Adenosine 3',5'-monophosphate-dependent phosphorylation of a 6000 and a 22,000 dalton protein from cardiac sarcoplasmic reticulum.

In canine cardiac sarcoplasmic reticulum, adenosine 3',5'-monophosphate (cyclic AMP)-dependent protein kinase specifically phosphorylates two proteins, as seen by sodium dodecyl sulfate-slab gel electrophoresis and autoradiography. One protein has a molecular weight ranging between 22,000 and 24,000 daltons and has previously been identified and named phospholamban (Tada, M., Kirchberger, M.A. and Katz, A.M. (1975) J. Biol. Chem. 250, 2640-2647). The other protein that the 32P label incorporates into has a molecular weight of approximately 6000. Like the 22,000 dalton protein, the 6000 dalton protein has characteristics of phosphoester bonding. The time-dependent course of phosphorylation shows that initially the 32P label is incorporated more rapidly into the 22,000 dalton protein than the 6000 dalton protein, with both proteins reaching a steady-state level of phosphorylation after 10 min of incubation. When both protein kinase and cyclic AMP are eliminated from the incubation medium, both the 22,000 and the 6000 dalton protein are still phosphorylated, but only to about a quarter of the activity found when cyclic AMP and protein kinases are included in the incubation mixture. The addition of phosphodiesterase completely eliminates the phosphorylation of both proteins. Treating the microsomes with trypsin prevents subsequent phosphorylation of either protein. Phosphorylating the microsomes first, then treating with trypsin, renders both the 22,000 and the 6000 dalton proteins resistant to even prolonged trypsin attack. Unphosphorylated, both proteins are solubilized by a very low concentration of deoxycholate. After phosphorylation the proteins cannot be solubilized by deoxycholate. Phosphorylation appears to alter greatly the physical properties of these proteins. Control experiments exclude the possibility that a lipid is being phosphorylated. After phosphorylation the phosphorylated 22,000 dalton protein is separated from the 6000 dalton protein by proteolipid extraction. After first treating the microsomes with methanol, the 22,000 dalton protein is then soluble in acidified chloroform/methanol, while the 6000 dalton protein remains insoluble. The finding that both proteins have much different biochemical properties when phosphorylated than when not, may be relevant in how they regulate calcium transport in the sarcoplasmic reticulum.