Development of phosphatase inhibitor-1 peptides acting as indirect activators of phosphatase 1

• Published in: Naunyn-Schmiedeberg's archives of pharmacology Vol. 388; no. 3; pp. 283 - 293
• Main Authors: Sotoud, Hannieh, Borgmeyer, Uwe, Schulze, Christian, El-Armouche, Ali, Eschenhagen, Thomas
• Format: Journal Article
• Language: English
• Published: Berlin/Heidelberg Springer Berlin Heidelberg 01-03-2015; Springer Nature B.V
• Subjects: Animals; Biomedical and Life Sciences; Biomedicine; Cells, Cultured; Cyclic AMP-Dependent Protein Kinases - metabolism; Intracellular Signaling Peptides and Proteins - genetics; Intracellular Signaling Peptides and Proteins - metabolism; Mutagenesis, Site-Directed; Myocytes, Cardiac - drug effects; Myocytes, Cardiac - metabolism; Neurosciences; Original Article; Peptides - pharmacology; Pharmacology/Toxicology; Protein Phosphatase 1 - metabolism; Rats; Decoy peptides; Neonatal rat cardiac myocytes; Phosphatase inhibitor-1; Phospholamban; Protein phosphatase type 1
• ISSN: 0028-1298; 1432-1912
• DOI: 10.1007/s00210-014-1065-2

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 IC
50
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.