Activation of Adenylyl Cyclase by Preincubation of Rat Cerebral‐Cortical Membranes Under Phosphorylating Conditions: Role of ATP, GTP, and Divalent Cations

Abstract: The effects of preincubation under phosphorylating conditions on adenylyl cyclase activity were studied in preparations containing synaptic membranes from rat cerebral cortex. Preincubation of the membranes with 2 mM ATP and 10 mM MgCl2 resulted in a 50% increase of adenylyl cyclase activity which withstood sedimentation and washing. This activation was maximal after 5 min of preincubation, was reversed after longer preincubations, and paralleled the time course of endogenous phosphorylation‐dephosphorylation of proteins observed under these conditions. The activation showed a critical requirement for Mg2+ ions and was dependent on ATP concentration. Similar activation was observed after preincubation of cerebral‐cortical membranes with adenosine‐5′‐0‐(3‐thiophosphate) (ATPγS), but this activation was not reversed by prolonged preincubation times. The activation by ATPγS was potentiated severalfold by including synaptoplasm in the preincubation. Further experiments indicated that the activity of nucleoside diphosphokinase, which converts ATPγS to guanosine‐5′‐0‐(3‐thiophosphate) (GTPγS), could account for this potentiation. Preincubation of washed membranes for 5 min with 10 μ.M GTP and 10 mM MgCl2 also produced a 50% activation of adenylyl cyclase which withstood sedimentation and washing and was reversed by longer preincubations. Endogenous phosphorylation of specific protein components in the membranes during the preincubation was examined by including radioactively labeled nucleoside thiophosphates in the preincubation medium. Incorporation of 35S from [35S]ATPγS into a protein component with apparent Mr of 54,000 daltons (54K) correlated significantly with the activation of adenylyl cyclase by ATPγS. Thiophosphorylation of the 54K protein was potentiated by addition of GDP to reactions carried out with [35S]ATPγS. Endogenous activity utilizing [γ‐32P]GTP as a phosphate donor also preferentially phosphorylated the 54K protein band. These results support previous suggestions that protein phosphorylation plays a role in the regulation of adenylyl cyclase activity. Among the numerous membrane‐bound phosphoproteins in rat brain, we have identified a specific protein component with an apparent Mr of 54,000 daltons as the most likely candidate for involvement in this mode of regulation. This 54K protein, which is a principal substrate for a GTP‐preferring protein kinase activity in brain membranes, can now be at the focus of investigations attempting to demonstrate a direct role for protein phosphorylation in adenylyl cyclase regulation.

[1]  H. Mahler,et al.  Calcium‐Stimulated Protein Phosphorylation in Synaptic Membranes , 1983, Journal of neurochemistry.

[2]  D. Brenneman,et al.  Induction of Cholinergic Expression in Developing Spinal Cord Cultures , 1983, Journal of neurochemistry.

[3]  W. Halpern,et al.  A high-spatial-resolution laser beam densitometer for quantitative analysis of polyacrylamide gels. , 1983, Analytical biochemistry.

[4]  S. Whittemore,et al.  GTP-preferring protein phosphorylation systems in brain membranes: possible role in adenylate cyclase regulation. , 1982, Biochemical and biophysical research communications.

[5]  C. Kahn,et al.  Insulin stimulates the phosphorylation of the 95,000-dalton subunit of its own receptor. , 1982, Science.

[6]  H. Mahler,et al.  Calcium‐Stimulated Adenosine Triphosphatases in Synaptic Membranes , 1981, Journal of neurochemistry.

[7]  J. M. Richards,et al.  ATP-dependent activation of adenylate cyclase. , 1981, Journal of Biological Chemistry.

[8]  J. Holderness,et al.  Benzodiazepine inhibition of the calcium-calmodulin protein kinase system in brain membrane. , 1981, Science.

[9]  Y. Salomon,et al.  Mechanism of desensitization of adenylate cyclase by lutropin. Impaired introduction of GTP into the regulatory site. , 1981, The Journal of biological chemistry.

[10]  L. Limbird.  Activation and attenuation of adenylate cyclase. The role of GTP-binding proteins as macromolecular messengers in receptor--cyclase coupling. , 1981, The Biochemical journal.

