adenosine-5--o-(3-thiotriphosphate) and thiophosphoric-acid

Protein phosphorylation is a ubiquitous posttranslational modification that regulates cell signaling in both prokaryotes and eukaryotes. Although the study of phosphorylation has made great progress, several major hurdles remain, including the difficulty of the assignment of endogenous substrates to a discrete kinase and of global phosphoproteomics investigations. We have developed a novel chemical strategy for detecting phosphorylated proteins. This method utilizes adenosine 5'-O-(3-thiotriphosphate) (ATPγS), which results in the transfer of a thiophosphate moiety by a kinase to its substrate(s). This group can subsequently be employed as a nucleophilic handle to promote protein detection. To selectively label thiophosphorylated proteins, cellular thiols (e.g., cysteine-containing proteins) must first be blocked. Most common cysteine-capping strategies rely upon the nucleophilicity of the sulfur group and would therefore also modify the thiophosphate moiety. We hypothesized that the radical-mediated thiol-ene reaction, however, would be selective for cysteine over thiophosphorylated amino acids due to the differences in the electronics and pKa values between these groups. Here, we report rapid and specific tagging of thiophosphorylated proteins in vitro following chemoselective thiol capping using the thiol-ene reaction.

Topics: Adenosine Triphosphate; Biological Assay; Cysteine; Free Radicals; Molecular Structure; Phosphates; Phosphorylation; Phosphotransferases; Proteins; Staining and Labeling; Sulfhydryl Compounds

Protein kinases catalyze the transfer of the gamma-phosphate group from ATP to a serine, threonine or tyrosine residue of an acceptor protein. These enzymes play an important role in signal transduction. New inhibitors for these enzymes are actively being sought. In this article, we present a novel approach for detecting the activity of protein kinases, which could be useful for the high-throughput screening of chemical libraries. The method is based on the use of ATP gamma S instead of ATP in the phosphorylation reaction. This results in the transfer of a thiophosphate group onto a fluorescein-labeled acceptor peptide substrate. The mixture is then treated with a sulfur-reactive iodoacetyl derivative of biotin, which leads to the modification of the nucleophilic sulfur of the thiophosphate group and the generation of a fluorescently labeled, biotinylated molecule. Finally, streptavidin is added to the mixture and it binds to all biotinylated molecules present. The binding of streptavidin to the thiophosphorylated and biotinylated kinase substrate can be conveniently detected by measuring the change in fluorescence polarization of the fluorescent dye attached to the peptide. The detection of kinase inhibitors is demonstrated. The method is completely homogeneous and does not require any separation steps.

Topics: Adenosine Triphosphate; Biotinylation; Cyclic AMP-Dependent Protein Kinases; Edetic Acid; Fluorescein; Fluorescence; Oligopeptides; Phosphates; Phosphorylation; Protein Kinases; Reproducibility of Results; Streptavidin

Calmodulin-dependent protein kinase II (CaM-kinase II) undergoes a very rapid autophosphorylation at Thr-286/Thr-287 in the presence of Ca2+/calmodulin and ATP/Mg2+, and this has greatly hampered studies on the role of the autophosphorylation in the regulation of the enzyme activity, because it has been difficult to measure the activity of the non-autophosphorylated enzyme in the presence of Ca2+/calmodulin. In the present study, this difficulty was overcome by using adenosine 5'-O-(3-thiotriphosphate) (ATP gamma S) in place of ATP. When the enzyme was assayed with 2 microM ATP gamma S and 200 microM syntide-2 at 5 degrees C in the presence of Ca2+/calmodulin, the linear reaction rate of thiophosphorylation of syntide-2 was much slower than that of the enzyme which had previously undergone autothiophosphorylation. Under the limiting assay conditions, thiophosphorylation of the enzyme did not occur significantly during assay. Using this assay condition, activation by autothiophosphorylation was examined. When CaM-kinase II was autothiophosphorylated at 5 degrees C, the concomitant stimulation of both activities in the presence and absence of Ca2+/calmodulin was observed. The activity of the recombinant wild-type enzyme in the presence of Ca2+/calmodulin as well as in its absence was also markedly activated upon autothiophosphorylation, whereas those of the recombinant mutated enzyme, whose Thr-287 was replaced by Ala, was not activated at all. These results provide strong support for the contention that CaM-kinase II initially possesses a basal low level of the total activity and that the initial rapid autophosphorylation on Thr-286/Thr-287 results in full activation of the enzyme.

Topics: Adenosine Triphosphate; Animals; Brain; Calcium; Electrophoresis, Polyacrylamide Gel; Enzyme Activation; Intercellular Signaling Peptides and Proteins; Kinetics; Mutation; Peptides; Phosphates; Phosphorylation; Rats; Recombinant Proteins; Threonine

Human polymorphonuclear neutrophils (PMN) respond to ATP with an elevation in intracellular calcium and a marked enhancement of O2-production in response to stimulation by the chemotactic peptide N'-formyl-Met-Leu-Phe (FMLP). These pertussis toxin-sensitive pathways appear to be mediated by a nucleotide receptor(s) on the surface of human PMN. In the current study, we have examined the binding to intact human PMN of the ATP analog, adenosine 5'-O-(3-thio[35S] triphosphate) [( 35S]ATP gamma S). On the basis of Scatchard analysis, the binding of [35S]ATP gamma S involves at least two sites, one of high and one of low affinity. In the presence of sodium thiophosphate, a compound which did not affect intracellular increases in calcium induced by ATP or N'-formyl-Met-Leu-Phe, a significant fraction of the [35S]ATP gamma S binding was eliminated. This reduction involved both high and low affinity binding of [35S]ATP gamma S and was related to a reduction in numbers of binding sites. The Kd values for the high affinity binding site were unaffected by the presence of sodium thiophosphate, although the low affinity Kd values were numerically increased by 2-fold. In the presence of thiophosphate, [35S]ATP gamma S binding was specific, saturable, and reversible, and was related to a single class of high affinity (Kd = 36 +/- 19 nM) binding sites (184 +/- 144 sites/cell), together with a second class of low affinity (Kd = 1110 +/- 503 nM) binding sites (13,562 +/- 6,851 sites/cells). Competitive binding experiments, based on the ability of nucleotides and ATP analogs to block [35S]ATP gamma S binding to PMN, revealed a rank order of ATP gamma S greater than ATP greater than 2-MeS-ATP = 8-Bromo ATP greater than ADP = ITP greater than AMP-PCP = GTP much greater than CTP. A comparison between the ability of nucleotides to compete with [35S]ATP gamma S binding and their ability to induce a biologic response (elevation of intracellular calcium) revealed a close correlation (r2 = 0.83). These findings support the possibility of a common nucleotide PMN receptor functionally linked to a cellular response which involves increases in intracellular calcium.

Topics: Adenosine Triphosphate; Binding, Competitive; Calcium; Humans; Intracellular Membranes; Neutrophils; Nucleotides; Phosphates; Receptors, Cell Surface