Development of an intracellularly acting inhibitory peptide selective for PKN.
PKNs form a subfamily of the AGC serine/threonine protein kinases, and have a catalytic domain homologous with that of PKC (protein kinase C) in the C-terminal region and three characteristic ACC (antiparallel coiled-coil) domain repeats in the N-terminal region. The preferred peptide phosphorylation motif for PKNs determined by a combinatorial peptide library method was highly similar to that of PKCs within a 10-amino-acid stretch. Previously reported PKN inhibitory compounds also inhibit PKCs to a similar extent, and no PKN selective inhibitors have been commercially available. We have identified a 15-amino-acid peptide inhibitor of PKNs based on amino acids 485-499 of the C-terminal region of the C2-like domain of PKN1. This peptide, designated as PRL, selectively inhibits the kinase activity of all isoforms of PKN (Ki=0.7 muM) towards a peptide substrate, as well as autophosphorylation activity of PKN in vitro, in contrast with PKC. Reversible conjugation by a disulfide bond of a carrier peptide bearing a penetration accelerating sequence to PRL, facilitated the cellular uptake of this peptide and significantly inhibited phosphorylation of tau by PKN1 at the PKN1-specific phosphorylation site in vivo. This peptide may serve as a valuable tool for investigating PKN activation and PKN-mediated responses.
Graduate School of Science, Kobe University, Kobe 657-8501, Japan.
Kazuhiro
K
Shiga
Kentaro
K
Takayama
Shiroh
S
Futaki
Jessica E
JE
Hutti
Lewis C
LC
Cantley
Katsuko
K
Ueki
Yoshitaka
Y
Ono
Hideyuki
H
Mukai
D002463:Cell Membrane Permeability; D006367:HeLa Cells; D006801:Humans; D010446:Peptide Fragments / Q000138:chemical synthesis / Q000493:pharmacokinetics / Q000494:pharmacology*; D019151:Peptide Library; D010766:Phosphorylation / Q000187:drug effects; D020033:Protein Isoforms; D011493:Protein Kinase C / Q000037:antagonists & inhibitors*; D013379:Substrate Specificity
D010446:Peptide Fragments; D019151:Peptide Library; D020033:Protein Isoforms; C061570:protein kinase N; D011493:Protein Kinase C