Rabbit skeletal muscle glycogen synthase (synthase a) can be phosphorylated by 0.2 to 2.5 μM catalytic subunit of cAMP-dependent protein kinase to a stoichiometry of 1.5 to 3 mol of 32PO4/subunit (90,000 x g). When a complete tryptic digest of this 32P-synthase was chromatographed on reverse phase high performance liquid chromatography, it was observed that, in addition to sites 1a, 1b, and 2, site 3 was phosphorylated. The peptide containing site 5 also contained 32PO4, but sequence analysis identified a new phosphorylation site, site 4 (Arg-His-Ser-Ser(PO4)-) which precedes site 5. Phosphorylation of sites 3 and 4 became significant when the total phosphorylation stoichiometry exceeded 1.5 mol/subunit. The heat-stable protein kinase inhibitor protein kinase decreased the phosphorylation of all sites, including sites 3 and 4. Phosphorylation of all sites by the holoenzyme form of cAMP-dependent kinase was highly dependent on the presence of cAMP. These results establish that phosphorylation of these sites is due to the cAMP-dependent protein kinase itself and not to a contaminating kinase(s). Synthase b from control rabbit muscle, containing 2 to 2.5 mol of phosphate/subunit, incorporated up to 2.0 mol of 32PO4 catalyzed in vitro by the cAMP-dependent protein kinase. Again, significant 32PO4 was detected in sites 3 and 4 as well as in 1a, 1b, and 2. These results suggest that the in vivo phosphorylation of sites 1a, 1b, 2, and 3 observed after injection of epinephrine in rabbits could be due solely to the known activation of the cAMP-dependent protein kinase.
|Original language||English (US)|
|Number of pages||6|
|Journal||Journal of Biological Chemistry|
|State||Published - 1985|
ASJC Scopus subject areas
- Molecular Biology
- Cell Biology