Insulin activates a rat liver peripheral plasma-membrane cyclic AMP phosphodiesterase by a phosphorylation mechanism.

The molecular events occurring at the level of the plasma membrane after insulin occupies its receptor remain an enigma. We can demonstrate that with purified rat liver plasma membranes the cyclic AMP phosphodiesterase activity (Marchmont & Houslay, 1980) can be increased some t t fo ld (at 0.4pM-CYCliC AMP) if the membranes are pretreated with bovine insulin (1OnM) together with cyclic AMP (0.1 mM), ATP ( 3 m ~ ) and MgCI, (5mM). This activation is apparently achieved within lmin of incubation at 37OC and was not reversed by washing the membranes, and was totally dependent on the presence of all three ligands. The peripheral cyclic AMP phosphodiesterase and not the intrinsic enzyme is entirely responsible for the increased activity that we observe. It can be released from the membrane in an activated state by using the high-ionic-strength treatment described by Marchmont & Houslay (1980). This activation appears to ensue through insulin triggering a cyclic AMP-dependent protein kinase to phosphorylate the enzyme. Consistent with such an hypothesis are our observations that polyacrylamide-gel electrophoresis of the peripheral membrane proteins from membranes pre-treated with insulin, cyclic AMP and [p3:P1ATP resolves three peaks of radioactivity, one of which co-migrates with the peak of cyclic AMP phosphodiesterase activity. If either ‘or both insulin and cyclic AMP were absent then no peaks of radioactivity were observed. Furthermore, if the specific inhibitor protein @pug/ ml) of cyclic AMP-dependent protein kinase (Walsh et al., 1971) was added to the incubation mixture, then both the peaks of radioactivity migrating on polyacrylamide-gel electrophoresis and the activation of the phosphodiesterase were abolished. Both the K, and V,,,,,, of the apparently negatively co-operative peripheral enzyme were relatively unchanged by the activation, but the Hill coefficient was markedly decreased from 0.6 to 0.45, yielding an increased activity at low substrate concentrations (Fig. 1). The stimulation of activity was dose-dependent on both insulin and cyclic AMP, which exhibited K , values of 0.1 nM and 2pM respectively. Under basal conditions, hepatocyte cyclic AMP concentrations are only 0 . 3 0 . 5 , ~ ~ (Exton et al., 1973; Smith et al., 1978) and as such are insufficient to allow insulin to activate the enzyme. However, aRer exposure to glucagon, cyclic AMP increases to 2 4 p M (Jefferson et a!., 1968; Blackmore et al., 1979), which would suffice for insulin to trigger the activation of the phosphodiesterase. This would be consistent with insulin not being able to lower basal cyclic AMP concentrations, yet be able to decrease those elevated by glucagon (Jefferson et al., 1968). At 1 ,UM-CYChC AMP, the plasma membrane comprises some 10% of the total cellular cyclic AMP phosphodiesterase activity (Westwood et al., 1979; Marchmont & Houslay, 1980), and this