Stephen A. Rudolph

Widespread occurrence of a specific protein in vertebrate tissues and regulation by cyclic AMP of its endogenous phosphorylation and dephosphorylation

A protein whose endogenous phosphorylation and dephosphorylation are affected by cAMP has been found in the soluble and particulate fractions of all vertebrate tissues studied. This phosphoprotein, which contained a substantial proportion of the radioactive phosphate observed on SDS-polyacrylamide gels, was estimated to have an apparent molecular weight of 49,000. In the presence of Zn++, cAMP inhibited the endogenous phosphorylation of this protein (protein 49) in the cytosol and microsomal fractions. In the presence of Mg++, cAMP stimulated the phosphorylation of protein 49 in the cytosol fractions, but had only slight effects in the microsomal fractions. The dephosphorylation of protein 49 by an endogenous protein phosphatase was markedly stimulated by cAMP in the cytosol and microsomal fractions of all tissues studied. The binding of 8-azido-cAMP (a photoaffinity analog of cAMP, which reacts specifically with cAMP-binding sites) to subcellular fractions was also studied. This binding was principally to a protein of molecular weight 49,000. These and other data suggest that a cAMP-binding protein with a molecular weight of 49,000 capable of undergoing cAMP-dependent phosphorylation and dephosphorylation, occurs in a variety of tissues.

Autophosphorylation of adenosine 3',5'-monophosphate-dependent protein kinase from bovine brain.

A highly purified adenosine 3′,5′-monophosphate-dependent protein kinase from bovine brain has been found to catalyze its own phosphorylation. The incorporated phosphate was shown to be associated with the cyclic AMP-binding subunit (R-protein) of the protein kinase. The catalytic subunit exhibited no detectable incorporation of phosphate into itself, but was required for the phosphorylation of R-protein. The molecular weight of R-protein was determined by polyacrylamide gel electrophoresis to be about 48,000 in the presence of sodium dodecyl sulfate. Cyclic AMP strikingly inhibited the rate of autophosphorylation observed in the presence of ZnCl2, CaCl2, NiCl2, or FeCl2, but had no significant effect in the presence of MgCl2 or CoCl2. The concentration of cyclic AMP required to give half-maximal inhibition of phosphorylation was 3 × 10−7m in the presence of either CaCl2 or ZnCl2. Guanosine 3′,5′-monophosphate was far less effective under the same experimental conditions than cyclic AMP. R-protein appears to be similar to a phosphoprotein recently discovered in synaptic membrane fractions from rat and bovine cerebral cortex.

Solubilization of a phosphoprotein and its associated cyclic AMP-dependent protein kinase and phosphoprotein phosphatase from synaptic membrane fractions, and some kinetic evidence for their existence as a complex.

Abstract Synaptic membrane preparations contain a protein with an apparent molecular weight of 49,000, designated Protein II, whose endogenous phosphorylation and dephosphorylation are regulated by cyclic AMP. Protein II and the enzymes which catalyze the cyclic AMP-dependent phosphorylation and dephosphorylation of Protein II have been obtained in a soluble form from these synaptic membrane preparations by treatment with either 0.25% Triton X-100 or 0.1 m NH4Cl. In the Triton X-100 solubilized enzyme system, the regulation of the endogenous phosphorylation of Protein II by cyclic AMP was similar to that observed in untreated synaptic membrane preparations. Thus, the effect of cyclic AMP on the phosphorylation of Protein II was stimulatory in the presence of Mg2+ and inhibitory in the presence of Zn2+. The stimulation by cyclic AMP of the phosphorylation of Protein II observed in the presence of Mg2+ could be converted to an inhibitory effect either by increased ionic strength, by Zn2+, or by a thiol reagent, dithionitrobenzoate. In the NH4Cl-solubilized enzyme system, cyclic AMP-dependent phosphorylation was also observed, but the mode of regulation by cyclic AMP of the phosphorylation of Protein II was partially altered: the effect of cyclic AMP was inhibitory in the presence of either Mg2+ or Zn2+. In both the Triton X-100-solubilized and the NH4Cl-solubilized enzyme systems, cyclic AMP facilitated the endogenous dephosphorylation of Protein II in the absence of any divalent cation, as it did in untreated membrane preparations. The kinetic data obtained for the endogenous phosphorylation and endogenous dephosphorylation of Protein II, in both the Triton extract and the NH4Cl extract, are compatible with the idea that Protein II, its cyclic AMP-dependent protein kinase, and its cyclic AMP-dependent protein phosphatase, may exist in, and be extractable from, synaptic membranes in the form of a complex.

Adenosine and Cyclic Nucleotides in Modulation of Immune Responses

The discovery of an immunodeficiency disease associated with the deficiency of adenosine deaminase focused attention on the relationship between adenosine metabolism and immune function [1]. Adenosine receptors similar to those described by Sattin and RaIl [2] in brain have been identified on mouse [3] and human lymphocytes [4, 5]. Adenosine activates lymphocyte adenylate cyclase and increases cAMP levels by its interaction with adenosine receptors [4, 5] with similar properties to R sites on other tissues [6]. Since β-adrenergic catecholamines, prostaglandins of the E series (PGE), histamine, and other pharmacologic agents that increase cAMP synthesis influence a wide variety of immune responses [7], it is not surprising that adenosine would have effects on immune function as well. In most instances, agents that increase cAMP levels inhibit immune functions such as lymphocyte proliferation [8], cell-mediated cytolysis [3, 9] and IgE-mediated release of histamine from mast cells [10]. However, in some instances adenosine acts differently from other agents that also increase cAMP levels. For example, adenosine potentiates mast cell histamine release, whereas isoproterenol inhibits it [11]. In this chapter, the immunoregulatory properties of adenosine will be discussed and, as in the foregoing example, adenosine’s immunologic actions are markedly different from those of other adenylate cyclase activating agents.