The preparation and use of pyridoxal [32P]phosphate as a labeling reagent for proteins on the outer surface of membranes.

Pyridoxal [32P] phosphate was prepared using [gamma-32P] ATP, pyridoxal, and pyridoxine kinase purified from Escherichia coli B. The pyridoxal [32P] phosphate obtained had a specific activity of at least 1 Ci/mmol. This reagent was used to label intact influenza virus, red blood cells, and both normal and transformed chick embryo fibroblasts. The cell or virus to be labeled was incubated with pyridoxal [32P] phosphate. The Schiff base formed between pyridoxal [32P] phosphate and protein amino groups was reduced with NaBH4. The distribution of pyridoxal [32P] phosphate in cell membrane or virus envelope proteins was visualized by autoradiography of the proteins separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis. The labeling of the proteins of both influenza and chick cells appeared to be limited exclusively to those on the external surface of the virus or plasma membrane. With intact red blood cells the major portion of the probe was bound by external proteins, but a small amount of label was found associated with the internal proteins spectrin and hemoglobin.     

Characterization of casein kinase II from a virally transformed macrophage-like cell line, RAW264

Casein kinase II from a virally-transformed macrophage cell line (RAW264) was purified by a sequential DEAE, Procion Red, phosvitin-Sepharose and heparin-Sepharose chromatography. With [tau-32P]GTP as a phosphate donor and casein as a substrate, the kinase was stimulated by polyamines and inhibited by heparin. The purified kinase had a specific activity of 1137 nmol/min/mg protein and exhibited three major protein bands of 40 K, 35 K, and 25 K. Under non-denaturing conditions in 50 mM Tris-50 mM NaCl the enzyme was eluted as a single peak with molecular weight of 110 K. Incubation of kinase in the presence of [tau-32P]GTP and Mg2+ resulted in phosphorylation of the 25 K protein band of the enzyme. In the presence of [tau-32P]GTP and Mg2+ the kinase was able to phosphorylate 55 K protein band in purified ornithine decarboxylase preparation from RAW264 cells and the rat-type II regulatory subunit of the cyclic AMP-dependent protein kinase.     

Autophosphorylation and autoactivation of spleen protein tyrosine kinase

Incubation of a highly purified bovine spleen protein tyrosine kinase with [gamma-32P]ATP and Mg2+ resulted in a gradual radioactive labeling of the protein kinase (50 kDa) with no change in the protein kinase activity toward angiotensin II. On the other hand, treatment of the protein tyrosine kinase with an immobilized alkaline phosphatase caused essentially complete loss in the kinase activity, which could be restored by incubation of the enzyme with ATP and Mg2+. By using the alkaline phosphatase-treated kinase, time courses of the protein phosphorylation and the enzyme activation were demonstrated to correlate closely. These results indicate that this protein tyrosine kinase relies on autophosphorylation for activity and that the purified enzyme usually exists in a fully phosphorylated state. The radioactive labeling of the purified kinase during incubation with [gamma-32P]ATP resulted from a phosphate exchange reaction: the exchange of [gamma-32P]phosphate of ATP with the protein bound phosphate as previously suggested (Kong, S.K., and Wang, J.H. (1987) J. Biol. Chem. 262, 2597-2603). It could be shown that the autophosphorylation of phosphatase-treated tyrosine kinase was strongly inhibited by the substrate angiotensin II, whereas the exchange reaction carried out with untreated tyrosine kinase was not. Autophosphorylation is suggested to be an intermolecular reaction since its initial rate is proportional to the square of the protein concentration.     

The preparation and use of pyridoxal [32P] phosphate as a labeling reagent for proteins on the outer surface of membranes

Pyridoxal [³²P] phosphate was prepared using [γ-³²P]ATP, pyridoxal, and pyridoxine kinase purified from Escherichia coli B. The pyridoxal [³²P] phosphate obtained had a specific activity of at least 1 Ci/mmol. This reagent was used to label intact influenza virus, red blood cells, and both normal and transformed chick embryo fibroblasts. The cell or virus to be labeled was incubated with pyridoxal [³²P] phosphate. The Schiff base formed between pyridoxal [³²P] phosphate and protein amino groups was reduced with NaBH₄. The distribution of pyridoxal [³²P] phosphate in cell membrane or virus envelope proteins was visualized by autoradiography of the proteins separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis.The labeling of the proteins of both influenza and chick cells appeared to be limited exclusively to those on the external surface of the virus or plasma membrane. With intact red blood cells the major portion of the probe was bound by external proteins, but a small amount of label was found associated with the internal proteins spectrin and hemoglobin.     

