Dephosphorylation of cAMP-dependent protein kinase regulatory subunit (type II) by calmodulin-dependent protein phosphatase. Determinants of substrate specificity.

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Publication Type Journal Article
School or College College of Pharmacy; School of Medicine
Department Biomedical Informatics; Biochemistry; Pharmacology & Toxicology
Creator Blumenthal, Donald K.
Other Author Takio, Koji; Hansen, R. Scott; Krebs, Edwin G.
Title Dephosphorylation of cAMP-dependent protein kinase regulatory subunit (type II) by calmodulin-dependent protein phosphatase. Determinants of substrate specificity.
Date 1985-06-25
Description Calmodulin-dependent protein phosphatase purified from bovine cardiac muscle catalyzed the rapid dephosphorylation of Ser-95 of bovine cardiac cAMP-dependent protein kinase regulatory subunit (RII). The kinetic constants determined for the reaction (Km = 20 microM; Vmax = 2 mumol min-1 mg-1) are comparable to those determined for other good substrates of this phosphatase. Because little is known about the determinants of substrate specificity for the calmodulin-dependent phosphatase, various phosphopeptides were used to investigate the structural features important for substrate recognition. Limited proteolysis of phospho-RII with trypsin and chymotrypsin yielded fragments (residues 93-400 and 91-400, respectively) that were poor substrates, whereas digestion with Staphylococcal aureus V8 protease produced three phosphopeptides that were all dephosphorylated as rapidly as intact RII. The sequence of the shortest phosphopeptide produced by S. aureus V8 protease was determined by sequence analysis to be Asp-Leu-Asp-Val-Pro-Ile-Pro-Gly-Arg-Phe-Asp-Arg-Arg-Val-Ser-Val-Cys-Ala-Glu, corresponding to residues 81-99 of RII. Synthetic phosphopeptides corresponding to residues 81-99, 85-99, 90-99, and 91-99 were prepared to determine the minimum sequence necessary for substrate recognition. Only the 19-residue peptide (81-99) was dephosphorylated with kinetics comparable to RII (Km = 26 microM, Vmax = 1.7 mumol min-1 mg-1). Structural analysis of this peptide indicates that an amphipathic beta-sheet structure may be an important structural determinant for some substrates of the calmodulin-dependent phosphatase.
Type Text
Publisher American Society for Biochemistry and Molecular Biology (ASBMB)
Volume 261
Issue 18
First Page 8140
Last Page 8145
Subject Metabolism; High Pressure Liquid; Enzymology
Subject MESH Calmodulin; Chromatography; Kinetics; Myocardium; Phosphoprotein Phosphatase; Protein Kinases; Substrate Specificity
Language eng
Bibliographic Citation Blumenthal DK, Takio K, Hansen RS, Krebs EG. Dephosphorylation of cAMP-dependent protein kinase regulatory subunit (type II) by calmodulin-dependent protein phosphatase. Determinants of substrate specificity. J Biol Chem. 1986 Jun 25;261(18):8140-5. Retrieved August 31, 2006 from http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?CMD=Display&DB=pubmed
Rights Management Copyright © American Society for Biochemistry and Molecular Biology, J Biol Chem., 261, 8140-8145, 1986
Format Medium application/pdf
Identifier ir-main,361
ARK ark:/87278/s6668xd1
Setname ir_uspace
Date Created 2012-06-13
Date Modified 2012-06-13
ID 703268
Reference URL https://collections.lib.utah.edu/ark:/87278/s6668xd1
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