Mechanism of the stimulation of Ca 2+-dependent ATPase of skeletal muscle sarcoplasmic reticulum by protein kinase

Abstract

Sarcoplasmic reticulum isolated from moderately fast rabbit skeletal muscle contains intrinsic adenosine 3′,5′-monophosphate (cAMP)-independent protein kinase activity and a substrate of 100 000 M r . Phosphorylation of skeletal sarcoplasmic reticulum by either endogenous membrane bound or exogenous cAMP-dependent protein kinase results in stimulation of the initial rates of Ca 2+ transport and Ca 2+-ATPase activity. To determine the molecular mechanism by which protein kinase-dependent phosphorylation regulates the calcium pump in skeletal sarcoplasmic reticulum, we examined the effects of protein kinase on the individual steps of the Ca 2+-ATPase reaction sequence. Skeletal sarcoplasmic reticulum vesicles were preincubated with cAMP and cAMP-dependent protein kinase in the presence (phosphorylated sarcoplasmic reticulum) and absence (control sarcoplasmic reticulum) of adenosine 5′-triphosphate (ATP). Control and phosphorylated sarcoplasmic reticulum were subsequently assayed for formation (5–100 ms) and decomposition (0–73 ms) of the acid-stable phosphorylated enzyme ( E ∼ P ) of Ca 2+-ATPase. Protein kinase mediated phosphorylation of skeletal sarcoplasmic reticulum resulted in pronounced stimulation of initial rates and levels of E ∼ P in sarcoplasmic reticulum preincubated with either ethylene glycol bis(β- aminoethyl ether)-N,N′- tetraacetic acid (EGTA) prior to assay (Ca 2+-free sarcoplasmic reticulum), or with calcium/EGTA buffer (Ca 2+-bound sarcoplasmic reticulum). These effects were evident within a wide range of ionized Ca 2+. Phosphorylation of skeletal sarcoplasmic reticulum by protein kinase also increased the initial rate of E ∼ P decomposition. These findings suggest that protein kinase-dependent phosphorylation of skeletal sarcoplasmic reticulum regulates several steps in the Ca 2+-ATPase reaction sequence which result in an overall stimulation of the active calcium transport observed at steady state.