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.     

Analysis of tyrosine kinase activity in cell extracts using nondenaturing polyacrylamide gel electrophoresis

A general procedure for detecting tyrosine kinase activity in crude or purified preparations using nondenaturing gel electrophoresis is presented. Samples are resolved by electrophoresis in minigels which are then incubated in an assay mixture containing [gamma-32P]ATP, poly(glutamic acid, tyrosine)4:1, and cofactors. Subsequently, gels are fixed and washed in trichloroacetic acid-pyrophosphate, dried, and analyzed by autoradiography or liquid scintillation counting. The procedure is simple and fast and allows analysis of different molecular weight forms of tyrosine kinase under linear kinetics at 37 degrees C without interference from phosphatases and proteases.     

Partial purification and characterization of a new p36/40 tyrosine protein kinase from HL-60

A major peak of tyrosine protein kinase activity was partially purified from a Triton X100 extract of HL-60. This preparation submitted to high pressure gel filtration was eluted at a volume corresponding to a mass of 35/40 kD. This activity was insensitive to EGF and insulin. Autoradiographs of the preparations incubated with [gamma P32]-ATP and separated by electrophoresis do not give any evidence that autophosphorylation occurs for that particular tyrosine protein kinase. Furthermore, we failed to immunoprecipitate the enzyme with a specific antiphosphotyrosine antibody and anti v-src antibody. All the data presented herein suggest that this enzyme has not been previously purified.     

Prostatic epithelial cells in culture: phosphorylation of protein tyrosyl residues and tyrosine protein kinase activity.

The ability of dividing canine prostatic epithelial cells in primary monolayers to phosphorylate protein tyrosyl residues was evaluated by metabolic studies performed through incorporation of [32P]-phosphate into alkali-resistant phosphoproteins and by the assay of their tyrosine protein kinase activity. The presence of sodium orthovanadate during cell incubation with [32P]-phosphate greatly enhanced the relative labelling intensity of a 44 kDa alkali-resistant phosphoprotein and the total cellular content of phosphotyrosine in proteins; in this respect, growth factors such as epidermal growth factor, insulin, and insulin-like growth factor I, and the steroids dihydrotestosterone and estradiol were inactive. When the cells were solubilized, sodium orthovanadate stimulated their tyrosine protein kinase activity and inhibited their phosphotyrosine phosphatase activity. To characterize the tyrosine protein kinase of these cultured cells, conditions for optimal activity were established using the substrate poly [Glu80Na, Tyr20]. The subcellular localization of the enzyme was determined upon cell fractionation: 88% of the kinase activity was associated with the particulate fraction and 30% of this activity was partially solubilized with 0.5% Triton X-100; this solubilization was improved to 83% in the presence of 0.25 M KCI. The enzyme directly solubilized from prostatic cells with Triton X-100 (38% of activity) mainly catalyzed the alkali-resistant phosphorylation of pp63, pp59, and pp44, which contained phosphotyrosine. These proteins were also phosphorylated by the major peak of kinase activity which was eluted at an apparent molecular weight of 300-350 kDa upon gel filtration.(ABSTRACT TRUNCATED AT 250 WORDS)     

Detection of protein kinase activity in sodium dodecyl sulfate-polyacrylamide gels

A procedure is described for identifying protein kinase activity in protein samples following electrophoresis on sodium dodecyl sulfate-polyacrylamide gels. Protein kinase activity is detected by renaturation of the enzymes within the gel followed by phosphorylation with [gamma-32P]ATP of either substrates included in the polyacrylamide gel or of the kinase itself. Then, after removal of the unreacted [gamma-32P]ATP by washing the gel in the presence of an anion-exchange resin, the positions (Mr) of the protein kinase activity are visualized by autoradiography. Studies using a purified catalytic subunit of cAMP-dependent protein kinase indicate that enzyme concentrations as low as 0.01 microgram can easily be detected on gels containing 1 mg/ml casein. The technique is also useful for identifying active subunits of multisubunit enzymes. The active subunit of casein kinase II, for example, can readily be determined by renaturing the dissociated enzyme in gels containing casein. Putative protein kinases present in crude mixtures of proteins can also be detected following separation by gel electrophoresis and can be characterized on the basis of molecular weight and identity of the phosphorylated amino acid. Using this technique, at least three major protein kinases were detected in a mixture of proteins prepared by subfraction of red blood cell membranes.     

