Evidence of the activity of tyrosine kinase(s) and of the presence of phosphotyrosine proteins in pea plantlets

Homogenate fractions from etiolated pea plantlets showed tyrosine kinase activity when incubated with [32P]ATP and substrates like polyamino acid polymer (Glu-Ala-Tyr)n or [Val5]angiotensin II. When these tyrosine kinase substrates were recovered by high voltage electrophoresis, and analyzed by high pressure liquid chromatography after alkaline hydrolysis, yielded radioactive phosphotyrosine. The same product was obtained after acid hydrolysis of either endogenous or exogenous substrates. Phosphorylated polypeptides were extracted after sodium dodecyl sulfate gel electrophoresis of a pellet fraction incubated with [32P]ATP. After acid hydrolysis and high voltage electrophoresis, [32P]phosphotyrosine was found in gel bands with polypeptides of about 92 and 57 kDa. These results suggest that tyrosine kinase(s) and phosphotyrosine proteins are also present in higher plants.     

Purification and characterization of a protein tyrosine kinase from bovine spleen

Protein tyrosine kinase was purified extensively from a 30,000 X g particulate fraction of bovine spleen by a procedure involving four column chromatographies: DEAE-Sepharose, polyamino acids affinity, hydroxylapatite, and Sephacryl S-200 molecular sieving. The purification resulted in more than 3,000-fold enrichment in [Val5]angiotensin II phosphorylation activity (specific activity 202 nmol/min/mg). All column chromatography profiles showed single protein tyrosine kinase activity peaks with the exception of that of affinity chromatography, where about 50% of the enzyme activity appeared with the breakthrough fraction; only the bound enzyme was further purified. Analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography of a purified sample phosphorylated in the presence of [gamma-32P]ATP revealed the presence of a single phosphorylated polypeptide of molecular weight 50,000 which represents about 40% of total protein. Analysis by polyacrylamide gel electrophoresis under nondenaturing conditions showed that protein tyrosine kinase activity co-migrated with the phosphoprotein. Stoichiometry of the phosphorylation of the 50-kDa polypeptide was found to be 1.0 mol/mol. The purified sample did not appear to contain phosphotyrosine protein phosphatase activity. Both casein and histone could be phosphorylated by the purified sample, and the phosphorylation occurred only at tyrosine residue, suggesting that there was no protein serine and threonine kinase contamination.     

Evidence of Tyrosine Kinase Activity in the Protozoan Parasite Trypanosoma brucei

ABSTRACT. Phosphorylation of proteins at tyrosine is an important mechanism for regulating cell growth and proliferation in metazoan organisms. In this report, we have demonstrated that Trypanosoma brucei , a protozoan parasite, possesses a tyrosine kinase that plays a role in regulation of proliferation of this protozoan. Genistein, a tyrosine kinase inhibitor, prevented multiplication of the parasite. An in vitro kinase assay demonstrated the presence of a kinase capable of phosphorylating an exogenous substrate at tyrosine, and genistein was able to reduce trypanosome-mediated phosphorylation of this substrate. An alkali digestion of ³²P-labeled trypanosome proteins separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis indicated several proteins phosphorylated at tyrosine. These results indicate that T. brucei has a tyrosine kinase that is involved in proliferation or growth regulation of the parasite and provide further evidence for the possibility of growth factor regulation and signal transduction in trypanosomes.     

Evidence of tyrosine kinase activity in the protozoan parasite Trypanosoma brucei.

Phosphorylation of proteins at tyrosine is an important mechanism for regulating cell growth and proliferation in metazoan organisms. In this report, we have demonstrated that Trypanosoma brucei, a protozoan parasite, possesses a tyrosine kinase that plays a role in regulation of proliferation of this protozoan. Genistein, a tyrosine kinase inhibitor, prevented multiplication of the parasite. An in vitro kinase assay demonstrated the presence of a kinase capable of phosphorylating an exogenous substrate at tyrosine, and genistein was able to reduce trypanosome-mediated phosphorylation of this substrate. An alkali digestion of 32P-labeled trypanosome proteins separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis indicated several proteins phosphorylated at tyrosine. These results indicate that T. brucei has a tyrosine kinase that is involved in proliferation or growth regulation of the parasite and provide further evidence for the possibility of growth factor regulation and signal transduction in trypanosomes.     

