title: Fluorescence studies on the interaction of inhibitor 2 and okadaic acid with the catalytic subunit of type 1 phosphoprotein phosphatases.
abstract: Phosphatase inhibitor 2 was mutagenized and expressed in Escherichia coli to produce a protein with a single cysteinyl residue at position 129. The newly introduced sulfhydryl group was labeled with a maleimide derivative of coumarin (CPM). The resulting fluorescent inhibitor 2 molecule (CPM-I2) retains biological activity and binds to the catalytic subunit of type 1 phosphatase (PP1-C) with a Kd similar to the Ki of native I2 (2-3 nM). Fluorescence anisotropy data indicate that kinase FA (glycogen synthase kinase 3) does not dissociate the CPM-I2.PP1-C complex but rather causes a conformational change in the I2 molecule that is retained even after the CPM-I2 is displaced by an excess of native I2. The fluorescence data presented here also indicate that okadaic acid and I2 are competitive for binding to PP1-C, even after kinase FA treatment of the CPM-I2.PP1-C complex.
authors.affiliation: Department of Chemistry and Biochemistry, University of Texas, Austin 78712.
authors.forename: W D
authors.initials: WD
authors.lastname: Picking
authors.forename: W
authors.initials: W
authors.lastname: Kudlicki
authors.forename: G
authors.initials: G
authors.lastname: Kramer
authors.forename: B
authors.initials: B
authors.lastname: Hardesty
authors.forename: J R
authors.initials: JR
authors.lastname: Vandenheede
authors.forename: W
authors.initials: W
authors.lastname: Merlevede
authors.forename: I K
authors.initials: IK
authors.lastname: Park
authors.forename: A
authors.initials: A
authors.lastname: DePaoli-Roach
mesh_terms: D000255:Adenosine Triphosphate / Q000494:pharmacology; D000818:Animals; D001667:Binding, Competitive; D002384:Catalysis; D004988:Ethers, Cyclic / Q000737:chemistry*; D005454:Fluorescence Polarization; D008274:Magnesium / Q000494:pharmacology; D019319:Okadaic Acid; D010749:Phosphoprotein Phosphatases / Q000037:antagonists & inhibitors / Q000737:chemistry*; D011494:Protein Kinases / Q000494:pharmacology; D011506:Proteins / Q000737:chemistry*; D011817:Rabbits; D000097847:Protein Phosphatase Inhibitory Proteins
chemical_list: D000255:Adenosine Triphosphate; D004988:Ethers, Cyclic; D008274:Magnesium; D019319:Okadaic Acid; D010749:Phosphoprotein Phosphatases; D011494:Protein Kinases; D011506:Proteins; C024833:protein phosphatase inhibitor-2; D000097847:Protein Phosphatase Inhibitory Proteins