Fluorescence Studies on the Interaction of Inhibitor 2 and Okadaic Acid with the Catalytic Subunit of Type 1 Phosphoprotein Phosphatases

William D. Picking, Wieslaw Kudlicki, Gisela Kramer, Boyd Hardesty, Jackie R. Vandenheede, Wilfried Merlevede, In Kyung Park, Anna DePaoli-Roach

Research output: Contribution to journalArticle

31 Scopus citations

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 12 (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 12 molecule that is retained even after the CPM-I2 is displaced by an excess of native 12. 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.

Original languageEnglish (US)
Pages (from-to)10280-10287
Number of pages8
JournalBiochemistry
Volume30
Issue number42
DOIs
StatePublished - Oct 1 1991

ASJC Scopus subject areas

  • Biochemistry

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    Picking, W. D., Kudlicki, W., Kramer, G., Hardesty, B., Vandenheede, J. R., Merlevede, W., Park, I. K., & DePaoli-Roach, A. (1991). Fluorescence Studies on the Interaction of Inhibitor 2 and Okadaic Acid with the Catalytic Subunit of Type 1 Phosphoprotein Phosphatases. Biochemistry, 30(42), 10280-10287. https://doi.org/10.1021/bi00106a028