title: Development of phosphatase inhibitor-1 peptides acting as indirect activators of phosphatase 1.
abstract: Phosphatase inhibitor-1 (I-1) inhibits the catalytic subunit of protein phosphatase type 1 (PP1c) in its protein kinase A (PKA)-phosphorylated form (I-1(P)). It thereby amplifies PKA signaling, which, in the heart, mediates both beneficial (acute) and adverse (chronic) effects of catecholamines. Genetic deletion of I-1 was associated with protection against catecholamine toxicity, making the PP1c-I-1(P) complex a potential therapeutic target for chronic heart disease. Here, we sought to define targetable interaction sites of I-1 and PP1c, concentrating on the N-terminal domain of I-1 which includes the PP1c binding motif ((9)KIQF(12)) as well as a poly-Arg stretch. Substitution of (9)KIQ(11) residues for analogous amino acids, (9)RLN(11), resulted in doubling of the IC50 values, deletion of (9)KIQF(12) prevented I-1 PKA-dependent phosphorylation and thus activation. Mutation of the Arg residues preceding the PKA phosphorylation site (Thr35) to Ala (R/A(30-33)) abolished I-1 phosphorylation and its binding to and inhibition of PP1c. A series of synthetic peptides (4-11 residues) indicated that the KIQF motif as well as the surrounding anchoring residues was essential for interfering with the inhibitory effect of I-1(P) on PP1c, whereas the four Arg residues were not. Unexpectedly, the most effective nonapeptide (SPRKIQFTV) also antagonized the inhibitory effect of the non-conditional PP1 inhibitor-2 with similar affinity. Incubation of neonatal rat cardiac myocytes with a poly-Arg-modified SPRKIQFTV (10 μM) reduced catecholamine-induced phosphorylation of phospholamban, a well-known PKA downstream target sensitive to PP1c. Our data reiterate the importance of the KIQF motif and provide a tool for antagonizing I-1 inhibitory effects on PP1c, i.e., activating PP1 in vivo.
authors.affiliation: Department of Experimental Pharmacology and Toxicology, University Medical Center Hamburg-Eppendorf (UKE) and DZHK (German Centre for Cardiovascular Research), partner site Hamburg/Kiel/Lübeck, Hamburg, Germany.
authors.forename: Hannieh
authors.initials: H
authors.lastname: Sotoud
authors.forename: Uwe
authors.initials: U
authors.lastname: Borgmeyer
authors.forename: Christian
authors.initials: C
authors.lastname: Schulze
authors.forename: Ali
authors.initials: A
authors.lastname: El-Armouche
authors.forename: Thomas
authors.initials: T
authors.lastname: Eschenhagen
mesh_terms: D000818:Animals; D002478:Cells, Cultured; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism; D047908:Intracellular Signaling Peptides and Proteins / Q000235:genetics / Q000378:metabolism*; D016297:Mutagenesis, Site-Directed; D032383:Myocytes, Cardiac / Q000187:drug effects / Q000378:metabolism; D010455:Peptides / Q000494:pharmacology*; D054645:Protein Phosphatase 1 / Q000378:metabolism*; D051381:Rats
chemical_list: D047908:Intracellular Signaling Peptides and Proteins; D010455:Peptides; C021042:protein phosphatase inhibitor-1; D017868:Cyclic AMP-Dependent Protein Kinases; D054645:Protein Phosphatase 1