On the mechanism of regulation of type I phosphoprotein phosphatase from bovine heart. Regulation by a novel intracyclic activation-deactivation mechanism via transient phosphorylation of the regulatory subunit by phosphatase-1 kinase (FA).
Adenosine 5'-(gamma-thio)triphosphate (ATP gamma S) can substitute for ATP in the activation of the ATP X Mg2+-dependent form of bovine heart type I protein phosphatase (Mr = 75,000) catalyzed by phosphatase-1 kinase (FA). ATP gamma S activates the enzyme to a lower level than ATP, but it phosphorylates the regulatory (R)-subunit to a much higher extent. An [35S]phosphatase-1 [( 35S]E-P) has been isolated, identified, and shown to be a key intermediate in the activation reaction. Treatment of [35S]E-P with dimethyl suberimidate results in cross-linking of the Mr = 34,000 [35S]R-subunit with the Mr = 40,000 catalytic (C)-subunit to form a Mr = 75,000 species, indicating that phosphorylation is not accompanied by dissociation of the holoenzyme. The catalytically active form (Ea) is not the phosphorylated enzyme intermediate. Instead, Ea is directly produced from the intermediate by a Mg2+-dependent, intramolecular autodephosphorylation reaction. The isolated Ea derived from [35S]E-P or from ATP-activated phosphatase-1 has the same half-life (23 min at 30 degrees C). It spontaneously deactivates, via an intramolecular process, to a resting state (Er) which can be fully reactivated by FA X ATP X Mg2+. The deactivation of Ea can be accelerated by chelators, PPi greater than ATP X Mg2+ blocks the PPi effect. Limited trypsinization selectively digests the R-subunit and the resulting C-subunit is Mg2+-dependent. Based on the present data, a novel intracyclic activation-deactivation mechanism via transient phosphorylation of the R-subunit is proposed for regulation of phosphatase-1. (formula; see text).
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D000255:Adenosine Triphosphate / Q000031:analogs & derivatives / Q000494:pharmacology; D000818:Animals; D002417:Cattle; D002614:Chelating Agents / Q000494:pharmacology; D004789:Enzyme Activation / Q000187:drug effects; D066298:In Vitro Techniques; D007700:Kinetics; D008274:Magnesium / Q000494:pharmacology; D008970:Molecular Weight; D009206:Myocardium / Q000201:enzymology*; D010749:Phosphoprotein Phosphatases / Q000302:isolation & purification / Q000378:metabolism*; D010766:Phosphorylation; D011494:Protein Kinases / Q000378:metabolism*; D054645:Protein Phosphatase 1; D013873:Thionucleotides / Q000494:pharmacology
D002614:Chelating Agents; D013873:Thionucleotides; C022571:adenosine 5'-O-(3-thiotriphosphate); D000255:Adenosine Triphosphate; D011494:Protein Kinases; C045455:phosphatase-1 kinase; D010749:Phosphoprotein Phosphatases; D054645:Protein Phosphatase 1; D008274:Magnesium