Study of the subunit interactions in myosin phosphatase by surface plasmon resonance

Attila Tóth, Enikö Kiss, Friedrich W. Herberg, P. Gergely, David J. Hartshorne, F. Erdődi

Research output: Contribution to journalArticle

62 Citations (Scopus)

Abstract

The interactions of the catalytic subunit of type 1 protein phosphatase (PP1c) and the N-terminal half (residues 1-511) of myosin phosphatase target subunit 1 (MYPT1) were studied. Biotinylated MYPT1 derivatives were immobilized on streptavidin-biosensor chips, and binding parameters with PP1c were determined by surface plasmon resonance (SPR). The affinity of binding of PP1c was: MYPT11-296 > MYPT11-38 > MYPT123-38. No binding was detected with MYPT11-34, suggesting a critical role for residues 35- 38, i.e. the PP1c binding motif. Binding of residues 1-22 was inferred from: a higher affinity binding to PP1c for MYPT11-38 compared to MYPT123- 38, as deduced from SPR kinetic data and ligand competition assays; and an activation of the myosin light chain phosphatase activity of PP1c by MYPT11-38, but not by MYPT123-38. Residues 40-296 (ankyrin repeats) in MYPT11-296 inhibited the phosphorylase phosphatase activity of PP1c (IC50 = 0.2 nM), whereas MYPT11-38, MYPT123-38 or MYPT11-34 were without effect. MYPT140-511, which alone did not bind to PP1c, showed facilitated binding to the complexes of PP1c-MYPT11-38 and PP1c- MYPT123-38. The inhibitory effect of MYPT140-511 on the phosphorylase phosphatase activity of PP1c also was increased in the presence of MYPT11-38. The binding of MYPT1304-511 to complexes of PP1c and MYPT11-38, or MYPT11-296, was detected by SPR. These results suggest that within the N-terminal half of MYPT1 there are at least four binding sites for PP1c. The essential interaction is with the PP1c-binding motif and the other interactions are facilitated in an ordered and cooperative manner.

Original languageEnglish
Pages (from-to)1687-1697
Number of pages11
JournalEuropean Journal of Biochemistry
Volume267
Issue number6
DOIs
Publication statusPublished - 2000

Fingerprint

Myosin-Light-Chain Phosphatase
Surface Plasmon Resonance
Surface plasmon resonance
Phosphorylase Phosphatase
Ankyrin Repeat
Protein Phosphatase 1
Streptavidin
Biosensing Techniques
Biosensors
Inhibitory Concentration 50
Assays
Chemical activation
Binding Sites
Ligands
Derivatives
Kinetics

Keywords

  • Myosin phosphatase
  • Myosin phosphatase target subunit-1
  • Protein phosphatase-1
  • Surface plasmon resonance

ASJC Scopus subject areas

  • Biochemistry

Cite this

Study of the subunit interactions in myosin phosphatase by surface plasmon resonance. / Tóth, Attila; Kiss, Enikö; Herberg, Friedrich W.; Gergely, P.; Hartshorne, David J.; Erdődi, F.

In: European Journal of Biochemistry, Vol. 267, No. 6, 2000, p. 1687-1697.

Research output: Contribution to journalArticle

Tóth, Attila ; Kiss, Enikö ; Herberg, Friedrich W. ; Gergely, P. ; Hartshorne, David J. ; Erdődi, F. / Study of the subunit interactions in myosin phosphatase by surface plasmon resonance. In: European Journal of Biochemistry. 2000 ; Vol. 267, No. 6. pp. 1687-1697.
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AU - Hartshorne, David J.

AU - Erdődi, F.

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AB - The interactions of the catalytic subunit of type 1 protein phosphatase (PP1c) and the N-terminal half (residues 1-511) of myosin phosphatase target subunit 1 (MYPT1) were studied. Biotinylated MYPT1 derivatives were immobilized on streptavidin-biosensor chips, and binding parameters with PP1c were determined by surface plasmon resonance (SPR). The affinity of binding of PP1c was: MYPT11-296 > MYPT11-38 > MYPT123-38. No binding was detected with MYPT11-34, suggesting a critical role for residues 35- 38, i.e. the PP1c binding motif. Binding of residues 1-22 was inferred from: a higher affinity binding to PP1c for MYPT11-38 compared to MYPT123- 38, as deduced from SPR kinetic data and ligand competition assays; and an activation of the myosin light chain phosphatase activity of PP1c by MYPT11-38, but not by MYPT123-38. Residues 40-296 (ankyrin repeats) in MYPT11-296 inhibited the phosphorylase phosphatase activity of PP1c (IC50 = 0.2 nM), whereas MYPT11-38, MYPT123-38 or MYPT11-34 were without effect. MYPT140-511, which alone did not bind to PP1c, showed facilitated binding to the complexes of PP1c-MYPT11-38 and PP1c- MYPT123-38. The inhibitory effect of MYPT140-511 on the phosphorylase phosphatase activity of PP1c also was increased in the presence of MYPT11-38. The binding of MYPT1304-511 to complexes of PP1c and MYPT11-38, or MYPT11-296, was detected by SPR. These results suggest that within the N-terminal half of MYPT1 there are at least four binding sites for PP1c. The essential interaction is with the PP1c-binding motif and the other interactions are facilitated in an ordered and cooperative manner.

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