Decreased incorporation of the photoaffinity probe 8N3-[gamma-32P]-GTP into a 45kD protein in lung tumors

Photoincorporation of 8N3-[gamma-32P]-GTP into tissue and cell extracts was examined using gel electrophoresis and autoradiography. Decreased photoincorporation into a 45kD band was observed in extracts from mouse lung tumors as compared to normal mouse lung, and in extracts from lung tumor-derived cell lines when compared to isolated bronchiolar epithelial cells. Decreased 45kD photolabelling was also observed in extracts of S49 lymphoma cyc- cells (deficient in Gs alpha, a 45kD GTP binding protein of receptor-coupled adenylate cyclase) when compared to wild type S49 cells. This, and the observation that there was no cholera toxin-catalyzed ADP-ribosylation in the 45kD band of lung tumor extracts, suggests that the 45kD band contains Gs alpha.     

Localization of the ATP binding site on alpha-tubulin

The binding site for ATP to tubulin was established by use of the photoaffinity label [gamma-32P]N3ATP. Photolysis of the analog in the presence of tubulin resulted in covalent modification of the protein as revealed by autoradiography of electropherograms. Scanning the autoradiograms showed that the ATP analog was bound mainly to the alpha subunit of the tubulin dimer; the alpha subunit was two to three times more radioactive than was the beta subunit. The location of a particular site on the alpha subunit was further defined by peptide maps. The alpha and beta subunits from affinity-labeled tubulin were separated and digested with Staphylococcus protease. Radioactivity was found predominantly in one peptide band from the alpha subunit. The location of the [gamma-32P]N3ATP binding site on the alpha subunit distinguishes it from the previously known exchangeable GTP binding site which is on the beta subunit. Moreover, excess GTP did not compete with [gamma-32P]N3ATP binding. The ATP binding site is distinct from the nonexchangeable GTP binding site. The GTP content of tubulin was the same after dialysis in 0.5 mM ATP as it was following dialysis against ATP-free buffer. Proof that the binding site for [gamma-32P]N3ATP is the same as that for ATP was obtained by competition experiments. In the presence of ATP, photolysis of the affinity analog did not label the alpha subunit preferentially.     

GTP-binding membrane proteins in activated and differentiating T cells

We have earlier reported changes in the GTP binding of several membrane proteins including Gs alpha and Gi alpha during thymic differentiation of T cells. Using an [alpha-32P]GTP-photoaffinity labeling technique we have studied the pattern of GTP binding proteins in activated and resting T lymphocytes and in T cells induced to differentiate by TPA. The GTP binding proteins in mitogen-activated T cells resembled those seen in leukemia T cell lines. Treatment of Jurkat, but not of CCRF-CEM, T cells with TPA caused increased GTP-labeling of a 34 kDa protein and Gi alpha. The GTP labeling pattern in TPA-treated Jurkat cells resembled that in resting T lymphocytes. TPA induced de novo expression of functional TCR/CD3 on CCRF-CEM and downregulation of TCR/CD3 on Jurkat cells but these changes did not correlate with the altered GTP-labeling patterns.     

Photoaffinity labeling of a viral induced protein from tobacco. Characterization of nucleotide-binding properties

We have used the photoaffinity analogs 8-azidoadenosine 5'-triphosphate (8-N3ATP) and 8-azidoguanosine 5'-triphosphate (8-N3GTP) to investigate the relationship between a viral induced protein (Mr = 120,000) in tobacco mosaic virus (TMV)-infected tobacco and the TMV-induced RNA-dependent RNA polymerase activity. When the radioactive analogs [gamma-32P]8-N3ATP and [gamma-32P]8-N3GTP were incubated with the tobacco tissue homogenate from TMV-infected plants, incorporation of label occurred into the viral induced protein in the presence of UV light. The incorporation was found to be totally dependent on UV-illumination and greatly enhanced by Mg2+. Saturation of photoincorporated label indicates an apparent Kd of 16 microM (+/- 3 microM) and 12 microM (+/- 3 microM) for 8-N3ATP and 8-N3GTP, respectively. Protection against photolabeling by [gamma-32P]8-N3ATP and [gamma-32P]8-N3GTP with various nonradioactive nucleotides and nucleosides suggests that the photolabeled site is protected best by nucleoside triphosphates. At 200 microM both deoxyribonucleoside triphosphates and ribonucleoside triphosphates were very effective at protecting the site from photolabeling. These data suggest that the photolabeled protein may be part of an RNA-dependent RNA polymerase. The utility of nucleotide photoaffinity analogs as a method to study viral induced nucleotide-binding proteins is discussed.     

