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

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: 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 affinity 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 inhibited the phosphorylase phosphatase activity of PP1c (IC 50 = 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 complexes of PP1c±MYPT1 1±38 and PP1c±MYPT1 23±38 . The inhibitory effect of MYPT1 40±511 on the phosphorylase phosphatase activity of PP1c also was increased in the presence of MYPT1 1±38 . The binding of MYPT1 304±511 to complexes 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-binding motif and the other interactions are facilitated in an ordered and cooperative manner.Keywords: myosin phosphatase; protein phosphatase-1; myosin phosphatase target subunit-1; surface plasmon resonance.Phosphorylation of the 20-kDa light chains of smooth muscle myosin (MLC20) on Ser19 plays a pivotal role in the regulation of contractile activity of smooth muscle and is also implicated in several functions of nonmuscle cells [1]. The phosphorylation level of myosin reflects the balance of activities of myosin light chain kinase and the myosin phosphatase (MP). Several different preparations of MP that are active with phosphorylated myosin and/or the phosphorylated form of MLC20 (P-MLC20) have been isolated from smooth muscle [2]. Recently several groups have suggested that the major form of MP in smooth muscle is a trimeric holoenzyme composed of the 38-kDa d isoform of type 1 protein phosphatase catalytic subunit (PP1cd), a large subunit of 110/133 kDa, and a small subunit of 20/21 kDa [3±6]. The 20/21 kDa subunit interacts with the large subunit and its function is not known [7,8]. The 110/133 kDa subunit binds to both the catalytic subunit of type 1 protein phosphatase (PP1c) and myosin [7±10] and thus is termed myosin phosphatase target subunit (MYPT) [11,12]. MP is not confined to smooth muscle. For example, PP1c complexed with a large subunit similar to MYPT that localized to the cytoskeletal and membrane fractions was identified in human platelets [13]. Recently,...

Structural basis of protein phosphatase 1 regulation

“…The present structure offers an interesting twist: the ankyrin repeats of MYPT1 seem to play only a limited part in binding, but a potentially key part in modulating the catalytic activity of PP1. This finding is in agreement with biochemical evidence that shows almost no binding to PP1 of MYPT1 constructs lacking the N-terminal 38 amino acids [4][5][6][7][8] . Constructs, however, that add the ankyrin repeat domain to the N terminus of MYPT1 display increased myosin specificity and reduced activity towards other substrates, comparable to full-length myosin phosphatase [4][5][6]8 .…”

“…9), and a site responsible for catalysis that engages PP1 and the N-terminal one-third of MYPT1 simultaneously. In a similar way to that of the intact myosin phosphatase holoenzyme, the complex between PP1 and the N-terminal one-third of MYPT1 displays ,15-fold increased catalytic activity and ,10-fold higher affinity for phosphorylated myosin (or P-RLC) than the isolated catalytic subunit [4][5][6][7]10 . In addition, this complex has reduced activity towards other substrates, such as phosphorylase a and glycogen synthase 4,5,7 .…”

“…MYPT1 plays a key part in determining the physical and functional integrity of the trimeric myosin phosphatase holoenzyme. It binds PP1d at the N terminus [4][5][6][7][8] and M20 at the C terminus 3,4 . Binding to myosin seems to take place through two separate sites: a site situated within the C-terminal one-third of MYPT1 (ref.…”

“…A peptide corresponding to the sequence Met B1 to Phe B38 of MYPT1 can confer upon PP1 some of the myosin specificity and increased activity characteristic of the myosin phosphatase holoenzyme 4,[6][7][8] . However, a shorter peptide (Asp B23 to Phe B38) binds to PP1 but has no effect on its catalytic activity 7 . Together these results emphasize the importance of the N terminus of MYPT1 for the myosin specificity of myosin phosphatase.…”

Myosin phosphatase target subunit: Many roles in cell function

“…binding of PP1cδ and the substrate, P-myosin (or, P-RLC) to MYPT1. PP1c is anchored by the RVXF PP1c-binding motif and forms secondary interactions with the N-terminal sequence, some of the ankyrin repeats and with a site within the sequence 304-501 [5]. It was suggested that the acidic patch (326-372) was involved in activation of phosphatase activity with P-RLC [6].…”