The activated human met gene encodes a protein tyrosine kinase

We have raised antibodies against a synthetic dodecapeptide corresponding to the carboxyl terminus of the predicted met gene product. Phosphorylation of 60 kDa and 65 kDa proteins on tyrosine residues was observed when immunoprecipitates of cells containing the activated human met gene were incubated with [gamma-32P]ATP. Phosphoproteins with the same molecular masses could be immunoprecipitated from cells metabolically labelled with [32P]orthophosphate. When considered together, these observations indicate that the activated human met gene encodes 60 kDa and 65 kDa proteins that can catalyse autophosphorylation on tyrosine residues.     

Phosphorylation of glucokinase from rat liver in vitro by protein kinase A with a concomitant decrease of its activity

Glucokinase, purified from rat liver, was phosphorylated to an extent of 1 mol [32P]-phosphate/mol of enzyme when incubated with [32P]ATP and protein kinase A from pig or rabbit muscle. The phosphate was bound to serine residues. K0.5 increased and Vmax decreased upon phosphorylation. The phosphate group was removed during incubation of the phosphorylated glucokinase with alkaline phosphatase. Enzymatically inactive glucokinase was not phosphorylated by the protein kinase.     

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.     

Tyrosine protein kinase activity of rat spleen and other tissues

Using a synthetic peptide (Glu-Asp-Ala-Glu-Tyr-Ala-Ala-Arg-Arg-Arg-Gly) as a substrate, various normal tissues from the rat were probed for tyrosine protein kinase activity. Spleen was shown to contain much higher tyrosine protein kinase activity than other rat tissues (lung, brain, testes, liver, kidney, heart, and thymus, in decreasing order of specific activity). Most of the tyrosine protein kinase activity of the various rat tissues (greater than 80%) was present in the particulate fraction, and Nonidet P-40, a nonionic detergent, could activate the particulate form of the enzyme 2-20-fold in many of the tissues. Epidermal growth factor (1 microgram/ml), cyclic AMP, cyclic GMP, or Ca2+ did not increase spleen tyrosine protein kinase activity. Half-maximal enzyme activity was observed at 60-80 microM MgATP and at 2.2 mM peptide, and both Mg2+ (10 mM) and Mn2+ (0.5-1.0 mM) were effective divalent metal ions for the expression of activity. When the particulate fraction of spleen was incubated with [gamma-32P]ATP followed by polyacrylamide gel electrophoresis in the presence of Na dodecyl SO4, a number of alkali-stable bands were identified by autoradiography. Two major bands at Mr = 53,000 and 56,000 were shown to contain phosphotyrosine. Two similar alkali-stable bands containing phosphotyrosine but with lower amounts of 32P labeling were also observed in the particulate fractions of various other tissues (lung, brain, kidney, and testes). The particulate form of tyrosine protein kinase of rat spleen could be solubilized by using high concentrations of Nonidet P-40 (5%) at an alkaline pH (pH 9.0). Partial purification and subsequent filtration on Sephacryl S-200 yielded a peak of tyrosine protein kinase activity with an apparent molecular weight of 55,000. The two major phosphorylated bands of Mr = 53,000 and 56,000 co-migrated with the peak of enzyme activity. The solubilized and partially purified enzyme preparation phosphorylated only tyrosine residues when either endogenous proteins or casein were used as substrates. These results suggest that relatively high activities of tyrosine protein kinase exist in a normal tissue (rat spleen). Major endogenous substrates of the enzyme(s) appear to be represented by two proteins of Mr = 53,000 and 56,000; one or both of these substrates may be the tyrosine protein kinase itself.     

Sites of in vivo phosphorylation of the T cell tyrosine protein kinase in LSTRA cells and their alteration by tumor-promoting phorbol esters

The LSTRA cell line contains an elevated level of a tyrosine protein kinase of apparent molecular weight of 56,000 (pp56Tcell). Analysis of the tryptic fragments of this protein labeled in vivo with 32P shows that it contains four sites of tyrosine phosphorylation and one site of serine phosphorylation. Two of the sites of in vivo tyrosine phosphorylation are also labeled in vitro when membranes are incubated with [gamma-32P]ATP. One of the sites that is labeled in vivo and in vitro (site 1) is identical in sequence with the major site of tyrosine phosphorylation in the transforming protein of the Rous sarcoma virus. Analysis of the sites of in vivo phosphorylation in pp56Tcell from LSTRA cells treated with 4 beta-phorbol 12 beta-myristate 13 alpha-acetate (PMA) reveals that this agent induces at least four new sites of serine phosphorylation. Treatment with PMA also causes a selective reduction in the level of tyrosine phosphorylation in site 1. Thus PMA causes new sites of serine phosphorylation in pp56Tcell and reduces the amount of phosphate in one of the sites of tyrosine phosphorylation.     