Identification of the guanine binding domain peptide of the GTP-binding site of glucagon.

Glucagon, a peptide hormone synthesized and secreted by alpha islet cells, regulates glucose homeostasis by several mechanisms. Using [gamma 32P]8N3GTP, a proven photoaffinity probe for GTP, a specific nucleotide binding site on human glucagon was detected that showed preference for GTP. Half-maximal saturation of photoinsertion into the polypeptide hormone was at 8-12 microM with either [alpha 32P]8N3GTP or [gamma 32P]8N3GTP. GTP protected photolabeling with an apparent kd of 15 microM, whereas ATP was less effective as a protector, exhibiting an apparent kd of about 30 microM. Maximal protection by GTP and ATP was over 90%. UTP, CTP, GDP, ADP, GMP, AMP, guanosine, adenosine, guanine, and adenine were much less effective protectors, indicating that binding is specific for purine nucleoside triphosphates, particularly GTP. Mg2+ at 150 microM enhanced photoinsertion (twofold), whereas at 2-10 mM, it inhibited photoinsertion. Both Ca2+ and Zn2+ at 0.2 mM decreased photoinsertion about 45%. Purification of chymotryptic and tryptic digests of photolabeled glucagon by reverse-phase high performance liquid chromatography (HPLC) revealed that the N-terminal peptide, HSQGTF, was the only peptide region covalently photomodified by [32P]8N3GTP. GTP, if present during photolysis, greatly reduced both photoinsertion into glucagon and the amount of radiolabeled peptide recovered on HPLC. The specificity of binding to the N-terminal region is suggestive of a physiological role for a glucagon-GTP complex in the mechanism of action of this hormone.     

ras p21 and other Gn proteins are detected in mammalian cell lines by [gamma-35S]GTP gamma S binding

The presence of guanine nucleotide binding proteins in mouse and human cell lines was investigated using [gamma-35S]GTP gamma S and [gamma-32P]GTP. Cell lysate polypeptides were separated by sodium dodecyl sulphate polyacrylamide gel electrophoresis and transferred to nitrocellulose. Incubation of the nitrocellulose blots with [gamma-35S]GTP gamma S identified 9 distinct GTP-binding polypeptides in all lysates. One of these is the ras oncogene product, p21, as demonstrated by subsequent immunochemical staining of the nitrocellulose blots. We have shown that this procedure provides a sensitive method for detection of p21 in culture cell lines.     

Use of 8-azidoguanosine 5'-[gamma-32P]triphosphate as a probe of the guanosine 5'-triphosphate binding protein subunits in bovine rod outer segments

In an in vitro incubation, 8-azidoguanosine 5'-[gamma-32P]triphosphate ( [gamma-32P]-8-azido-GTP) labeled bleached rhodopsin independent of ultraviolet light. Characterization of this labeling indicated that rhodopsin was phosphorylated with [gamma-32P]-8-azido-GTP as a phosphate donor. At low concentrations, ATP increased this labeling activity 5-fold. In the same incubation, [gamma-32P]-8-azido-GTP also labeled G alpha (Mr 40 000). This labeling was ultraviolet light dependent. G beta (Mr 35 000) was also labeled dependent for the most part upon ultraviolet light, but a smaller component of labeling appeared to result from phosphorylation. Differential labeling of G alpha and G beta was found to vary intricately with experimental conditions, especially prebleaching of rhodopsin, tonicity of the medium, and the presence or absence of 2-mercaptoethanol. Affinity labeling of G alpha and G beta by [gamma-32P]-8-azido-GTP in competition with ATP or GTP was kinetically complex, consistent with possible multiple binding sites for GTP on both subunits. Independent evidence for two or more binding sites on G alpha has been offered by other laboratories, and recently, at least one binding site on G beta and its analogues among the N proteins of adenylate cyclases has been identified.     

