phosphorus-radioisotopes and 8-azidoadenosine-3--5--monophosphate

Phosphorylation of the RII regulatory subunits of cyclic AMP-dependent protein kinases (PKAs) was examined during the HeLa cell cycle. Three RIIalpha isoforms of 51, 54, and 57 kDa were identified by RIIalpha immunodetection and labeling with 8-azido[32P]cAMP in different cell cycle phases. These isoforms were characterized as different phosphorylation states by the use of selective PKA and cyclin-directed kinase inhibitors. Whereas RIIalpha autophosphorylation by PKA caused RIIalpha to shift from 51 to 54 kDa, phosphorylation of RIIalpha by one other or a combination of several kinases activated during mitosis caused RIIalpha to shift from 51 to 57 kDa. In vivo incorporation of [32P]orthophosphate into mitotic cells and RIIalpha immunoprecipitation demonstrated that RIIalpha was hyperphosphorylated on a different site than the one phosphorylated by PKA. Deletion and mutation analysis demonstrated that the cyclin B-p34(cdc2) kinase (CDK1) phosphorylated human recombinant RIIalpha in vitro on Thr54. Whereas RIIalpha was associated with the Golgi-centrosomal region during interphase, it was dissociated from its centrosomal localization at metaphase-anaphase transition. Furthermore, particulate RIIalpha from HeLa cell extracts was solubilized following incubation with CDK1 in vitro. Our results suggest that at the onset of mitosis, CDK1 phosphorylates RIIalpha, and this may alter its subcellular localization.

Topics: Affinity Labels; Amino Acid Substitution; Antibodies, Monoclonal; Azides; Calcium-Calmodulin-Dependent Protein Kinases; Cell Cycle; Cyclic AMP; Cyclic AMP-Dependent Protein Kinases; Cyclin-Dependent Kinases; Enzyme Inhibitors; HeLa Cells; Humans; Macromolecular Substances; Mitosis; Mutagenesis, Site-Directed; Phosphorus Radioisotopes; Phosphorylation; Polymerase Chain Reaction; Recombinant Proteins; Subcellular Fractions; Transfection

Previously, we have reported a defect in the cAMP-dependent protein kinases (cAMP-PK) in psoriatic cells (i.e., a decrease in 8-azido-[32P]cAMP binding to the regulatory subunits and a decrease in phosphotransferase activity) which is rapidly reversed with retinoic acid (RA) treatment of these cells. This led us to examine a possible direct interaction between retinoids and the RI and RII regulatory subunits through retinoylation. Retinoylation of RI and RII present in normal and psoriatic human fibroblasts was analysed by [3H]RA treatment of these cells, followed either by chromatographic separation of the regulatory subunits or by their specific immunoprecipitation. These studies indicated that RI and RII can be retinoylated. [3H]RA labeling of the RII subunit was significantly (P < 0.005) greater in psoriatic fibroblasts (nine subjects; mean 7.47 relative units +/- 1.37 SEM) compared to normal fibroblasts (eight subjects; mean 2.46 relative +/- 0.49 SEM). [3H]RA labeling of and the increase in 8-azido-[32P]-binding to the RI and RII subunit in psoriatic fibroblasts showed a similar time course. This suggests that the rapid effect of retinoic acid treatment to enhance 8-azido-[32P]-cAMP binding to the RI and RII in psoriatic fibroblasts may be due, in part, to covalent modification of the regulatory subunits by retinoylation.

