The quantity of protein-bound [32P]phosphotyrosine in hepatocytes and fibroblasts. The effects of tyrosine protein kinase activating agents

Tyrosine protein kinase activities have been demonstrated in transformed and normal cell systems. So far, few data on the quantity of protein-bound phosphotyrosine in intact cells have been published. A knowledge of the stoichiometric increase in phosphotyrosine in cells after hormonal induction could be of interest when evaluating the importance of the tyrosine protein kinase activities found. By the addition of a known amount of unlabeled phosphotyrosine to the precipitated protein of 32P-phosphate-labeled cells it was possible after alkaline hydrolysis to spectrophotometrically follow the phosphotyrosine during consecutive chromatographies of the material. From the specific radioactivity of the purified phosphotyrosine the initial concentration of [32P]phosphotyrosine could be calculated. The method proved to be useful for the determination of [32P]phosphotyrosine is small amounts of cells. The minimum detectable amount of [32P]phosphotyrosine was about 1 pmol, and as an example, only 2.5 X 10(6) fibroblasts were needed. By this method it was shown that platelet-derived growth factor increased protein-bound [32P]phosphotyrosine from 600 to 3,200 pmol/g of fibroblasts, while insulin only increased the [32P]phosphotyrosine from 110 to 120 pmol/g of hepatocytes.     

Phosphotyrosine as a substrate of acid and alkaline phosphatases

A new spectrophotometric method for following dephosphorylation of phosphotyrosine has been described. The absorption spectra of phosphotyrosine and tyrosine were plotted over the pH range from 3 to 9. The change in absorbance accompanying the conversion of phosphotyrosine to tyrosine was the greatest at 286 nm. The difference absorption coefficients were calculated for several pH values. Dephosphorylation of phosphotyrosine by acid phosphatases from human prostate gland, from wheat germ and potatoes obeys the Michaelis-Menten equation, whereas alkaline phosphatases calf intestine and E. coli are inhibited by excess of substrate.     

Regulation of protein phosphotyrosine content by changes in tyrosine kinase and protein phosphotyrosine phosphatase activities during induced granulocytic and monocytic differentiation of HL-60 leukemia cells

About 1.5% of phosphorylated amino acid residues of HL-60 promyelocytic leukemia cells are phosphotyrosine. Induction of granulocytic differentiation by exposure to dimethylsulfoxide decreased tyrosine phosphorylation to 0.2%. A maximum 3-fold increase in tyrosine kinase activity and a 7-fold increase in protein phosphotyrosine phosphatase activity accompanied this change. Monocytic differentiation induced by 12-O-tetradecanoylphorbol-13-acetate, caused a decrease in phosphotyrosine levels to 0.1%; tyrosine kinase activity maximally increased 2-fold, and protein phosphotyrosine phosphatase activity increased 11-fold in these differentiated cells. Thus, although total tyrosine kinase activity markedly increased during differentiation, this was counteracted by an even greater elevation in protein phosphotyrosine phosphatase activity. The findings support the concept that tyrosine phosphorylation is important in the regulation of growth and differentiation of leukemia cells.     

A novel approach for evaluating tyrosine kinase activity based on the radioimmunological determination of phosphotyrosine.

A novel technique was designed to conveniently determine substrate phosphorylation by tyrosine kinase. The technique is based on quantitation of phosphotyrosine content of the phosphoproteins, generated during the enzyme reaction, by radioimmunoassay. Here, we utilized high-titer monoclonal antibodies to phosphotyrosine, and radioiodinated bovine serum albumin-phosphotyrosine conjugate. The radiolabeled antigen was displaced from the complex formed in the assay by unlabeled phosphotyrosine, phosphotyrosine derivatives or phosphotyrosine-containing protein substrates. Half-maximal displacement was achieved at 0.4 +/- 0.05 microM by free phosphotyrosine, and at 40 +/- 3 and 45 +/- 4 nM by acetyl-phosphotyrosine and acetyl-phosphotyrosyl-glycine ethyl ester, respectively. Neither phosphoserine, phosphothreonine nor ATP cross-reacted with the phosphotyrosine antibodies. None of the components of the enzyme reaction interfered in the RIA. The method allows quantitation of the incorporated phosphate into tyrosyl residues without interference of serine/threonine phosphorylation. This technique avoids the use of short-lived [gamma-32P]ATP and omits the separation of the phosphorylated substrate from excess nucleotide.     

