Interaction of myosin phosphatase target subunit 1 with the catalytic subunit of type 1 protein phosphatase.
In the investigation of the sequences of myosin phosphatase target subunit 1 (MYPT1) involved in binding the substrate and catalytic subunit of protein phosphatase type 1 (PP1c), fragments of MYPT1 were prepared and characterized. The shortest fragment capable of full activation of PP1c contained the sequence of residues 1-295. Within this fragment, the N-terminal sequence of residues 1-38 is involved in activation of PP1c (kcat) and the ankyrin repeats (residues 39-295) were involved in substrate binding (Km). The ankyrin repeats alone (residues 39-295) and the C-terminal fragment of residues 667-1004 did not activate PP1c. Using gel filtration, an interaction with PP1c was detected for the sequences of residues 1-295, 17-295, and 1-170. Affinity columns were prepared with various fragments to assess binding of PP1c. Binding to the column with residues 1-295 was strongest, followed by the binding to the column with residues 1-170. A weak interaction was observed with the column with residues 1-38. The column with residues 1-295 was used to isolate PP1c from gizzard. The purified PP1c was activated by MYPT1 and fragments to a greater extent than previous preparations. These results suggest that the N-terminal sequence (residues 1-38) and the ankyrin repeats are involved in binding PP1c. The C-terminal ankyrin repeats appear to be dominant, but there is an interaction of PP1c with the N-terminal ankyrin repeats. The N-terminal peptide has two apparent functions, the binding of PP1c via the consensus binding sequence and activation of PP1c by the sequence of residues 1-16.
Tanaka
J
J
First Department of Internal Medicine, Mie University School of Medicine, Japan.
Ito
M
M
Feng
J
J
Ichikawa
K
K
Hamaguchi
T
T
Nakamura
M
M
Hartshorne
D J
DJ
Nakano
T
T
D000818:Animals; D002384:Catalysis; D002645:Chickens; D002846:Chromatography, Affinity; D002850:Chromatography, Gel; D004789:Enzyme Activation / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics; D005895:Gizzard, Avian; D007527:Isoenzymes / Q000235:genetics / Q000378:metabolism / Q000502:physiology; D043263:Myosin-Light-Chain Phosphatase; D010446:Peptide Fragments / Q000235:genetics / Q000378:metabolism / Q000502:physiology; D010749:Phosphoprotein Phosphatases / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D054645:Protein Phosphatase 1; D011994:Recombinant Proteins / Q000378:metabolism / Q000494:pharmacology
D007527:Isoenzymes; D010446:Peptide Fragments; D011994:Recombinant Proteins; D010749:Phosphoprotein Phosphatases; D054645:Protein Phosphatase 1; D043263:Myosin-Light-Chain Phosphatase