Topics: Affinity Labels; Autoradiography; Azides; Blotting, Western; Cell Fractionation; Chromatography; Cyclic AMP; Cyclic AMP-Dependent Protein Kinase Type II; Cyclic AMP-Dependent Protein Kinases; Fibroblasts; Humans; Phosphorus Radioisotopes; Protein Binding; Psoriasis; Skin; Time Factors; Tretinoin; Tritium

Little is known of the regulatory steps in the cellular action of vasopressin (AVP) on the renal epithelium, subsequent to the cAMP generation. We studied cAMP-binding proteins in the medullary collecting tubule (MCT) and the thick ascending limb of Henle's loop (MTAL) microdissected from the rat kidney by use of photoaffinity labeling. Microdissected tubules were homogenized and photoaffinity labeled by incubation with 1 microM 32P-labeled 8-azido-adenosine 3',5'-cyclic monophosphate (N3-8-[32P]-cAMP); the incorporated 32P was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography. Both in MCT and MTAL preparations, the analyses showed incorporation of N3-8-[32P]cAMP into two bands (Mr = 49,000 and Mr = 55,000) that comigrated with standards of the cAMP-dependent protein kinase regulatory subunits RI and RII. In MCT, most of the 32P (80%) was incorporated into RI, whereas in MTAL the 32P incorporated into RI and RII was equivalent. When freshly dissected MCT segments were incubated with 10(-12)-10(-6) M AVP, the subsequent photoaffinity labeling of RI with N3-8-[32P]cAMP was markedly diminished in a dose-dependent manner compared with controls. Our results suggest that cAMP binds in MCT and MTAL to regulatory subunits RI and RII of cAMP-dependent protein kinase. However, in MCT the dominant type of cAMP-dependent protein kinase appears to be type I. The outlined procedure is suitable to indirectly measure the occupancy of RI by endogenous cAMP generated in MCT cells in response to physiological levels (10(-12) M) of AVP.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Affinity Labels; Animals; Arginine Vasopressin; Azides; Carrier Proteins; Cyclic AMP; Cyclic AMP Receptor Protein; In Vitro Techniques; Kidney Medulla; Kidney Tubules; Kidney Tubules, Collecting; Kinetics; Loop of Henle; Male; Phosphorus Radioisotopes; Photolysis; Protein Binding; Protein Kinases; Rats; Rats, Inbred Strains; Reference Values

Topics: Affinity Labels; Aluminum Oxide; Animals; Azides; Chromatography; Chromatography, Affinity; Chromatography, Gel; Chromatography, Ion Exchange; Cyclic AMP; Drosophila; Kinetics; Macromolecular Substances; Molecular Weight; Phosphorus Radioisotopes; Protamine Kinase; Protein Kinases; Radioisotope Dilution Technique

Topics: Affinity Labels; Azides; Binding, Competitive; Cell Membrane; Cyclic AMP; Dictyostelium; Kinetics; Ligands; Phosphorus Radioisotopes; Radioisotope Dilution Technique; Receptors, Cyclic AMP

We have previously identified and demonstrated reversible ligand-induced modification of the major cell surface cAMP receptor in Dictyostelium discoideum. The receptor, or a subunit of it, has been purified to homogeneity by hydroxylapatite chromatography followed by two-dimensional preparative sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The purification was monitored by following 32Pi incorporated by photoaffinity labeling with 8-azido-[32P]cAMP or by in vivo labeling with 32Pi. Two interconvertible forms of the receptor, designated R (Mr 40,000) and D (Mr 43,000), co-purified. Two-dimensional peptide maps of independently purified and 125I-iodinated R and D forms of the receptor were nearly identical but did have several distinct peptides. The estimated 6000-fold purification required is consistent with the number of cell surface binding sites assuming there are not multiple binding sites/polypeptide. In the accompanying article we report the generation of a monospecific polyclonal antiserum which has helped to further elucidate the physical properties and developmental regulation of the cAMP receptor.