Pharmacological doses of insulin equalize insulin receptor phosphotyrosine content but not tyrosine kinase activity in plasmalemmal and endosomal membranes.

Following insulin administration to intact rats, the insulin receptor kinase activity of subsequently isolated cell fractions was significantly augmented. Of interest was the observation that the endosomal insulin receptor tyrosine kinase displayed four- to six-fold greater autophosphorylation activity than that of plasma membrane. Surprisingly, the endosomal insulin receptor tyrosine kinase displayed a decrease in beta-subunit phosphotyrosine content compared with that seen in the plasma membrane. These observations prompted the suggestion that insulin receptor tyrosine kinase phosphotyrosine dephosphorylation mediated by an endosome-specific phosphotyrosine phosphatase(s) yields activation of the endosomal insulin receptor tyrosine kinase. In a previous study we examined the effect of subsaturating doses of injected insulin. In this work we evaluated insulin receptor tyrosine kinase activity and phosphotyrosine content in plasma membrane and endosomes after a receptor-saturating pharmacological dose of insulin (150 micrograms/100 g body weight). At this dose the phosphotyrosine content per receptor was reduced compared with that seen earlier at insulin doses of 1.5 and 15 micrograms/100 g body weight. Endosomal insulin receptor tyrosine kinase was greater than that seen at the lower nonsaturating insulin doses. Furthermore, endosomal insulin receptor tyrosine kinase activity exceeded that of the plasma membrane, despite retaining about the same phosphotyrosine content per receptor. These data are consistent with the view that insulin receptor tyrosine kinase activity may be regulated by a particular pattern of phosphotyrosine content on the beta-subunit wherein both activating and inhibitory phosphotyrosine residues play a role.     

Exogenously added free phosphotyrosine induces aggregation of circulating platelets in rabbits.

When free phosphotyrosine is injected into rabbits, circulating aggregated platelets are readily observed in concomitance with altered electrocardiographic profiles. Since phosphotyrosine is also able to induce platelet aggregation in vitro, altogether these results suggest that free phosphotyrosine in blood could be meaningful for in vivo platelet activation.     

Fluorescence of phosphotyrosine--terbium(III) complexes.

Phosphotyrosine, a biologically important protein residue, was investigated for the ability to enhance terbium (Tb3+) fluorescence. Spectroscopic analysis of the Tb3+: phosphotyrosine interaction indicated the development of a new excitation peak at 275 nm and strong Tb+ fluorescence enhancement at 488 and 540 nm that was linear over a range from 0.5 to 100 microM amino acid. Subsequent experiments comparing the ability of phosphotyrosine, phosphothreonine, phosphoserine and 20 other common non-phosphorylated amino acids showed that only phosphotyrosine produced significant Tb3+ fluorescence enhancement. Analysis of various phospho-sugars and nucleotides showed (with the expected exception of GMP) that they produced little or no significant fluorescence enhancement, indicating a further selectiveness for the phosphotyrosine: Tb3+ fluorescence enhancement event. These results establish a basis for the future use of Tb3+ fluorescence enhancement as a unique probe for the investigation of phosphotyrosine residues.     

Impact of epididymal maturation, ejaculation and in vitro capacitation on tyrosine phosphorylation patterns exhibited of boar (Sus domesticus) spermatozoa

Mammalian spermatozoa acquire functionality during epididymal maturation and ability to penetrate and fertilize the oocyte during capacitation. The aim of this study was to investigate the impact of epididymal maturation, ejaculation and capacitation on phosphotyrosine content of sperm proteins. Western blot, immunocytochemical and flow cytometry analyses demonstrated that epididymal maturation in vivo is associated with a progressive loss of phosphotyrosine residues of the sperm head followed by a subtle increase after in vitro capacitation. As cells pass from caput to cauda epididymis, tyrosine phosphorylation becomes confined to a triangular band over the posterior part of midacrosome region, whereas in vitro capacitation causes a spread labeling over the whole head. Different bands with phosphotyrosine residues were detected during epididymal maturation and after in vitro capacitation: 1) 93, 66 and 45 kDa bands with specific phosphotyrosine expression in immature spermatozoa; 2) 76, 23 and 12 kDa bands with specific phosphotyrosine expression in mature spermatozoa, being significantly increased in their expression after in vitro capacitation; 3) 49, 40, 37, 30, 26 and 25 kDa constitutive bands that increased their phosphotyrosine expression after maturation and/or in vitro capacitation; and 4) 28 and 20 kDa bands with a specific phosphotyrosine expression in in vitro capacitated spermatozoa. These results provided integral novel data of expression and location of phosphotyrosine residues during epididymal maturation, ejaculation and in vitro capacitation of boar spermatozoa. Two new constitutive proteins bands of 26 and 25 kDa with phosphotyrosine residues were also identified.     

