Demonstration of the Control of Rabbit Skeletal-Muscle Phosphorylase Kinase Activity in vivo by Adenosine 3′5′-Cyclic Monophosphate-Dependent Protein Kinase

Abstract

Rabbit skeletal-muscle phosphorylase kinase has an activity at physiological pH (6.8) only 1-2% of that observed at the pH optimum (8.2), but may be activated @fold at pH6.8 after incubation with cyclic AMP-dependent protein kinase, cyclic AMP and ATP-MgZ+ in vitro (Walsh et al., 1971 ; Cohen, 1973). Two molecules of covalently bound phosphate are incorporated during the reaction. The phosphorylation at the primary site on the 8-subunit parallels the increase in activity (Hayakawa et al., 1973; Cohen, 1973), whereas the slower phosphorylation of a second site, on the a-subunit, controls the rate of dephosphorylation of the 8-subunit catalysed by phosphorylase kinase phosphatase (Cohen & Antoniw, 1973). The specificity of this reaction in vitro has been confirmed by the isolation of a nine-residue tryptic phosphopeptide from the 8-subunit (Cohen, 1974) and a 39-residue tryptic phosphopeptide from the a-subunit (P. Cohen, unpublished work). Although phosphorylase kinase activity previously has been shown to increase after an intravenous injection of adrenalin (Posner et al., 1965; Drummond et al., 1969), this need not necessarily be a consequence of the activation of cyclic AMP-dependent protein kinase. Phosphorylase kinase activity can also be stimulated by two further mechanisms in vitro, by a calcium-dependent phosphorylation of phosphorylase kinase by itself (Walsh et al., 1971) and by proteolysis (Huston & Krebs, 1968). In the present paper, phosphorylase kinase was activated by an intravenous injection of adrenalin, and purified through the (NH4)2S04 step by the procedure previously described for the non-activated enzyme (Cohen, 1973). The pH6.8/8.2 activity ratio in the muscleextract was0.22 incontrast with thevalueof0.03f0.01 observedin theabsenceof adrenalin. NaF (50m~) was included in the purification to inhibit phosphorylase kinase phosphatase, but there was nevertheless a slight decline of the activity ratio to 0.16 during the 8 h isolation. Phosphorylase kinase (1 .Oms), which had been phosphorylated in vitro at both the aand 8-subunit sites, was then added to phosphorylase kinase activated tn vivo (200mg), and the aand 8-subunit phosphopeptides were isolated by using the trace of radioactivemarker protein to locate the position of the peptides and to calculate the yield at each step.