Study of the subunit interactions in myosin phosphatase by surface plasmonresonance

The interactions of the catalytic subunit of type 1 protein phosphatase (PP 1c) and the N-terminal half (residues 1-511) of myosin phosphatase target s ubunit 1 (MYPT1) were studied. Biotinylated MYPT1 derivatives were immobili zed on streptavidin-biosensor chips, and binding parameters with PP1c were determined by surface plasmon resonance (SPR). The affinity of binding of P P1c was: MYPT1(1-296) > MYPT1(1-38) > MYPT1(23-38). No binding was detected with MYPT1(1-34), suggesting a critical role for residues 35-38, i.e. the PP1c binding motif. Binding of residues 1-22 was inferred from: a higher af finity binding to PP1c for MYPT1(1-38) compared to MYPT1(23-38), as deduced from SPR kinetic data and ligand competition assays; and an activation of the myosin light chain phosphatase activity of PP1c by MYPT1(1-38), but not by MYPT1(23-38). Residues 40-296 (ankyrin repeats) in MYPT1(1-296) inhibit ed the phosphorylase phosphatase activity of PP1c (IC50 = 0.2 nm), whereas MYPT1(1-38), MYPT1(23-38) or MYPT1(1-34) were without effect. MYPT1(40-511) , which alone did not bind to PP1c, showed facilitated binding to the compl exes of PP1c-MYPT1(1-38) and PP1c-MYPT1(23-38). The inhibitory effect of MY PT1(40-511) on the phosphorylase phosphatase activity of PP1c also was incr eased in the presence of MYPT1(1-38). The binding of MYPT1(304-511) to comp lexes of PP1c and MYPT1(1-38), or MYPT1(1-296), 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-bi nding motif and the other interactions are facilitated in an ordered and co operative manner.