Photoaffinity labeling of the major nucleosidetriphosphatase of rat liver nuclear envelope

We employed the photoaffinity probe 8-azido-adenosine 5'-triphosphate (aATP) to identify the nuclear envelope (NE) nucleosidetriphosphatase activity (NTPase) implicated in control of RNA transport. The photoprobe was hydrolyzed at rates comparable to those for ATP, with a Michaelis constant of 0.225 mM. Photolabeling was dependent upon UV irradiation (300-nm max) and was not affected by quercetin. Unlabeled ATP or GTP competed with [32P]aATP in photolabeling experiments, and UTP was a less effective competitor, paralleling the substrate specificity of the NTPase. Incubation of NE with aATP led to a UV, time, and concentration dependent irreversible inactivation of NTPase. The inactivation could be blocked by ATP or GTP. Polyacrylamide gel electrophoresis and autoradiography of photolabeled NE showed selective, UV-dependent labeling of a 46-kDa protein with both [gamma-32P]aATP and [alpha-32P]aATP. This band was not labeled with [gamma-32P]ATP. Since the NE NTPase implicated in RNA transport is modulated by RNA, we examined the effects of RNA on the labeling process. Removal of RNA from the NE preparations (by RNase/DNase digestion) reduced NTPase by 30-40% and eliminated photolabeling of the 46-kDa band. Addition of yeast RNA to such preparations increased NTPase activity to control levels and selectively reinstated photolabeling of the 46-kDa band. These results suggest that the 46-kDa protein represents the major NTPase implicated in RNA transport.     

Cell adhesion-dependent differences in endogenous protein phosphorylation on the surface of various cell lines

Endogenous phosphorylation of intact cells was studied with four mouse, hamster and human cell lines using [gamma-32P]ATP and [gamma-32P]GTP as exogenous substrates. With all four cell lines distinct differences in the phosphoprotein patterns could be demonstrated for cells grown in suspension culture compared to cells grown in monolayers. Two major, apparently ubiquitous phosphoproteins with molecular weights of 135 000 (128 000 in HeLa cells) and 105 000, representing up to 60% of total phosphorylation, were phosphorylated only in cells grown in suspension. These phosphoproteins and the kinase(s) were located on the surface of the suspension cells. Evidence showed that phosphorylation was apparently not a true endogenous reaction, that rather it occurred by cell-cell collision, showing exponentially increasing 32P incorporation with increasing cell population density. Phosphorylation of pp135 and pp105 was established with ATP as well as with GTP and was not dependent on cyclic nucleotides cyclic AMP, cyclic GMP and cyclic CMP. The substrate-attached cells of all four cell lines have protein kinases on the cell surface. The lack of pp135 and pp105 phosphorylation may be due to the fact that these phosphoproteins are not expressed at all on the surface of substrate-attached cells or that these phosphoproteins are already fully phosphorylated.     

DNA methyltransferase levels in tumorigenic and nontumorigenic cells in culture

The levels of DNA methyltransferase in nuclei from 9 tumorigenic and 9 nontumorigenic cell lines were examined. In all but 2 cases, the extractable methyltransferase activity was 4-3000-fold higher in tumorigenic than in nontumorigenic cells. Tumorigenic and nontumorigenic cells from four species were grown in the presence of various concentrations (10(-8)-10(-6) M) of an inhibitor of the methylase enzyme, 5-aza-2'-deoxycytidine (5-aza-dCyd). The reduction of 5-methylcytosine content in newly replicated DNA in the presence of 5-aza-dCyd was used to determine the relative methylase activity in each cell line. In all 4 cases, tumorigenic cells required larger doses of drug to inhibit DNA methylation to the same extent as their nontumorigenic counterparts. The relative rates of incorporation of [3H]5-aza-dCyd were determined for each cell line, and tumorigenic cells were shown to incorporate equal or greater amounts of 5-aza-dCyd into DNA compared to nontumorigenic cells. These results showed that the differences in the inhibition of DNA methylation in response to 5-aza-dCyd were not due to differences in the ability of these cells to incorporate the drug. Thus, it was demonstrated by two independent methods that tumorigenic cells contained higher levels of methylating capacity than nontumorigenic cells. This overabundance of methyltransferase may alter DNA methylation patterns and affect phenotypic stability.     

