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 De Paoli-Roach

Research output: Contribution to journalArticle

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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
Pages (from-to)10280-10287
Number of pages8
JournalBiochemistry
Volume30
Issue number42
StatePublished - 1991

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Okadaic Acid
Phosphoprotein Phosphatases
Catalytic Domain
Phosphotransferases
Fluorescence
Glycogen Synthase Kinase 3
Coumarins
Molecules
Fluorescence Polarization
Competitive Binding
Bioactivity
Phosphoric Monoester Hydrolases
Escherichia coli
Anisotropy
Proteins
maleimide
(2-carboxy-3-phenylpropyl)methylsulfodiimide
protein phosphatase inhibitor-2

ASJC Scopus subject areas

  • Biochemistry

Cite this

Picking, W. D., Kudlicki, W., Kramer, G., Hardesty, B., Vandenheede, J. R., Merlevede, W., ... De Paoli-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.

Fluorescence studies on the interaction of inhibitor 2 and okadaic acid with the catalytic subunit of type 1 phosphoprotein phosphatases. / Picking, William D.; Kudlicki, Wieslaw; Kramer, Gisela; Hardesty, Boyd; Vandenheede, Jackie R.; Merlevede, Wilfried; Park, In Kyung; De Paoli-Roach, Anna.

In: Biochemistry, Vol. 30, No. 42, 1991, p. 10280-10287.

Research output: Contribution to journalArticle

Picking, WD, Kudlicki, W, Kramer, G, Hardesty, B, Vandenheede, JR, Merlevede, W, Park, IK & De Paoli-Roach, A 1991, 'Fluorescence studies on the interaction of inhibitor 2 and okadaic acid with the catalytic subunit of type 1 phosphoprotein phosphatases', Biochemistry, vol. 30, no. 42, pp. 10280-10287.
Picking WD, Kudlicki W, Kramer G, Hardesty B, Vandenheede JR, Merlevede W et al. Fluorescence studies on the interaction of inhibitor 2 and okadaic acid with the catalytic subunit of type 1 phosphoprotein phosphatases. Biochemistry. 1991;30(42):10280-10287.
Picking, William D. ; Kudlicki, Wieslaw ; Kramer, Gisela ; Hardesty, Boyd ; Vandenheede, Jackie R. ; Merlevede, Wilfried ; Park, In Kyung ; De Paoli-Roach, Anna. / Fluorescence studies on the interaction of inhibitor 2 and okadaic acid with the catalytic subunit of type 1 phosphoprotein phosphatases. In: Biochemistry. 1991 ; Vol. 30, No. 42. pp. 10280-10287.
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AB - 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.

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