Phosphorylation of skeletal and cardiac muscle C-proteins by the catalytic subunit of cAMP-dependent protein kinase

Catecholamines are known to influence the contractility of cardiac and skeletal muscles, presumably via cAMP-dependent phosphorylation of specific proteins. We have investigated the in vitro phosphorylation of myofibrillar proteins by the catalytic subunit of cAMP-dependent protein kinase of fast- and slow-twitch skeletal muscles and cardiac muscle with a view to gaining a better understanding of the biochemical basis of catecholamine effects on striated muscles. Incubation of canine red skeletal myofibrils with the isolated catalytic subunit of cAMP-dependent protein kinase and Mg-[γ-32P]ATP led to the rapid incorporation of [32P]phosphate into five major protein substrates of subunit molecular weights (MWs) 143 000, 60 000, 42 000, 33 000, and 11 000. The 143 000 MW substrate was identified as C-protein; the 42 000 MW substrate is probably actin; the 33 000 MW substrate was shown not to be a subunit of tropomyosin and, like the 60 000 and 11 000 MW substrates, is an unidentified myofibrillar protein. Isolated canine red skeletal muscle C-protein was phosphorylated to the extent of ~0.5 mol Pi/mol C-protein. Rabbit white skeletal muscle and bovine cardiac muscle C-proteins were also phosphorylated by the catalytic subunit of cAMP-dependent protein kinase, both in myofibrils and in the isolated state. Cardiac C-protein was phosphorylated to the extent of 5–6 mol Pi/mol C-protein, whereas rabbit white skeletal muscle C-protein was phosphorylated at the level of ~0.5 mol Pi/mol C-protein. As demonstrated earlier by others, C-protein of skeletal and cardiac muscles inhibited the actin-activated myosin Mg2+-ATPase activity at low ionic strength in a system reconstituted from the purified skeletal muscle contractile proteins (actin and myosin). Phosphorylation of skeletal or cardiac C-proteins had no effect on their inhibition of this actomyosin Mg2+-ATPase activity. Furthermore, cardiac C-protein inhibited the Mg2+-ATPase activity of desensitized cardiac actomyosin; in this case, phosphorylation of cardiac C-protein enhanced its inhibitory effect on the actomyosin Mg2+-ATPase. These observations suggest that C-proteins of fast- and slow-twitch skeletal muscle fibers and cardiac muscle fibers are phosphorylated in response to catecholamines and other agents which induce cAMP formation and that, at least in the heart, this phosphorylation may affect actin–myosin interaction and the contractile state of the muscle.