Abstract
The c-Jun N-terminal kinases (JNKs) are a subfamily of the mitogen-activated protein kinases (MAPKs). Although progress in evaluating the functions of other MAPKs has been facilitated by the characterization of specific inhibitors, no JNK-directed inhibitor is commercially available. We have identified a 21-amino acid peptide inhibitor of activated JNKs, based on amino acids 143-163 of the JNK-binding domain (JBD) of the JNK scaffolding protein, JNK-interacting protein-1 (JIP-1). This peptide, I-JIP (Inhibitor of JNK-based on JIP-1), inhibited JNK activity in vitro toward recombinant c-Jun, Elk, and ATF2 up to 90%. A truncated I-JIP (TI-JIP), the C-terminal 11 amino acids of I-JIP, directly interacted with recombinant JNKs but not its substrates as shown by surface plasmon resonance analysis. Scanning alanine replacement within truncated I-JIP identified 4 residues (Arg-156, Pro-157, Leu-160, or Leu-162) as independently critical for inhibition. JBD peptide sequences from JIP-2 and JIP-3 shared these critical residues and accordingly were effective JNK inhibitors. In contrast, peptides based on the JBDs of ATF2 and c-Jun inhibited JNK activity by <40%, which agreed with their lack of homology to the critical Arg-156 and Pro-157. These studies thus define a small peptide inhibitor sequence of JNKs based on the JIP proteins.
Highlights
The mitogen-activated protein kinases (MAPKs)1 act within signal transduction cascades that can relay information from extracellular signals to the cytoplasm and nucleus of the cell
SB203580, but not truncated I-JNK-interacting protein (JIP) (TI-JIP), significantly inhibited arseniteinduced p38 activity (Fig. 4C). These results indicated that the TI-JIP inhibition appeared relatively specific for Jun N-terminal kinases (JNKs), given that the related ERK and p38 MAPKs were not inhibited by an equivalent concentration of this peptide
We present evidence in this study that small peptides based on the JNK-binding domain (JBD) of JNK-interacting protein-1 (JIP-1) are effective inhibitors of previously activated JNK
Summary
During the course of our study, Bonny and colleagues (23) reported a similar 21-amino acid JIP-1-derived peptide could be delivered by a cell-permeable peptide delivery system to a pancreatic -cell line, to markedly inhibit interleukin-1-induced c-jun and c-fos expression and protect against interleukin-1-induced apoptosis This highlights the high potency of an inhibitory motif based on JIP-1 to act as a JNK inhibitor intracellularly. By further characterizing the properties of peptide inhibitors of JNK, we have shown that this peptide alone is sufficient for inhibition of activity of activated JNK in vitro toward c-Jun and ATF2 This peptide can inhibit JNK-mediated phosphorylation of Elk, a substrate that lacks a JBD but requires the Elk D-domain for interaction with JNKs (24).
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