Isoform differences in substrate recognition by glycogen synthase kinases 3 alpha and 3 beta in the phosphorylation of phosphatase inhibitor 2.
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-3 alpha and GSK-3 beta. GSK-3 beta was a significantly better inhibitor 2 kinase than was GSK-3 alpha. The Vmax/Km value for GSK-3 beta was approximately 10-fold higher than that for GSK-3 alpha. GSK-3 beta phosphorylated inhibitor 2 to a stoichiometry of approximately 1.0 mol of phosphate/mol of inhibitor 2. The phosphorylation by GSK-3 beta 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-3 alpha in keeping with earlier reports using undefined GSK-3 preparations. Phosphorylation by GSK-3 beta, 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 other do not.(ABSTRACT TRUNCATED AT 250 WORDS)
Wang
Q M
QM
Department of Biochemistry and Molecular Biology, Indiana University School of Medicine, Indianapolis 46202-5122.
Park
I K
IK
Fiol
C J
CJ
Roach
P J
PJ
DePaoli-Roach
A A
AA
D000595:Amino Acid Sequence; D000818:Animals; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000378:metabolism*; D017362:Cyclic AMP Response Element-Binding Protein / Q000378:metabolism; D004791:Enzyme Inhibitors / Q000378:metabolism*; D038341:Glycogen Synthase Kinases; D007527:Isoenzymes / Q000378:metabolism*; D007700:Kinetics; D008969:Molecular Sequence Data; D009132:Muscles / Q000201:enzymology; D016297:Mutagenesis, Site-Directed; D010446:Peptide Fragments / Q000138:chemical synthesis / Q000378:metabolism; D010455:Peptides / Q000138:chemical synthesis / Q000378:metabolism; D010766:Phosphorylation; D017354:Point Mutation; D011506:Proteins / Q000378:metabolism*; D011817:Rabbits; D011994:Recombinant Proteins / Q000378:metabolism; D013379:Substrate Specificity; D000097847:Protein Phosphatase Inhibitory Proteins
D017871:Calcium-Calmodulin-Dependent Protein Kinases; D017362:Cyclic AMP Response Element-Binding Protein; D004791:Enzyme Inhibitors; D038341:Glycogen Synthase Kinases; D007527:Isoenzymes; D010446:Peptide Fragments; D010455:Peptides; D011506:Proteins; D011994:Recombinant Proteins; C024833:protein phosphatase inhibitor-2; D000097847:Protein Phosphatase Inhibitory Proteins