Interactions and properties of smooth muscle myosin phosphatase.
Interactions of the type 1 phosphatase catalytic subunit (PP1c) and the myosin phosphatase holoenzyme (MBP) were compared using affinity columns. In the absence of ATP, MBP bound to dephosphorylated myosin, heavy meromyosin (HMM), and subfragment 1. In contrast, PP1c was not bound. In the presence of ATP, the binding of MBP occurred only with phosphorylated protein. The interaction of MBP with phosphorylated proteins also was demonstrated using thiophosphorylated proteins as competitive inhibitors. Kinetics parameters were determined. With phosphorylated light chains (P-LC20), the major difference between PP1c and MBP was a lower K(m) for the latter. With myosin, MBP showed a marked increase in kcat, compared to PP1c. ATP did not affect these parameters. To investigate the role of the large phosphatase subunit, two recombinant proteins representing the N-terminal two-thirds of the molecule were expressed. These activated PP1c, and activation was maximum at approximately an equimolar ratio. The equimolar mixture of recombinant fragment and PP1c exhibited K(m) values similar to MBP and increased kcat values, compared to PP1c alone. An affinity column was prepared using the recombinant fragment. Phosphorylated HMM and P-LC20 were bound in the presence and absence of ATP. The interaction of P-LC20 was not ATP-dependent. Dephosphorylated HMM did not bind in the presence of ATP. The N-terminal fragment of the large subunit also contained a binding site for PP1c. These results indicate that the N-terminal portion of the large subunit of MBP contained binding sites for P-LC20 and PP1c.
Ichikawa
K
K
Muscle Biology Group, University of Arizona, Tucson 85721, USA.
Hirano
K
K
Ito
M
M
Tanaka
J
J
Nakano
T
T
Hartshorne
D J
DJ
D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D002417:Cattle; D002846:Chromatography, Affinity; D017931:DNA Primers / Q000235:genetics; D066298:In Vitro Techniques; D007700:Kinetics; D008297:Male; D008969:Molecular Sequence Data; D009130:Muscle, Smooth / Q000201:enzymology*; D043263:Myosin-Light-Chain Phosphatase; D009218:Myosins / Q000378:metabolism; D010446:Peptide Fragments / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D010749:Phosphoprotein Phosphatases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D011487:Protein Conformation; D054645:Protein Phosphatase 1; D011817:Rabbits; D014422:Turkeys
D017931:DNA Primers; D010446:Peptide Fragments; D000255:Adenosine Triphosphate; D010749:Phosphoprotein Phosphatases; D054645:Protein Phosphatase 1; D043263:Myosin-Light-Chain Phosphatase; D009218:Myosins