A 3-aminobenzamide-resistant labeled protein in [32P]NAD+-labeled cells

A series of proteins are covalently labeled when human lymphocytes are incubated with [32P]NAD+. The majority of this labeling is effectively inhibited when the lymphocytes are coincubated with 3-aminobenzamide, a potent inhibitor of poly(ADP-ribose) polymerase. However, labeling of a 72 000 molecular weight protein was resistant to the inhibitory effect of 3-aminobenzamide. Labeling of this protein from [32P]NAD+ was shown to be Mg2+-dependent. The 72 000 molecular weight protein could also be labeled on incubation with [alpha-32P]ATP, [gamma-32P]ATP and [32P]orthophosphate, but not from [3H]NAD+ or [14C]NAD+. In the present study, we show that the 72 000 molecular weight protein is not ADP-ribosylated but rather, phosphorylated on incubation with [32P]NAD+. This phosphorylation appears to occur via an Mg2+-dependent conversion of NAD+ to AMP with the eventual utilization of the alpha-phosphate for phosphorylation of the 72 000 molecular weight protein.     

Ca2+-dependent protein phosphorylation associated with microsomal fraction of rat pancreas

Microsomes isolated from cat pancreas were incubated with [gamma-32P]ATP in the presence or absence of Ca2+. Following fractionation of phosphoproteins by sodium dodecyl sulfate-polyacrylamide gel electrophoresis a single microsomal protein with an apparent molecular mass of 77,000 dalton (77K) was found to be phosphorylated in a Ca2+-dependent mechanism. Maximal phosphate incorporation into the 77K protein was observed at 10(-6) mol/l [Ca2+] and was 4-fold higher than in the absence of Ca2+. The 77K phosphoprotein showed characteristic of a stable phosphoester rather than an acyl phosphate. Measurable phosphate incorporation into the 77K protein was noted 5 s following addition of [gamma-32P]ATP and reached maximum at 9-10th min. The lack of effect of exogenous cyclic AMP, cyclic AMP-dependent protein kinase, calmodulin, the calmodulin antagonist trifluoperazine, leupeptin and the suppression of phosphorylation by some phospholipid-interacting drugs suggested that the 77K protein is a substrate for cyclic AMP- and calmodulin-independent, Ca2+-activated phospholipid-sensitive kinase activity. Centrifugation of the pancreatic homogenate in a ficoll-sucrose density gradient indicated that both the 77K protein and enzyme were associated in a fraction enriched in rough endoplasmic reticulum.     

Studies on the active site of yeast hexokinase. Specific phosphorylation of a serine residue induced by D-xylose and ATPMg.

Yeast hexokinase A(ATP:D-hexose 6-phosphotransferase) is inactivated when incubated in the presence of xylose and ATPMg, or in the presence of D-lyxose in a reaction medium in which ATPMg is being continuously regenerated (phosphoenolpyruvate and pyruvate kinase). The inactivation is due to the phorphorylation of the protein. A linear relationship was observed between the inactivation and the incorporation of 32P from [gamma-32P] ATP. All hexokinase and ATPase activity of the enzyme is lost when one phosphoryl group is incorporated per enzyme subunit (molecular weight 51,000). The phosphoryl group is covalently bound by a ester linkage with a serine residue of the protein.     

Phosphorylation of highly purified growth hormone receptors by a growth hormone receptor-associated tyrosine kinase