C2 can do it, too

In this issue of Cell, report that the C2 domain of the serine/threonine protein kinase Cdelta is a phosphotyrosine binding domain and present the crystal structure of this C2 domain bound to a peptide containing phosphotyrosine. Prior to this work, C2 domains were thought to bind only to phospholipids or to unphosphorylated proteins, and the SH2 and PTB domains were the only signaling domains known to recognize phosphotyrosine. This new role for the C2 domain links phosphotyrosine recognition directly to serine/threonine kinase activity and reveals an unexpected mechanism for crosstalk between distinct signaling pathways.     

Comparison of three quantitative phosphoproteomic strategies to study receptor tyrosine kinase signaling

There are three quantitative phosphoproteomic strategies most commonly used to study receptor tyrosine kinase (RTK) signaling. These strategies quantify changes in: (1) all three forms of phosphosites (phosphoserine, phosphothreonine and phosphotyrosine) following enrichment of phosphopeptides by titanium dioxide or immobilized metal affinity chromatography; (2) phosphotyrosine sites following anti- phosphotyrosine antibody enrichment of phosphotyrosine peptides; or (3) phosphotyrosine proteins and their binding partners following anti-phosphotyrosine protein immunoprecipitation. However, it is not clear from literature which strategy is more effective. In this study, we assessed the utility of these three phosphoproteomic strategies in RTK signaling studies by using EphB receptor signaling as an example. We used all three strategies with stable isotope labeling with amino acids in cell culture (SILAC) to compare changes in phosphoproteomes upon EphB receptor activation. We used bioinformatic analysis to compare results from the three analyses. Our results show that the three strategies provide complementary information about RTK pathways.     

The C2 domain of PKCdelta is a phosphotyrosine binding domain

In eukaryotic cells, the SH2 and PTB domains mediate protein-protein interactions by recognizing phosphotyrosine residues on target proteins. Here we make the unexpected finding that the C2 domain of PKCdelta directly binds to phosphotyrosine peptides in a sequence-specific manner. We provide evidence that this domain mediates PKCdelta interaction with a Src binding glycoprotein, CDCP1. The crystal structure of the PKCdelta C2 domain in complex with an optimal phosphopeptide reveals a new mode of phosphotyrosine binding in which the phosphotyrosine moiety forms a ring-stacking interaction with a histidine residue of the C2 domain. This is also the first example of a protein Ser/Thr kinase containing a domain that binds phosphotyrosine.     

Src kinase regulation by phosphorylation and dephosphorylation

Src and Src-family protein-tyrosine kinases are regulatory proteins that play key roles in cell differentiation, motility, proliferation, and survival. The initially described phosphorylation sites of Src include an activating phosphotyrosine 416 that results from autophosphorylation, and an inhibiting phosphotyrosine 527 that results from phosphorylation by C-terminal Src kinase (Csk) and Csk homologous kinase. Dephosphorylation of phosphotyrosine 527 increases Src kinase activity. Candidate phosphotyrosine 527 phosphatases include cytoplasmic PTP1B, Shp1 and Shp2, and transmembrane enzymes include CD45, PTPalpha, PTPepsilon, and PTPlambda. Dephosphorylation of phosphotyrosine 416 decreases Src kinase activity. Thus far PTP-BL, the mouse homologue of human PTP-BAS, has been shown to dephosphorylate phosphotyrosine 416 in a regulatory fashion. The platelet-derived growth factor receptor protein-tyrosine kinase mediates the phosphorylation of Src Tyr138; this phosphorylation has no direct effect on Src kinase activity. The platelet-derived growth factor receptor and the ErbB2/HER2 growth factor receptor protein-tyrosine kinases mediate the phosphorylation of Src Tyr213 and activation of Src kinase activity. Src kinase is also a substrate for protein-serine/threonine kinases including protein kinase C (Ser12), protein kinase A (Ser17), and CDK1/cdc2 (Thr34, Thr46, and Ser72). Of the three protein-serine/threonine kinases, only phosphorylation by CDK1/cdc2 has been demonstrated to increase Src kinase activity. Although considerable information on the phosphoprotein phosphatases that catalyze the hydrolysis of Src phosphotyrosine 527 is at hand, the nature of the phosphatases that mediate the hydrolysis of phosphotyrosine 138 and 213, and phosphoserine and phosphothreonine residues has not been determined.     