Modelling of insulin receptor tyrosine kinase in its active form: a case study for validation of theoretical methods

An active form of an insulin receptor tyrosine kinase (IRK) catalytic core was modelled based on its experimentally known inactive form and the active form of a serine/threonine kinase, protein kinase A (PKA). This theoretical model was compared with the crystallographic structure of the active form of IRK reported later. The structures are very similar, which shows that all the most important features and interactions have been taken into account in the modelling procedure. The elaborated procedure can be applied to other tyrosine kinases. This would allow designing of a wide class of tyrosine kinase inhibitors, very important potential anti-cancer and/or anti-viral drugs.     

Tyrosine protein kinase in boar spermatozoa: identification and partial characterization.

A protein-tyrosine kinase has been isolated from a detergent-soluble extract of boar spermatozoa, using poly(Glu, Tyr)4:1 as a substrate. The purification procedure involves sequential column chromatographies on phosphocellulose, polyamino acid affinity and Sephadex G-100 molecular sieving, and results in more than a 1200-fold enrichment. Analysis of the most purified preparation by sodium dodecyl sulfate polyacrylamide gel electrophoresis revealed a major Coomassie blue-stained band of molecular mass 42 kDa. The Tyr-protein kinase does not seem to be autophosphorylable. The Km value for poly(Glu, Tyr)4:1 is relatively low, 2.3 microM, and the tyrosine-polymer phosphorylating activity is apparently inhibited by tyrphostin. The characteristics shown by this new tyrosine kinase--the first to be described in mature male germ cells--support the hypothesis that it belongs to the group of non-receptor-associated tyrosine kinases.     

Western blot analysis of Src kinase assays using peptide substrates ligated to a carrier protein

We have applied intein-mediated peptide ligation (IPL) to the use of peptide substrates for kinase assays and subsequent Western blot analysis. IPL allows for the efficient ligation of a synthetic peptide with an N-terminal cysteine residue to an intein-generated carrier protein containing a cysteine reactive C-terminal thioester through a native peptide bond. A distinct advantage of this procedure is that each carrier protein molecule ligates only one peptide, ensuring that the ligation product forms a sharp band on sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). We demonstrate the effectiveness of this approach by mutational analysis of peptide substrates derived from human cyclin-dependent kinase, Cdc2, which contains a phosphorylation site of human c-Src protein tyrosine kinase.     

Novel yeast protein kinase (YPK1 gene product) is a 40-kilodalton phosphotyrosyl protein associated with protein-tyrosine kinase activity.

Extracts of bakers' yeast (Saccharomyces cerevisiae) contain protein-tyrosine kinase activity that can be detected with a synthetic Glu-Tyr copolymer as substrate (G. Schieven, J. Thorner, and G.S. Martin, Science 231:390-393, 1986). By using this assay in conjunction with ion-exchange and affinity chromatography, a soluble tyrosine kinase activity was purified over 8,000-fold from yeast extracts. The purified activity did not utilize typical substrates for mammalian protein-tyrosine kinases (enolase, casein, and histones). The level of tyrosine kinase activity at all steps of each preparation correlated with the content of a 40-kDa protein (p40). Upon incubation of the most highly purified fractions with Mn-ATP or Mg-ATP, p40 was the only protein phosphorylated on tyrosine. Immunoblotting of purified p40 or total yeast extracts with antiphosphotyrosine antibodies and phosphoamino acid analysis of 32P-labeled yeast proteins fractionated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis indicated that the 40-kDa protein is normally phosphorylated at tyrosine in vivo. 32P-labeled p40 immunoprecipitated from extracts of metabolically labeled cells by affinity-purified anti-p40 antibodies contained both phosphoserine and phosphotyrosine. The gene encoding p40 (YPK1) was cloned from a yeast genomic library by using oligonucleotide probes designed on the basis of the sequence of purified peptides. As deduced from the nucleotide sequence of YPK1, p40 is homologous to known protein kinases, with features that resemble known protein-serine kinases more than known protein-tyrosine kinases. Thus, p40 is a protein kinase which is phosphorylated in vivo and in vitro at both tyrosine and serine residues; it may be a novel type of autophosphorylating tyrosine kinase, a bifunctional (serine/tyrosine-specific) protein kinase, or a serine kinase that is a substrate for an associated tyrosine kinase.     