Topics: Affinity Labels; Azides; Cell Membrane; Chromatography; Chymotrypsin; Cyclic AMP; Dictyostelium; Durapatite; Electrophoresis, Polyacrylamide Gel; Hydroxyapatites; Peptide Fragments; Phosphorus Radioisotopes; Photochemistry; Receptors, Cyclic AMP

The recent observation that ammonium sulfate stabilizes cell-surface [3H]cyclic AMP binding in Dictyostelium discoideum (Van Haastert, P., and Kien, E. (1983) J. Biol. Chem. 258, 9636-9642) led us to attempt to identify the surface cAMP receptor by photoaffinity labeling with 8-azido-[32P]cAMP using this stabilization technique. 8-azido-[32P]cAMP specifically labeled a polypeptide which migrates as a closely spaced doublet (Mr = 40,000 to 43,000) on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Greater than 60% of the labeled polypeptide was found associated with membranes. This protein was distinguished from the cytosolic regulatory subunit of the cAMP-dependent protein kinase (Mr = 41,000) by differences in developmental regulation, specificity, and subcellular localization. No kinase regulatory subunit was detected in membranes by western blot analysis. Our preliminary observations show that labeling of this doublet correlates closely with cAMP-binding activity, suggesting that it is the surface receptor which mediates chemotaxis and cAMP signaling.

Topics: Azides; Cell Membrane; Cyclic AMP; Dictyostelium; Macromolecular Substances; Phosphorus Radioisotopes; Protein Kinases; Receptors, Cyclic AMP; Tritium

The photoaffinity label 8-azido[32P]adenosine 3':5'-monophosphate and affinity chromatography on N6-(2-aminoethyl)-cAMP-Sepharose were used to analyze the cAMP-binding proteins present in cell-free extracts of Blastocladiella emersonii zoospores. In the presence of a mixture of protease inhibitors, 8-azido[32P]cAMP was specifically and quantitatively incorporated into a major protein band of Mr = 58,000, and three minor protein bands of Mr = 50,000, Mr = 43,000, and Mr = 36,000 respectively, after autoradiography following sodium dodecyl sulfate-polyacryl-amide gel electrophoresis. In the absence of the protease inhibitors, the Mr = 58,000 protein band was converted into the lower molecular weight cAMP-binding proteins, indicating a high sensitivity of the intact Mr = 58,000 protein band to endogenous proteases. The Mr = 58,000 protein corresponded to the regulatory subunit (R), of the cAMP-dependent protein kinase of zoospores, as shown by their identical behavior on DEAE-cellulose chromatography. The partially purified protein kinase incorporated 32P from [gamma-32P] ATP . Mg2+ into R as demonstrated by the specific adsorption of the 32P-labeled protein with N6-(2-aminoethyl)-cAMP-Sepharose. The incorporated 32P group was rapidly removed by endogenous phosphoprotein phosphatases in the presence of cAMP, as shown by pulse-chase experiments with [gamma-32P]ATP. Dephosphorylation of R-cAMP and rapid proteolysis may indicate two other mechanisms, in addition to cAMP, for the control of this protein kinase in vivo.

Topics: Affinity Labels; Azides; Blastocladiella; Cyclic AMP; Fungi; Molecular Weight; Phosphorus Radioisotopes; Phosphorylation; Protein Kinases; Spores, Fungal

Topics: Affinity Labels; Animals; Azides; Carrier Proteins; Cattle; Cyclic AMP; Cytosol; Intracellular Signaling Peptides and Proteins; Kinetics; Myocardium; Phosphorus Radioisotopes; Phosphorylation; Photolysis; Protein Binding; Protein Kinases

Topics: Animals; Azides; Carrier Proteins; Cyclic AMP; Cyclic IMP; Intracellular Signaling Peptides and Proteins; Isoenzymes; Kinetics; Phosphorus Radioisotopes; Protein Kinases; Tritium

Topics: Affinity Labels; Animals; Azides; Brain; Carrier Proteins; Cattle; Cyclic AMP; Cyclic AMP Receptor Protein; Electrophoresis, Polyacrylamide Gel; Microtubules; Molecular Weight; Phosphorus Radioisotopes

Topics: Adenine Nucleotides; Adenosine Triphosphate; Affinity Labels; Animals; Azides; Binding Sites; Blood Proteins; Brain; Catalysis; Cyclic AMP; Erythrocyte Membrane; Humans; Membrane Proteins; Membranes; Osmolar Concentration; Phosphorus Radioisotopes; Photochemistry; Rats