Tyrosine phosphorylation of interleukin 2 receptor-related proteins in phytohemagglutinin-activated human lymphocytes

Three classes of proteins (mol wts 70k, 64k and 45k) having the characteristics of interleukin 2 receptor were detected in phytohemagglutinin-activated human lymphocytes using two monoclonal antibodies which recognize distinct epitopes on the receptor. It was shown that at least portions of these proteins were phosphorylated on tyrosine by analyses for phosphotyrosine by immunoblotting and by immunoaffinity chromatography with antibodies to phosphotyrosine. In addition an iodinated phosphotyrosine derivative was identified in partial hydrolysates of these proteins iodinated in vitro.     

The human red cell acid phosphatase is a phosphotyrosine protein phosphatase which dephosphorylates the membrane protein band 3

Human red cell cytosol acid phosphatase activity is supported by a main enzyme which can be extracted by DEAE and phosphocellulose chromatography. It uses pNPP as a substrate and is a protein phosphatase specific to phosphotyrosine. It dephosphorylates the tyrosine-phosphorylated cytosolic fragment of membrane protein 3. When taken together, these results suggest that the physiological role of red cell acid phosphatase is the FB3 phosphotyrosine dephosphorylation. Whatever it may be phosphotyrosine protein phosphatase activity is the first role of red cell acid phosphatase to be demonstrated.     

Regulation of the phosphotyrosine content of human sperm proteins by intracellular Ca2+: role of Ca2+-adenosine triphosphatases

An increase in the concentration of intracellular free Ca2+ and in the phosphotyrosine content of specific proteins characterizes human sperm capacitation. Whether tyrosine phosphorylation regulates the intracellular free Ca2+ concentration through modulation of Ca2+-ATPase activity or the phosphotyrosine content is under Ca2+ regulation was investigated using Ca2+-ATPase modulators and tyrosine kinase inhibitors. The presence of the Ca2+-ATPase-inhibitor thapsigargin during human sperm capacitation caused an increase in the cytoplasmic free Ca2+ concentration and was associated with an increase in the phosphotyrosine content of specific sperm proteins. Conversely, a decrease in protein tyrosine phosphorylation was observed when gingerol, a Ca2+-ATPase activator, was present during the incubation period. On the other hand, thapsigargin had no effect on the phosphotyrosine content or the cytoplasmic Ca2+ concentration when spermatozoa were incubated in the presence of the phosphodiesterase-inhibitor 3-isobutyl-1-methylxanthine (IBMX). However, the effect of IBMX on phosphotyrosine-containing proteins appears to be a Ca2+-dependent phenomenon, because it was partly inhibited in spermatozoa pretreated with 1,2-bis-(o-aminophenoxy)-ethane-N,N,N,N-tetraacetic acid tetra-(acetoxymethyl)-ester (BAPTA-AM) even though, by itself, BAPTA-AM caused an increase in sperm protein phosphotyrosine content. Tyrosine kinase inhibitors prevented the increase in the phosphotyrosine content without affecting the cytoplasmic free Ca2+ concentration. Based on these findings, the present study suggests that Ca2+-ATPases are involved in the filling of internal Ca2+ stores, such as the acrosome, and are inhibited later during capacitation. Their inhibition allows an increase in cytoplasmic free Ca2+, which is involved in the subsequent increase in the phosphotyrosine content of specific sperm proteins.     

Epidermal growth factor induces rapid tyrosine phosphorylation of proteins in A431 human tumor cells

Addition of EGF to A431 cells at physiological concentrations causes a rapid three- to four-fold increase in the abundance of phosphotyrosine in cellular protein. The increase is essentially complete within 1 min and is maintained for several hours. No change in phosphotyrosine levels is found with fibroblast growth factor or insulin. Two phosphoproteins (molecular weights of 39 and 81 kd) containing phosphotyrosine appear de novo upon administration of EGF to A431 cells. The EGF receptor itself is a phosphoprotein containing phosphotyrosine as well as phosphoserine and phosphothreonine. Changes in the phosphorylation pattern of the EGF receptor are seen upon treatment of A431 cells with EGF. Increased phosphorylation of tyrosine is the most rapid response of cells to EGF known, and may play an important role in the biological effects of EGF.     

