Study of the subunit interactions in myosin phosphatase by surface plasmon resonance
Study of the subunit interactions in myosin phosphatase by surface plasmon resonance
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DOI：
10.1046/j.1432-1327.2000.01158.x
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发表时间：
2000-03-01
期刊：
影响因子：
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通讯作者：
Erdödi, F
中科院分区：
文献类型：
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作者：
Tóth, A;Kiss, E;Erdödi, F
研究了I型蛋白磷酸酶催化亚基(PP1c)与肌球蛋白磷酸酶靶标亚基1(MYPT1)N末端部分(残基1~511)的相互作用。将生物素化的MYPT1衍生物固定在链霉亲和素生物传感器芯片上，用表面等离子体共振法(SPR)测定其与PP1c的结合参数。PP1c的亲和力为：MYPT1(1-296)>；MYPT1(1-38)>；MYPT1(23-38)。未检测到与MYPT1(1-34)的结合，表明35-38残基，即PP1c结合基序具有关键作用。根据SPR动力学数据和配体竞争分析，推测残基1-22与PP1c的结合比MYPT1(23-38)与PP1c的亲和力更高；MYPT1(1-38)激活了PP1c的肌球蛋白轻链磷酸酶活性，但MYPT1(23-38)没有激活。MYPT1(1-296)的40-296位残基(Ankyrin Repeats)抑制PP1c的磷酸化磷酸酶活性(IC50=0.2 nm)，而MYPT1(1-38)、MYPT1(23-38)或MYPT1(1-34)对PP1c的磷酸化磷酸酶活性无影响。MYPT1(40-511)单独不与PP1c结合，与PP1c-MYPT1(1-38)和PP1c-MYPT1(23-38)的络合物有易化结合。在MYPT1(1-38)的存在下，MYPT1(40-511)对PP1c磷酸化磷酸酶活性的抑制作用也增强。SPR检测到MYPT1(304-511)与PP1c和MYPT1(1-38)或MYPT1(1-296)的络合物结合。这些结果表明，在MYPT1的N-末端一半中，至少有四个PP1c结合位点。主要的相互作用是与PP1c结合基序，其他相互作用是以有序和合作的方式促进的。
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 (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 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.