Evidence for a rabbit luteal ADP-ribosyltransferase activity which appears to be capable of activating adenylyl cyclase.

1. An ADP-ribosyltransferase activity which appears to be capable of activating adenylyl cyclase was identified in a plasma membrane fraction from rabbit corpora lutea and partially characterized by comparing the properties of the luteal transferase with those of cholera toxin. 2. Incubation of luteal membranes in the presence of GTP and varying concentrations of NAD resulted in concentration-dependent increases in adenylyl cyclase activity. 3. Stimulation of adenylyl cyclase by NAD and cholera toxin plus NAD was observed in the presence of GTP but not in the presence of guanosine-5'-O-(2-thiodiphosphate) or guanyl-5'-yl imidodiphosphate. 4. NAD or cholera toxin plus NAD reduced the Kact values for luteinizing hormone to activate adenylyl cyclase 3- to 3.5-fold. 5. NAD or cholera toxin plus NAD increased the extent to which cholate extracts from luteal membranes were able to reconstitute adenylyl cyclase activity in S49 cyc- mouse lymphoma membranes. 6. It was necessary to add ADP-ribose and arginine to the incubation mixture in order to demonstrate cholera toxin-specific ADP-ribosylation of a protein corresponding to the alpha subunit of the stimulatory guanine nucleotide-binding regulatory component (alpha Gs). 7. Treatment of luteal membranes with NAD prior to incubation in the presence of [32P]NAD plus cholera toxin resulted in reduced labeling of alpha Gs. 8. Endogenous ADP-ribosylation of alpha Gs was enhanced by Mg but was not altered by guanine nucleotide, NaF or luteinizing hormone and was inhibited by cAMP. 9. Incubation of luteal membranes in the presence of [32P]ADP-ribose in the absence and presence of cholera toxin did not result in the labeling of any membrane proteins.     

Angiogenesis-inducing ability of human bladder epithelium cell lines and "spontaneously" transformed murine fibroblasts

Ten human bladder epithelium cell lines were tested for their ability to induce blood vessel formation after intradermal injection into irradiated ST/a mice. Cell lines that were shown to be tumorigenic in nude mice, were able to evoke angiogenesis of a higher intensity than nontumorigenic cell lines. No difference was observed between the angiogeneic ability of tumorigenic cells originating from tumors and from in vitro transformed urothelium of nontumor origin. Similarly the origin of nontumorigenic urothelial cell lines did not show any influence on their angiogeneic abilities, but nontumorigenic cell lines which had undergone "infinite growth transformation" exhibited a higher angiogeneic activity than nontumorigenic cell lines with a finite life. The angiogeneic reaction evoked by human bladder epithelium cell lines showed cell dose- and time-dependence; but it was unrelated to the growth potential of the cultured cells. Two "spontaneously" altered sarcoma-producing murine cell lines showed a higher angiogeneic activity than tumorigenic human bladder epithelial cells. The angiogeneic response to these two murine cell lines was unrelated to morphological signs of transformation and to differences in growth rate, serum requirement, saturation density, anchorage dependence, and isoimmunizing properties.     

Mechanism of inhibition of Escherichia coli RNA polymerase by captan.