Anti-IgM-mediated growth inhibition of a human B lymphoma cell line is independent of phosphatidylinositol turnover and protein kinase C activation and involves tyrosine phosphorylation

The RL cell line is an EBV-negative, surface IgM, IgD-positive B lymphoma line, which is significantly growth arrested in the presence of acrylamide-linked antibodies to the surface IgM receptor. We demonstrate here that activation of protein kinase C (PKC) with PMA abrogates anti-IgM-induced phosphoinositide turnover and Ca2+ mobilization; however, growth inhibition is not affected. In addition, inhibitors of PKC are unable to reverse the anti-IgM-mediated growth inhibition. Two-dimensional gel electrophoresis reveals a different pattern of protein phosphorylation after treatment of RL with PMA or anti-IgM. These data strongly suggest that anti-IgM-induced growth inhibition does not rely on phospholipase C-mediated phosphoinositide turnover, Ca2+ mobilization, or PKC activation. On the other hand, the phosphatase inhibitor orthovanadate results in an augmentation of proteins phosphorylated on tyrosine and the growth inhibition which follows anti-IgM treatment. Furthermore, protein tyrosine kinase inhibitors, genistein and herbimycin A, are able to reverse the anti-IgM-induced inhibition of growth. These data demonstrate that multiple signaling pathways are activated by the interaction of anti-IgM with its ligand, and suggest that tyrosine kinase activation is a critical component of the inhibitory response.     

Preganglionic stimulation increases the phosphorylation of tyrosine hydroxylase in the superior cervical ganglion by both cAMP-dependent and Ca2+-dependent protein kinases

Electrical stimulation of the preganglionic cervical sympathetic trunk increases the phosphorylation of tyrosine hydroxylase in the superior cervical ganglion of the rat by a nicotinic mechanism and by a noncholinergic mechanism. We have measured the incorporation of [32P]Pi into specific tryptic phosphopeptides in tyrosine hydroxylase in order to identify the protein kinases that phosphorylate this enzyme in electrically stimulated ganglia. 32P-labeled tyrosine hydroxylase was isolated from the ganglion by immunoprecipitation and polyacrylamide gel electrophoresis and was subjected to tryptic hydrolysis. Seven tryptic peptides were resolved from these hydrolysates by two-dimensional thin-layer electrophoresis and chromatography. Preganglionic stimulation (20 Hz, 5 min) increased the incorporation of 32P into four of these peptides. In the presence of cholinergic antagonists, however, electrical stimulation increased the labeling of only one phosphopeptide. From a comparison of the effects of preganglionic stimulation with the effects of agonists that activate specific protein kinases, we conclude that electrical stimulation increases the phosphorylation of tyrosine hydroxylase by both a cAMP-dependent protein kinase and a Ca2+/calmodulin-dependent protein kinase. The nicotinic component of preganglionic stimulation appears to be mediated by a Ca2+/calmodulin-dependent protein kinase, while the noncholinergic component appears to be mediated by cAMP-dependent protein kinase. Although protein kinase C can phosphorylate tyrosine hydroxylase, this kinase does not appear to participate in the stimulation-induced phosphorylation of tyrosine hydroxylase in the superior cervical ganglion.     

