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 MgC12 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+ and was dependent on ATP concentration. Similar activation was observed after preincubation of cerebral-cortical membranes with adenosine-5''-O-(3-thiophosphate) (ATP.gamma.S), but this activation was not reversed by prolonged preincubation times. The activation by ATP.gamma.S was potentiated severalfold by including synaptoplasm in the preincubation. Further experiments indicated that the activity of nucleoside diphosphokinase, which converts ATP.gamma.S to guanosine-5''-O-(3-thiophosphate) (GTP.gamma.S), could account for this potentiation. Preincubation of washed membranes for 5 min with 10 .mu.M GTP and 10 mM MgC12 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]ATP.gamma.S into a protein component with apparent Mr [molecular ratio] of 54,000 daltons (54K) correlated significantly with the activation of adenylyl cyclase by ATP.gamma.S. Thiophosphorylation of the 54K protein was potentiated by addition of GDP to reactions carried out with [35S]ATP.gamma.S. Endogenous activity utilizing [.gamma.-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, a specific protein component was identified 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.