We have shown previously that growth hormone (GH) promotes the phosphorylation of its receptor on tyrosyl residues (Foster, C. M., Shafer, J. A., Rozsa, F. W., Wang, X., Lewis, S. D., Renken, D. A., Natale, J. E., Schwartz, J., and Carter-Su, C. (1988) Biochemistry 27, 326-334). In the present study, we investigated the possibility that a tyrosine kinase is specifically associated with the GH receptor. GH-receptor complexes were first partially purified from GH-treated 3T3-F442A fibroblasts, a GH-responsive cell, by immunoprecipitation using anti-GH antiserum. 35S-Labeled proteins of Mr = 105,000-125,000 were observed in the immunoprecipitate from GH-treated cells labeled metabolically with 35S-amino-acids. These proteins were not observed in immunoprecipitates from cells not exposed to GH or when non-immune serum replaced the anti-GH antiserum, consistent with the proteins being GH receptors. GH receptors appeared to be phosphorylated, as evidenced by the presence of 32P-labeled bands, comigrating with the 105-125 kDa 35S-labeled proteins, in the immunoprecipitate of GH-treated cells labeled metabolically with [32P]Pi. When partially purified GH receptor preparation was incubated with [gamma-32P]ATP (7-15 microM) for 10 min at 30 degrees C in the presence of MnCl2, a protein of Mr = 121,000 was phosphorylated exclusively on tyrosyl residues. As expected for the GH receptor, this protein was not observed in immunoprecipitates when cells had not been treated with GH nor when non-immune serum replaced the anti-GH antiserum. GH-receptor complexes were also purified to near homogeneity by sequential immunoprecipitation with phosphotyrosyl-binding antibody followed by anti-GH antiserum. When cells were labeled metabolically with 35S-amino acids, the 35S label migrated almost exclusively as an Mr = 105,000-125,000 protein. This protein also incorporated 32P into tyrosyl residues when incubated in solution with [gamma-32P]ATP. These results show that highly purified GH receptor preparations undergo tyrosyl phosphorylation, suggesting that either the GH receptor itself is a tyrosine kinase or is tightly associated with a tyrosine kinase.     

Phosphorylation of Okazaki-like DNA fragments in mammalian cells and role of polyamines in the processing of this DNA.

In mammalian cells DNA synthesis is more complicated than in prokaryotes and less well understood. Here we incubated intact mammalian cells (polyamine auxotrophic Chinese hamster ovary cells and primary human fibroblasts) with [32P]orthophosphate and found that, besides high molecular weight DNA, a species of low molecular weight DNA, approximately 450 bp in size, became efficiently labeled. The short DNA was labeled first, and in pulse-chase experiments the labeling was transient. The isolated small DNA fragments (RNase A-treated) were phosphorylated by T4 polynucleotide kinase specific for polynucleotides with 5'-OH ends. A polynucleotide kinase phosphorylating these DNA pieces was also detected in nuclear extracts of the cells. Treatment with alkaline phosphatase removed most of the 32P label incorporated into the small DNA in vivo. Labeling with deoxyribonucleosides did not reveal these fragments. We hypothesize that the low molecular weight DNA represents Okazaki fragments and that the mammalian DNA replication machinery includes a polynucleotide kinase phosphorylating the 5'-termini of Okazaki fragments. This would imply a novel step in DNA synthesis. We also show that depriving cells of polyamines reversibly blocks synthesis of high molecular weight DNA and leads to accumulation of the short DNA pieces, suggesting a role for polyamines in joining the Okazaki fragments.     

Tyrosine phosphorylation of insulin receptor beta subunit activates the receptor tyrosine kinase in intact H-35 hepatoma cells

The phosphorylation characteristics of insulin receptor from control and insulin-treated rat H-35 hepatoma cells 32P-labeled to equilibrium have been documented. The 32P-labeled insulin receptor is isolated by immunoprecipitation with patient-derived insulin receptor antibodies in the presence of phosphatase and protease inhibitors to preserve the native phosphorylation and structural characteristics of the receptor. The unstimulated insulin receptor contains predominantly [32P] phosphoserine and trace amounts of [32P]phosphothreonine in its beta subunit. In response to insulin, the insulin receptor beta subunit exhibits marked tyrosine phosphorylation and a 2-fold increase in total [32P]phosphoserine contents. High pressure liquid chromatography of the tryptic hydrolysates of the 32P-labeled receptor beta subunit from quiescent cells results in the resolution of up to 9 fractions containing [32P]phosphoserine. The insulin-stimulated tyrosine phosphorylation is concentrated in two of these receptor phosphopeptide fractions, whereas the increase in [32P]phosphoserine content is scattered in low abundance over all receptor tryptic fractions. Insulin receptors affinity-purified by lectin- and insulin-agarose chromatographies from insulin-treated, 32P-labeled cells exhibit a 22-fold increase in the Vmax of receptor tyrosine kinase activity toward histone when compared to controls. The elevated kinase activity of the insulin receptor derived from insulin-treated cells is not due to the presence of hormone bound to the receptor because the receptor kinase activity is assayed while immobilized on insulin-agarose. Furthermore, the insulin-activated receptor kinase activity is reversed following dephosphorylation of the receptor beta subunit with alkaline phosphatase in vitro. The correlation between the insulin-stimulated site specific tyrosine phosphorylation on receptor beta subunit and the elevation of receptor tyrosine kinase activity strongly suggests that the insulin receptor kinase is activated by hormone-stimulated autophosphorylation on tyrosine residues in intact cells, as previously demonstrated for the purified receptor.