Phosphorylation of sites 3 and 4 in rabbit skeletal muscle glycogen synthase by cAMP-dependent protein kinase

V. S. Sheorain, J. D. Corbin, Thomas Soderling

Research output: Contribution to journalArticle

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Abstract

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 languageEnglish (US)
Pages (from-to)1567-1572
Number of pages6
JournalJournal of Biological Chemistry
Volume260
Issue number3
StatePublished - 1985
Externally publishedYes

Fingerprint

Glycogen Synthase
Phosphorylation
Cyclic AMP-Dependent Protein Kinases
Muscle
Skeletal Muscle
Rabbits
Stoichiometry
Phosphotransferases
Holoenzymes
High performance liquid chromatography
Reverse-Phase Chromatography
Protein Kinase Inhibitors
Protein Kinases
Epinephrine
Sequence Analysis
Catalytic Domain
Hot Temperature
Chemical activation
Phosphates
High Pressure Liquid Chromatography

ASJC Scopus subject areas

  • Biochemistry

Cite this

Phosphorylation of sites 3 and 4 in rabbit skeletal muscle glycogen synthase by cAMP-dependent protein kinase. / Sheorain, V. S.; Corbin, J. D.; Soderling, Thomas.

In: Journal of Biological Chemistry, Vol. 260, No. 3, 1985, p. 1567-1572.

Research output: Contribution to journalArticle

Sheorain, V. S. ; Corbin, J. D. ; Soderling, Thomas. / Phosphorylation of sites 3 and 4 in rabbit skeletal muscle glycogen synthase by cAMP-dependent protein kinase. In: Journal of Biological Chemistry. 1985 ; Vol. 260, No. 3. pp. 1567-1572.
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