Characterization of dual specificity protein kinase from maize seedlings

A protein kinase of 57 kDa, able to phosphorylate tyrosine in synthetic substrates pol(Glu4,Tyr1) and a fragment of Src tyrosine kinase, was isolated and partly purified from maize seedlings (Zea mays). The protein kinase was able to phosphorylate exogenous proteins: enolase, caseins, histones and myelin basic protein. Amino acid analysis of phosphorylated casein and enolase, as well as of phosphorylated endogenous proteins, showed that both Tyr and Ser residues were phosphorylated. Phosphotyrosine was also immunodetected in the 57 kDa protein fraction. In the protein fraction there are present 57 kDa protein kinase and enolase. This co-purification suggests that enolase can be an endogenous substrate of the kinase. The two proteins could be resolved by two-dimensional electrophoresis. Specific inhibitors of typical protein-tyrosine kinases had essentially no effect on the activity of the maize enzyme. Staurosporine, a nonspecific inhibitor of protein kinases, effectively inhibited the 57 kDa protein kinase. Also, poly L-lysine and heparin inhibited tyrosine phosphorylation by 57 kDa maize protein kinase. The substrate and inhibitor specificities of the 57 kDa maize protein kinase phosphorylating tyrosine indicate that it is a novel plant dual-specificity protein kinase.     

In vitro phosphorylation of bovine adrenal tyrosine hydroxylase by adenosine 3':5'-monophosphate-dependent protein kinase.

We have studied the effects of adenosine 3':5'-monophosphate (cAMP)-dependent protein kinase on the phosphorylative and functional modification of bovine adrenal tyrosine hydroxylase. Incubation of partially purified tyrosine hydroxylase with cAMP-dependent protein kinase in the presence of [gamma32P]ATP and 5 micron cAMP led to a 3- to 5-fold activation of tyrosine hydroxylase and to incorporation of [32P]phosphate into protein. When tyrosine hydroxylase preparations activated by exposure to enzymatic phosphorylating conditions were analyzed by sucrose density gradient centrifugation, polyacrylamide gel electrophoresis, and gel electrofocusing, the radioactivity of 32P was coincident with the activity of tyrosine hydroxylase, suggesting incorporation of 32P from [gamma-32P]ATP into tyrosine hydroxylase. Polyacrylamide gel electrophoresis of the phosphorylated tyrosine hydroxylase preparation in the presence of 0.1% sodium dodecyl sulfate revealed that the 60,000-dalton polypeptide subunit of tyrosine hydroxylase served as the phosphate acceptor.     

Multiple site phosphorylation of tyrosine hydroxylase. Differential regulation in situ by a 8-bromo-cAMP and acetylcholine

Suspension cultures of purified bovine adrenal chromaffin cells incorporated 32P from exogenous 32Pi into a protein of approximately M4 = 60,000 (isolated by discontinuous, sodium dodecyl sulfate-polyacrylamide slab gel electrophoresis). Phosphorylated tyrosine hydroxylase, purified from chromaffin cell supernatants by immunoprecipitation, co-migrated with the Mr = 60,000 band. Tryptic fragments prepared fom either the Mr congruent to 60,000 band or the immunoprecipitated tyrosine hydroxylase band were analyzed after separation with two-dimensional electrophoresis/chromatography. Two distinct 32P-peptides were present in either sample. After a 2-3-min lag period. 32P incorporation into both peptides was relatively linear with time for at least 20 min. In the presence of calcium, exogenous acetylcholine (100 microM) increased 32P incorporation into both of the 32P-labeled tryptic peptides whereas 8-bromo-cAMP (1 mM) increased 32P incorporation into only one of the two. Ethylene glycol bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid and MnCl2 inhibited the acetylcholine-induced phosphorylation of both tryptic peptides. Thus, tyrosine hydroxylase is phosphorylated in situ at more than one site, and the phosphorylation of these sites is affected differently by acetylcholine and 8-bromo-cAMP. The data imply that kinase activity other than (or in addition to) cAMP-dependent protein kinase activity attends tyrosine hydroxylase in the intact chromaffin cells and that multiple kinase activities may be involved in the short term regulation of catecholamine biosynthesis by afferent activity.     