Captan (N-trichloromethylthiocyclohex-4-ene-1,2-dicarboximide) was shown to inhibit RNA synthesis in vitro catalysed by Escherichia coli RNA polymerase. Incorporation of [gamma-32P]ATP and [gamma-32P]GTP was inhibited by captan to the same extent as overall RNA synthesis. The ratio of [3H]UTP incorporation to that of [gamma-32P]ATP or of [gamma-32P]GTP in control and captan-treated samples indicated that initiation was inhibited, but the length of RNA chains being synthesized was not altered by captan treatment. Limited-substrate assays in which re-initiation of RNA chains did not occur also showed that captan had no effect on the elongation reaction. Studies which measured the interaction of RNA polymerase with template DNA revealed that the binding of enzyme to DNA was inhibited by captan. Glycerol-gradient sedimentation of the captan-treated RNA polymerase indicated that the inhibition of the enzyme was irreversible and did not result in dissociation of its subunits. These data are consistent with a mechanism in which RNA polymerase activity was irreversibly altered by captan, resulting in an inability of the enzyme to bind to the template. This interaction was probably at the DNA-binding site on the polymerase and did not involve reaction of captan with the DNA template.     

ATP nucleotidylation of creatine kinase.

Creatine kinase (CK) will autoincorporate radiolabel from [gamma32P]ATP and has thus been reported to be autophosphorylated. Also, in contrast to normal brain enzyme, CK in Alzheimer-diseased brain homogenate shows greatly decreased activity, abolished photolabeling with [32P]8N3ATP, and no detectable autoincorporation of radiolabel by [gamma32P]ATP. Surprisingly, our studies with both human brain and purified CK showed that [alpha32P]ATP, [gamma32P]ATP, [alpha32P]ADP, [2,8H3]ATP, [gamma32P]2',3'-O-(2,4, 6-trinitrophenyl)-ATP, and [gamma32P]benzophenone-gammaATP all autoincorporate radiolabel into CK with good efficiency. This demonstrates that the gamma-phosphate and the 2' and 3' hydroxyls are not involved in the covalent linkage and that all three phosphates, the ribose and base of the ATP molecule are retained upon autoincorporation (nucleotidylation). Treatment with NaIO3 to break the 2'-3' linkage effected total loss of radiolabel indicating that nucleotidylation resulted in opening of the ribose ring at the C1' position. Nucleotidylation with increasing [alpha32P]ATP at 37 degrees C gives an approximate k0.5 of 125 microM and saturates at 340 microM nucleotide. Modification of 8-10% of the copy numbers occurs at saturation, and CK activity is inhibited to approximately the same degree. Low micromolar levels of native substrates such as ADP, ATP, and phosphocreatine substantially reduce [alpha32P]ATP nucleotidylation. In contrast, AMP, GTP, GMP, NADH, and creatine did not effectively reduce nucleotidylation. When [alpha32P]ATP-nucleotidylated or [alpha32P]8N3ATP-photolabeled CK is treated with trypsin a single, identical radiolabeled peptide (V279-R291) is generated that comigrates on reverse phase HPLC and Tris-tricine electrophoresis. Nucleotidylation into this peptide was prevented 86% by the presence of ATP. We conclude that CK is nucleotidylated within the active site by modification at the C1'position and that autophosphorylation of this enzyme does not occur.     

Phosphorylation of DNA topoisomerase II in vivo and in total homogenates of Drosophila Kc cells. The role of casein kinase II

The phosphorylation of DNA topoisomerase II in Drosophila Kc tissue culture cells was characterized by in vivo labeling studies and in vitro studies that examined the modification of exogenous enzyme in total homogenates of these embryonic cells. Several lines of evidence identified casein kinase II as the kinase primarily responsible for phosphorylating DNA topoisomerase II. First, the only amino acyl residue modified in the enzyme was serine. Second, partial proteolytic maps of topoisomerase II which had been labeled with [32P]phosphate by Drosophila cells in vivo, by cell homogenates in vitro, or by purified casein kinase II were indistinguishable from one another. Third, phosphorylation in cell homogenates was inhibited by micrograms/ml concentrations of heparin, micromolar concentrations of nonradioactive GTP, or anti-Drosophila casein kinase II antiserum. Fourth, cell homogenates were able to employ [gamma-32P]GTP as a phosphate donor nearly as well as [gamma-32P]ATP. Although topoisomerase II was phosphorylated in homogenates under conditions that specifically stimulate protein kinase C, calcium/calmodulin-dependent protein kinase, or cAMP-dependent protein kinase, modification was always sensitive to anti-casein kinase II antiserum or heparin. Thus, under a variety of conditions, topoisomerase II appears to be phosphorylated primarily by casein kinase II in the Drosophila embryonic Kc cell system.     

