Phosphorylation of inhibitor 2, the regulatory subunit of the ATP-Mg-dependent protein phosphatase, by glycogen synthase kinase 3 (GSK-3) causes activation of the phosphatase. Prior phosphorylation by casein kinase II has been shown to enhance both phosphorylation and activation of the phosphatase by GSK-3 (DePaoli-Roach, A. A. (1984) J. Biol. Chem. 259, 12144-12152). Reported here is a comparison of the phosphorylation of inhibitor 2 by two defined isoforms of GSK-3, GSK-3α and GSK-3β. GSK-3β was a significantly better inhibitor 2 kinase than was GSK-3α. The Vmax/Km value for GSK-3β was ∼10-fold higher than that for GSK-3α. GSK-3β phosphorylated inhibitor 2 to a stoichiometry of ∼1.0 mol of phosphate/mol of inhibitor 2. The phosphorylation by GSK-3β was determined to be exclusively at Thr-72 on the basis of the inability of the enzyme to modify a mutant inhibitor 2 in which Thr-72 was changed to alanine. Prior phosphorylation by casein kinase II promoted the action of GSK-3α in keeping with earlier reports using undefined GSK-3 preparations. Phosphorylation by GSK-3β, in contrast, was unaffected by the previous action of casein kinase II. These results suggest that there can be important differences in substrate recognition by different isoforms of the same protein kinase and may help explain why some reported GSK-3 substrates require prior phosphorylation whereas others do not. It is proposed that substrate recognition by GSK-3 involves multiple determinants, the relative importance of each depending on both the substrate and the GSK-3 isoform in question.
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