[11]  R. Iyengar,et al.  Divalent cation-induced desensitization of glucagon-stimulable adenylyl cyclase in rat liver plasma membrane. GTP-dependent stimulation by glucagon. , 1980, The Journal of biological chemistry.

[12]  E. Neer,et al.  Increase in the size of soluble brain adenylate cyclase with activation by guanosine 5'-(beta, gamma-imino)triphosphate. , 1980, The Journal of biological chemistry.

[13]  K. Wirtz,et al.  Modulation of brain polyphosphoinositide metabolism by ACTH-sensitive protein phosphorylation , 1980, Nature.

[14]  M. Rasenick,et al.  Partial purification and characterization of a macromolecule which enhances fluoride activation of adenylate cyclase. , 1980, Proceedings of the National Academy of Sciences of the United States of America.

[15]  L. G. Davis,et al.  Endorphin-regulated protein phosphorylation in brain membranes. , 1980, Life sciences.

[16]  G. Carpenter,et al.  Epidermal growth factor-receptor-protein kinase interactions. Co-purification of receptor and epidermal growth factor-enhanced phosphorylation activity. , 1980, The Journal of biological chemistry.

[17]  E. Brunngraber,et al.  Transient Changes in the Phosphorylation of Cortical Membrane Proteins After Electroconvulsive Shock , 1980, Journal of neurochemistry.

[18]  Martin Rodbell,et al.  The role of hormone receptors and GTP-regulatory proteins in membrane transduction , 1980, Nature.

[19]  D. Gill,et al.  Cholera toxin activation of adenylate cyclase. Roles of nucleoside triphosphates and a macromolecular factor in the ADP ribosylation of the GTP-dependent regulatory component. , 1980, The Journal of biological chemistry.

[20]  Y. Salomon,et al.  Mechanism of desensitization of adenylate cyclase in lutropin. GTP-dependent uncoupling of the receptor. , 1980, The Journal of biological chemistry.

[21]  R. Iyengar,et al.  Guanyl nucleotide regulation of hormonally-responsive adenylyl cyclases , 1979, Molecular and Cellular Endocrinology.

[22]  W. Kerrick,et al.  Irreversible thiophosphorylation and activation of tension in functionally skinned rabbit ileum strips by [35S]ATP gamma S. , 1979, The Journal of biological chemistry.

[23]  J. Chamberlain,et al.  Fluorographic detection of radioactivity in polyacrylamide gels with the water-soluble fluor, sodium salicylate. , 1979, Analytical biochemistry.

[24]  A. Matus,et al.  Long duration phosphorylation of synaptic membrane proteins , 1979, Neuroscience.

[25]  W. Anderson,et al.  Isoproterenol-induced desensitization of adenylate cyclase responsiveness in a cell-free system. , 1979, The Journal of biological chemistry.

[26]  L. Birnbaumer,et al.  Resensitization of the desensitized follicular adenylyl cyclase system to luteinizing hormone. , 1979, Endocrinology.

[27]  J. Davis,et al.  beta-Adrenergic receptor regulation by agonists and membrane depolarization in rat brain slices. , 1979, Proceedings of the National Academy of Sciences of the United States of America.

[28]  T. Walseth,et al.  The enzymatic preparation of [α-32P]nucleoside triphosphates, cyclic [32P]AMP, and cyclic [32P]GMP☆ , 1979 .

[29]  E. Neer.  Interaction of soluble brain adenylate cyclase with manganese. , 1979, The Journal of biological chemistry.

[30]  M. Brostrom,et al.  Calcium-dependent adenylate cyclase from rat cerebral cortex: activation by guanine nucleotides. , 1978, Archives of biochemistry and biophysics.

[31]  D. Hartshorne,et al.  Roles of calcium and phosphorylation in the regulation of the activity of gizzard myosin. , 1978, Biochemistry.

[32]  P. Greengard,et al.  Stimulation of brain membrane protein phosphorylation by calcium and an endogenous heat-stable protein , 1978, Nature.

[33]  P. Greengard.  Phosphorylated proteins as physiological effectors. , 1978, Science.

