Conversion of protein phosphatase 1 catalytic subunit to a Mn(2+)-dependent enzyme impairs its regulation by inhibitor 1.
The phosphorylase phosphatase activity of protein phosphatase 1 (PP1) catalytic subunit from freshly purified rabbit skeletal muscle was inhibited by MnCl2. Prolonged storage or inhibition by nonspecific phosphatase inhibitors ATP, sodium pyrophosphate, and NaF converted the muscle PP1 to a form that required Mn2+ for enzyme activity. Recombinant PP1 catalytic subunit expressed in Escherichia coli was also a Mn2+-dependent enzyme. While native PP1 was inhibited by the phosphoprotein inhibitor I (I-1), with an IC50 of 1 nM, 40-50-fold higher concentrations of I-1 were required to inhibit the Mn2+-dependent PP1 enzymes. Conversion to the Mn2+-dependent state was accompanied by a 20-fold increase in PP1's ability to dephosphorylate and inactivate I-1. Inhibition by thiophosphorylated I-1 established that dephosphorylation does not play a significant role in I-1's reduced potency as an inhibitor of Mn2+-dependent PP1. The Mn2+-dependent PP1 enzymes were poorly inhibited by N-terminal phosphopeptides of I-1, indicating their impaired interaction with the I-1 functional domain. Mutation of a residue conserved in I-1 and DARPP-32, a structurally related PP1 inhibitor, preferentially attenuated I-1's activity as an inhibitor of Mn2+-dependent PP1. These data showed that, in addition to changes in its catalytic properties, Mn2+-dependent PP1 was modified in its interaction with I-1 at a site that was distinct from its catalytic domain. Our studies suggest that conversion to a Mn2+-dependent state alters multiple structural elements in PP1 catalytic subunit that together define its regulation by I-1.
Endo
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Department of Pharmacology, Duke University Medical Center, Durham, North Carolina 27710-0001, USA.
Connor
J H
JH
Forney
B
B
Zhang
L
L
Ingebritsen
T S
TS
Lee
E Y
EY
Shenolikar
S
S
D000595:Amino Acid Sequence; D000818:Animals; D002352:Carrier Proteins; D002384:Catalysis; D002712:Chlorides / Q000494:pharmacology; D004722:Endoribonucleases; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins; D008345:Manganese / Q000378:metabolism*; D017895:Manganese Compounds / Q000494:pharmacology; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010749:Phosphoprotein Phosphatases / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000378:metabolism*; D010766:Phosphorylation; D011487:Protein Conformation; D054645:Protein Phosphatase 1; D011506:Proteins / Q000235:genetics / Q000494:pharmacology*; D016601:RNA-Binding Proteins; D011817:Rabbits; D013379:Substrate Specificity
D002352:Carrier Proteins; D002712:Chlorides; D004791:Enzyme Inhibitors; D047908:Intracellular Signaling Peptides and Proteins; D017895:Manganese Compounds; D011506:Proteins; D016601:RNA-Binding Proteins; C021042:protein phosphatase inhibitor-1; D008345:Manganese; D004722:Endoribonucleases; D010749:Phosphoprotein Phosphatases; D054645:Protein Phosphatase 1; C097081:PPP1R8 protein, human; C025340:manganese chloride