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

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.