Evidence of tyrosine kinase activity in the photosynthetic bacterium Rhodospirillum rubrum

The photosynthetic bacterium Rohodospirillum rubrum evidenced tyrosine protein phosphorylation under photoautotrophic conditions in the presence of [32P]Pi. The stability to alkaline treatment of the [32P] bound to the cell-free extract proteins suggested that tyrosine residues were carrying the labelling. One- and two-dimensional high voltage paper electrophoresis analysis revealed that such extracts do contain [32P]-phosphotyrosine residues. Furthermore, the association of alkali stable [32P] bound to specific proteins of the cell-free extract was confirmed by sodium dodecyl sulfate polyacrylamide gel electrophoresis combined with KOH treatment of the gel. A definite argument in favor of protein kinase(s) phosphorylating tyrosine residues in R.rubrum proteins was obtained by partial purification of a tyrosine kinase activity from cell-free extract capable of phosphorylating synthetic peptides that only contain a single tyrosine residue as phosphate acceptor.     

Insulin-mimetic effect of trypsin on the insulin receptor tyrosine kinase in intact adipocytes

It has previously been demonstrated that the insulin-mimetic agent trypsin stimulates autophosphorylation of purified insulin receptors and activates the insulin receptor tyrosine kinase in vitro. We now report the effects of trypsin on whole cell tyrosine kinase activation and insulin receptor autophosphorylation. Trypsin treatment of intact adipocytes produces a time-dependent stimulation of tyrosine kinase activity as measured in lectin extracts containing the insulin receptor, or specifically immunoprecipitated insulin receptor samples. Trypsin treatment of adipocytes also results in a loss of insulin binding capacity, and a linear correlation exists between loss of binding and stimulation of tyrosine kinase activity. Exposure of adipocytes to trypsin is known to result in a time- and dose-dependent activation of intracellular glycogen synthase. Examination of the time courses of stimulation of tyrosine kinase and glycogen synthase activation in our system indicates that the stimulation of tyrosine kinase activity by trypsin occurs with sufficient rapidity and magnitude to be consistent with a role of phosphorylation in the activation of glycogen synthase. Trypsin has further been demonstrated to stimulate autophosphorylation of the beta-subunit of the insulin receptor in intact adipocytes. Cells prelabeled with [32P]PO4 for 2 h were exposed to trypsin, and receptors were partially purified over wheat germ agglutinin-agarose columns. Receptors were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and the beta-subunit was identified by autoradiography. The protein was extracted and hydrolyzed, and the phosphoamino acids were separated by electrophoresis and quantitated. Two- and five-fold increases in phosphotyrosine were observed with 3 and 10 min of trypsin treatment, respectively. We conclude that trypsin-induced cleavage of the insulin receptor alpha-subunit is relevant to the ability of trypsin to activate the insulin receptor tyrosine kinase in intact adipocytes. We further conclude that autophosphorylation of the insulin receptor and activation of its tyrosine kinase by trypsin may be important to the insulin-mimetic anabolic effects of trypsin.     

Tyrosine phosphorylation of a 94-kDa protein of human fibroblasts stimulated by streptococcal lipoteichoic acid

Lipoteichoic acid (LTA) is an amphipathic component of Gram-positive bacteria. Previous studies from this laboratory have shown that at low concentrations, ranging from 0.1 to 10.0 micrograms/ml, LTA binds to mammalian cells and stimulates mitogenic responses as demonstrated by increased DNA and RNA synthesis. Tyrosine kinase appears to be involved in the action of a number of mitogens including epidermal growth factor, platelet-derived growth factor, and insulin. In the present study, we report the novel finding that tyrosine protein kinase activity is increased in human fibroblasts treated with LTA. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography of the whole cell lysate of fibroblasts cultured with 32Pi showed increased phosphorylation of a 94-kDa polypeptide. Alkali treatment of the gel resulted in a decreased intensity of the 94-kDa phosphorylated protein in control cells, but not in LTA-treated cells, suggesting the addition of phosphate groups to threonine or tyrosine residues. High voltage electrophoresis of the acid hydrolysate of the excised and eluted 94-kDa protein revealed that LTA stimulated the phosphorylation of tyrosine but not threonine residues. These results suggest that LTA acts on mammalian cells by phosphorylating tyrosine residues of certain proteins and thereby may regulate diverse functions of these cells.     