Functional evidence for a second tumor suppressor gene on human chromosome 17.

The development and progression of human tumors often involves inactivation of tumor suppressor gene function. Observations that specific chromosome deletions correlate with distinct groups of cancer suggest that some types of tumors may share common defective tumor suppressor genes. In support of this notion, our initial studies showed that four human carcinoma cell lines belong to the same complementation group for tumorigenic potential. In this investigation, we have extended these studies to six human soft tissue sarcoma cell lines. Our data showed that hybrid cells between a peripheral neuroepithelioma (PNET) cell line and normal human fibroblasts or HeLa cells were nontumorigenic. However, hybrid cells between the PNET cell line and five other soft tissue sarcoma cell lines remained highly tumorigenic, suggesting at least one common genetic defect in the control of tumorigenic potential in these cells. To determine the location of this common tumor suppressor gene, we examined biochemical and molecular polymorphic markers in matched pairs of tumorigenic and nontumorigenic hybrid cells between the PNET cell line and a normal human fibroblast. The data showed that loss of the fibroblast-derived chromosome 17 correlated with the conversion from nontumorigenic to tumorigenic cells. Transfer of two different chromosome 17s containing a mutant form of the p53 gene into the PNET cell line caused suppression of tumorigenic potential, implying the presence of a second tumor suppressor gene on chromosome 17.     

Specific in vitro guanylylation of a 43-kilodalton membrane-associated protein of Streptomyces coelicolor.

Incubation of [alpha-32P]GTP with cellular extracts or membranes of Streptomyces coelicolor labels a protein of 43 kDa, which was also labeled with [8,5'-3H]GTP but not with [alpha-32P]ATP or [gamma-32P]GTP. Radioactivity remained associated with this protein after boiling in 0.1 N NaOH, but it was dissociated after incubation in 0.1 N HCl or hydroxylamine. Chromatographic analysis of the HCl-dissociated compound showed that GMP was the covalently bound nucleotide. Furthermore, guanylylation appeared to be reversible and to take place by a pyrophosphorylytic mechanism. Guanylylation was more efficient at low temperatures. Several Streptomyces species showed a guanylylated protein with a similar molecular mass.     

Conversion of GDP into GTP by nucleoside diphosphate kinase on the GTP-binding proteins

A direct interaction of alpha beta gamma trimeric GTP binding proteins (G proteins; G0 and Gs) with nucleoside diphosphate kinase (NDP kinase) was investigated with homogeneously purified proteins. There was a progressive release of 32Pi from [gamma-32P]ATP when GDP-bound G0 was incubated together with NDP kinase. The Pi release induced by the interaction of G0 with NDP kinase was not accompanied by the dissociation of GDP bound to the alpha-subunit of G0. This was a sharp contrast to G protein-catalyzed GTP hydrolysis observed with GTP as the substrate; the dissociation of bound GDP was essentially required for the following binding of the substrate, GTP, to be hydrolyzed. A kinetic analysis displayed different properties for the substrate of NDP kinase between free GDP and G protein-bound GDP. NDP kinase-dependent phosphorylation of GDP on G0 was indeed demonstrated with adenosine 5'-(3-O-thio)triphosphate as the phosphate donor; there was a formation of guanosine 5'-(3-O-thio)triphosphate-bound G0 from the ATP analogue. Moreover, purified Gs was readily ADP-ribosylated by cholera toxin in the presence of NDP kinase, ATP, and an ADP-ribosylation factor, also suggesting that the nucleotide form on Gs was certainly GTP. These results indicate that NDP kinase can transfer the gamma-phosphate of ATP directly to GDP bound to G proteins and that this phosphorylation results in the activation of the signal-coupling proteins. A possible role of the new activation mechanism of G proteins is discussed in comparison with the previously characterized GDP-GTP exchange pathway by the agonist-receptor complex.