Multisite phosphorylation of glycogen synthase. Molecular basis for the substrate specificity of glycogen synthease kinase-3 and casein kinase-II (glycogen synthase kinase-5)

James R. Woodgett, Philip Cohen

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    153 Citations (Scopus)

    Abstract

    Glycogen synthase kinase-3 was isolated rabbit skeletal muscle by an improved procedure. The purification was estimated to be 67 000-fold and 0.2 mg of enzyme was isolated from 5000 g muscle, corresponding to an overall yield of 7%. The preparation was homogeneous by ultracentrifugal and electrophoretic criteria. The enzyme had a relative molecular mass of 47 kDa by sedimentation equilibrium centrifugation and 51 kDa by SDS-polyacrylamide gel electrophoresis. These values demonstrate that glycogen synthase kinase-3 is monomeric. The stokes radius of 37 nm suggests the molecule to be asymmetric. The activating factor of the Mg-ATP dependent form of protein phosphatase-1 coeluted with glycogen synthase kinase-3 activity at the final step, establishing that these two activities reside in the same protein. Glycogen synthase kinase-3 phosphorylates glycogen synthase at sites-3, while casein kinase-II phosphorylates site-5, just C-terminal to sites-3 (Picton, C., Aitken, A., Bilham, T. and Cohen, P. (1982) Eur. J. Biochem. 124, 37-45). The basis for the substrate specificities of these protein kinases was investigated using chymotryptic peptides that contain the sites phosphorylated by each enzyme. These studies showed that efficient phosphorylation of sites-3, required the presence of phosphate in site-5 and a region of polypeptide more than 20 residues C-terminal to site-5. In contrast, efficient phosphorylation by casein kinase-II does not require this C-terminal region, and the rsults are consistent with the view that the enzyme recognises acidic residues immediately C-terminal to site-5.

    Original languageEnglish
    Pages (from-to)339-347
    Number of pages9
    JournalBiochimica et Biophysica Acta (BBA)/Protein Structure and Molecular
    Volume788
    Issue number3
    DOIs
    Publication statusPublished - 14 Aug 1984

    Keywords

    • Casein kinase
    • Enzyme specificity
    • Glycogen synthase
    • Glycogen synthase kinase
    • Multisite phosphorylation

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