Structural characterization of the membrane-associated regulatory subunit of type I cAMP-dependent protein kinase by mass spectrometry:: Identification of Ser81 as the in vivo phosphorylation site of RIα

The mechanism by which the type I alpha regulatory subunit (RI alpha) of cAMP-dependent protein kinase is localized to cell membranes is unknown. To determine if structural modification of RI alpha is important for membrane association, both beef skeletal muscle cytosolic RI and beef heart membrane-associated RI were characterized by electrospray ionization mass spectrometry. Total sequence coverage was 98% for both the membrane-associated and cytosolic forms of RI after digestion with AspN protease or trypsin. Sequence data indicated that membrane-associated and cytosolic forms of RI were the same RI alpha gene product. A single RI alpha phosphorylation site was identified at Ser81 located near the autoinhibitory domain of both membrane-associated and cytosolic RI alpha. Because both R subunit preparations were 30-40% phosphorylated, this post-translational modification could not be responsible for the membrane compartmentation of the majority of RI alpha. Mass spectrometry also indicated that membrane-associated RI alpha had a higher extent of disulfide bond formation in the amino-terminal dimerization domain. No other structural differences between cytosolic and membrane-associated RI alpha were detected. Consistent with these data, masses of the intact proteins were identical by LCQ mass spectrometry. Lack of detectable structural differences between membrane-associated and cytosolic RI alpha strongly suggests an interaction between RI alpha and anchoring proteins or membrane lipids as more likely mechanisms for explaining RI alpha membrane association in the heart.