An ecto-protein tyrosine phosphatase of Entamoeba histolytica induces cellular detachment by disruption of actin filaments in HeLa cells

Actin cytoskeleton disruption in host cells has been demonstrated for PTPases from pathogenic microorganisms. In this work, we analysed whether the secreted acid phosphatase from Entamoeba histolytica has phosphotyrosine phosphatase activity and the possibility that this activity may participate in damaging host cells. The secreted acid phosphatase of E. histolytica, which catalyses p-nitrophenyl phosphate hydrolysis at acid pH values, was found to have phosphotyrosine phosphatase activity. The enzymatic properties of phosphotyrosine phosphatase and acid phosphatase were virtually identical and included: Km values of 10 x 10(-4) M, no requirement for divalent cations, and sensitivity to molybdate, vanadate, and tungstate. The phosphotyrosyl phosphatase activity caused significant levels of cell rounding and detachment correlating with disruption of the actin stress fibres in HeLa cells. Thus, our data suggest that secreted phosphotyrosine phosphatase could play a cytotoxic role during amoebic infection.     

Dopaminergic inhibition of DNA synthesis in pituitary tumor cells is associated with phosphotyrosine phosphatase activity.

Dopaminergic D2 receptor agonists, such as bromocriptine, are potent anti-proliferative agents in the treatment of human pituitary adenomas. We have reproduced the anti-proliferative effect of dopamine in an established pituitary cell line stably transfected with the rat D2 dopamine receptor cDNA. We found that dopaminergic inhibition of DNA synthesis parallels the stimulation of a phosphotyrosine phosphatase activity. Both actions are blocked by pertussis toxin and by the phosphotyrosine phosphatase inhibitor, vanadate. We suggest that the anti-proliferative action of dopamine is mediated, at least in part, by the dopaminergic stimulation of a phosphotyrosine phosphatase.     

Protein tyrosine phosphorylation in response to fertilization

The sea urchin egg contains one or more protein tyrosine kinases which are active during the response of the egg to fertilization. In the present study, we have used an antibody specific for phosphotyrosine to determine which egg proteins are phosphorylated on tyrosine in response to fertilization. Analysis of immunoblots prepared from fertilized and unfertilized eggs revealed that fertilization results in a major increase in the phosphotyrosine content of a 350-kDa egg protein. Increased phosphorylation of this protein was detected as early as 1 min after fertilization, at which time it represented the most prominent phosphotyrosine containing protein in the egg. Tyrosine phosphorylation of this protein was transient however, and after 5 min post-insemination, the protein was dephosphorylated or otherwise degraded. Egg membrane proteins of approximately 40, 75, and 145 kDa were also found to act as substrates for protein tyrosine kinases in vitro, but did not exhibit significant changes in phosphotyrosine content during egg activation.     

Preparation of monoclonal antibodies to phosphotyrosine and their use for identification of phosphotyrosine-containing proteins

Protein kinases phosphorylating proteins at tyrosine residues play an essential role in the cell growth regulation and neoplastic transformation. However, the functions of the majority of tyrosine protein kinases are still obscure, thus creating hindrances in the identification and isolation of phosphotyrosine-containing proteins. The use of the phosphotyrosine structural analog, aminobenzyl phosphonate, as a hapten group enabled the preparation of monoclonal antibodies capable of reacting to phosphotyrosine. The phosphotyrosine specificity of six clones of monoclonal antibodies was tested by a competitive solid phase immunoenzymatic assay. Using fluorescence quenching, the values of constants of binding for antibodies of four clones to phosphotyrosine (2.5-4.0 x 10(6) M-1) were determined. Using two independent methods, it was shown that clone B4 antibodies reveal the highest specificity towards phosphotyrosine. An immunoadsorbent based on clone B4 antibodies was obtained; this immunoadsorbent possessed an ability to selectively interact with an EFR receptor phosphorylated at tyrosine residue. Using eluate acid hydrolysis from the immunoadsorbent, it was demonstrated that clone B4 antibodies interact only with the phosphotyrosine-containing proteins. The experimental results are suggestive of clone B4 monoclonal antibody specificity to phosphotyrosine and of the feasibility of their application for the isolation and identification of tyrosine protein kinases and their substrates.