosphocompounds, including p-nitrophenylphosphate and several glycolytic intermediates. Both phosphoserine and phosphothreonine are not hydrolyzed by the enzyme. The phosphatase activity is not affected by high concentrations of chelating agents and does not require metal ions. Molybdate, orthovanadate, Zn2+, and Hg2+ are all potent inhibitors of the phosphatase activity. The ability of the phosphatase to dephosphorylate protein phosphotyrosine has been investigated. [32P-Tyr]poly(Glu,Tyr)n, [32P-Tyr]alkylated bovine serum albumin, [32P-Tyr]angiotensin-I, and [32P-Tyr]band 3 (from human erythrocyte) are all substrates of the phosphatase. On the other hand, the enzyme has no activity toward protein phosphoserine and phosphothreonine. Our result further indicates that the neutral phosphatase is distinct from the wheat germ acid phosphatase. The latter enzyme is found to dephosphorylate phosphotyrosyl as well as phosphoseryl and phosphothreonyl groups in proteins. In light of the many similarities in properties to phosphotyrosyl protein phosphatases isolated from several sources, it is suggested that the wheat seedling phosphatase may participate in cellular regulation involving protein tyrosine phosphorylation.     

Quantitative proteomic approaches for studying phosphotyrosine signaling

Protein tyrosine phosphorylation is a fundamental mechanism for controlling many aspects of cellular processes, as well as aspects of human health and diseases. Compared with phosphoserine and phosphothreonine, phosphotyrosine signaling is more tightly regulated, but often more challenging to characterize, due to significantly lower levels of tyrosine phosphorylation (i.e., a relative abundance of 1800:200:1 was estimated for phosphoserine/phosphothreonine/phosphotyrosine in vertebrate cells). In this review, we outline recent advances in analytical methodologies for enrichment, identification and accurate quantitation of tyrosine-phosphorylated proteins and peptides. Advances in antibody-based technologies, capillary liquid chromatography coupled with mass spectrometry, and various stable isotope labeling strategies are discussed, as well as non-mass spectrometry-based methods, such as those using protein/peptide arrays. As a result of these advances, powerful tools now have the power to crack signal transduction codes at the system level, and provide a basis for discovering novel drug targets for human diseases.