Differential tyrosine phosphorylation of leukemic cells during apoptosis as a result of treatment with imatinib mesylate

Bcr-Abl fusion tyrosine kinase contributes to leukemic transformation. Imatinib mesylate inhibits Bcr-Abl tyrosine kinase, resulting in a blockage of tyrosine phosphorylation in its downstream pathways. We analyzed the alteration of tyrosine phosphorylation, on BCR/ABL+ chronic myelogenous leukemia cells, after treatment with imatinib mesylate. Data were collected using a two-dimensional gel electrophoresis followed by Western blot and mass spectrometry. The inhibition of Bcr-Abl tyrosine kinase by 2.5 microM imatinib mesylate caused both cell cycle arrest in the G0/G1 phase and increased the portion of apoptotic cells. As a result, the population of leukemic cells decreased by 30% and 70% compared to controls at 24 and 72 h, respectively. Furthermore, treatment with imatinib mesylate altered tyrosine phosphorylation of 24 protein spots as the incubation time proceeded from 0 to 24 and 72 h. Ten of the 24 protein spots are visible at all three times. Four are detectable at both the 0 and 24 h points in time. Eight were detectable only at time 0.     

Tyrosine phosphorylation of a membrane protein from Pseudomonas solanacearum.

We have investigated a tyrosine kinase activity from Pseudomonas solanacearum, an economically important plant pathogen. In vitro incubation of membrane fractions with [gamma-32P]ATP and subsequent sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed an 85-kDa phosphoprotein. Phosphorylation of this protein on tyrosine residues was demonstrated by phosphoamino acid analysis of base hydrolysis products and by immunoanalysis of Western blots (immunoblots) with antiphosphotyrosine monoclonal antibody. In vitro incubation of membranes with ATP was not required for recognition by the antibody, indicating that the 85-kDa protein is phosphorylated in vivo. These results demonstrate that membranes from P. solanacearum exhibit a tyrosine kinase activity toward an endogenous membrane protein. This bacterium provides an opportunity to study the structure and function of a prokaryotic tyrosine kinase.     

A tyrosine kinase related to pp60c-src is associated with membranes of Electrophorus electricus electric organ

A tyrosine-specific protein kinase immunologically related to pp60c-src, the cellular homolog of the Rous sarcoma virus-transforming protein, was expressed at elevated levels in the electric organ of the electric eel Electrophorus electricus. The electric organ kinase phosphorylated antibodies reactive with pp60c-src at tyrosine residues in immune complex protein kinase assays and was associated with electric organ membranes enriched in acetylcholine receptors. The protein recognized by anti-pp60c-src antibodies was phosphorylated in endogenous membrane phosphorylation reactions and was shown to have a relative molecular mass of 57 kDa by two-dimensional gel electrophoresis. In immune complex protein kinase assays the 57-kDa protein was phosphorylated at threonine by a distinct threonine kinase from the electric organ. The tyrosine kinase was purified 844-fold from electric organ membranes by chromatography on omega-aminohexyl agarose, phosphocellulose, and casein-Sepharose. Threonine kinase activity in immunoprecipitates was not observed in the tyrosine kinase fractions after the first step. Incubation of the casein Sepharose fraction with [gamma-32P]ATP-Mn2+ in solution resulted in phosphorylation of only the 57-kDa protein. Phosphorylation occurred solely at tyrosine, suggesting that the kinase is capable of autophosphorylation. The structural and functional properties of the 57-kDa electric organ kinase indicate that the 57-kDa electric organ protein is a member of the src subfamily of tyrosine kinases and is closely related to pp60c-src.     

Isolation and identification of tyrosine kinase from the cytosolic fraction of bovine retinal rod outer segments]

It was shown that the cytosol fraction of bovine retinal rod outer segments contains three forms of tyrosine kinase. One of them was purified 171-fold to attain a specific activity of 1.6 nmol/min per mg protein. The isolated protein had a molecular weight of about 54,000 in SDS electrophoresis. It was shown that this protein is a tyrosine-specific protein kinase, capable of autophosphorylation at the tyrosine residues and restoration of kinase activity upon denaturation-renaturation.