[34]  M. Brostrom,et al.  Calcium-dependent adenylate cyclase from rat cerebral cortex. Reversible activation by sodium fluoride. , 1977, The Journal of biological chemistry.

[35]  D. Cassel,et al.  Activation of turkey erythrocyte adenylate cyclase and blocking of the catecholamine-stimulated GTPase by guanosine 5'-(gamma-thio) triphosphate. , 1977, Biochemical and biophysical research communications.

[36]  R. Delorenzo,et al.  Calcium-dependent phosphorylation of specific synaptosomal fraction proteins: possible role of phosphoproteins in mediating neurotransmitter release. , 1976, Biochemical and biophysical research communications.

[37]  I. Ohad,et al.  Phosphorylation and dephosphorylation of membrane proteins as a possible mechanism for structural rearrangement of membrane components. , 1976, Biochimica et biophysica acta.

[38]  Y. Salomon,et al.  Evidence for specific binding sites for guanine nucleotides in adipocyte and hepatocyte plasma membranes. A difference in fate of GTP and guanosine 5'-(beta, gamma-imino) triphosphate. , 1975, The Journal of biological chemistry.

[39]  A. Routtenberg,et al.  Endogenous phosphorylation of four cerebral cortical membrane proteins: Role of cyclic nucleotides, ATP and divalent cations , 1975, Brain Research.

[40]  A. Routtenberg,et al.  Cyclic AMP regulates phosphorylation of three protein components of rat cerebral cortex membranes for thirty minutes , 1974, FEBS letters.

[41]  V. Najjar,et al.  The activation of adenylate cyclase. II. The postulated presence of (A) adenylate cyclase in a phospho (inhibited) form (B) a dephospho (activated) form with a cyclic adenylate stimulated membrane protein kinase. , 1973, Biochemical and biophysical research communications.

[42]  R. Rodnight,et al.  Protein kinase activity in membrane preparations from ox brain. Stimulation of intrinsic activity by adenosine 3':5'-cyclic monophosphate. , 1973, The Biochemical journal.

[43]  Y. Cheng,et al.  Demonstration of the heterogeneity of nucleoside diphosphokinase in rat tissues. , 1973, Biochemistry.

[44]  R. Rodnight,et al.  Turnover of protein-bound phosphorylserine in membrane preparations from ox brain catalysed by intrinsic kinase and phosphatase activity. , 1971, The Biochemical journal.

[45]  Oliver H. Lowry,et al.  Protein measurement with the Folin phenol reagent. , 1951, The Journal of biological chemistry.

[46]  R. Rodnight.  Protein Kinases and Phosphatases , 1983 .

[47]  S. Whittemore,et al.  Protein phosphorylation in the regulation and adaptation of receptor function. , 1982, Progress in brain research.

[48]  Y. Salomon,et al.  The role of GTP in lutropin-induced desensitization of the GTP regulatory cycle and adenylate cyclase in the rat ovary. , 1981, Advances in cyclic nucleotide research.

[49]  A. Gilman,et al.  Biochemical properties of hormone-sensitive adenylate cyclase. , 1980, Annual review of biochemistry.

[50]  A. Fabiato,et al.  Calculator programs for computing the composition of the solutions containing multiple metals and ligands used for experiments in skinned muscle cells. , 1979, Journal de physiologie.

[51]  Y. Salomon.  Adenylate cyclase assay. , 1979, Advances in cyclic nucleotide research.

[52]  Y. Ehrlich.  Phosphoproteins as specifiers for mediators and modulators in neuronal function. , 1979, Advances in experimental medicine and biology.

[53]  W. Gispen,et al.  The behaviorally active neuropeptide ACTH as neurohormone and neuromodulator: the role of cyclic nucleotides and membrane phosphoproteins. , 1979, Advances in experimental medicine and biology.

[54]  T. Walseth.  The enzymatic preparation of [γ-^ P] nucleotide triphosphate, cyclic [^ P] AMP, and cyclic GMP , 1979 .

[55]  G. Johnson,et al.  Drug-induced modification of the responsiveness of adenylate cyclase to hormones. , 1978, Advances in cyclic nucleotide research.

[56]  J. Drummond.  Observations on the Phosphotungstates of certain Bases and Amino-acids. , 1918, The Biochemical journal.