Rational design of a selective antagonist of ε protein kinase C derived from the selective allosteric agonist, pseudo-RACK peptide

Abstract

We have previously shown that domains involved in binding of protein kinase C (PKC1) isozymes to their respective anchoring proteins (RACKs2) and short peptides derived from these domains are PKC isozyme-selective antagonists. We also identified PKC isozyme-selective agonists, named ψRACK3 peptides, derived from a sequence within each PKC with high homology to its respective RACK. We noted that all the ψRACK sequences within each PKC isozyme have at least one nonhomologous amino acid difference from their corresponding RACK that constitutes a charge change. Based on this information, we have devised here a new approach to design an isozyme-selective PKC antagonist, derived from the ψRACK sequence. We focused on εPKC ψRACK peptide, where the pseudo-εRACK sequence (ψεRACK; HDAPIGYD; corresponding to εPKC85-92) is different in charge from the homologous RACK-derived sequence (NNVALGYD; corresponding to εRACK285-292) in the second amino acid. Here we show that changing the charge of the ψεRACK peptide through a substitution of only one amino acid (aspartate to asparagine) resulted in a peptide with an opposite activity on the same cell function and a substitution for aspartate with an alanine resulted in an inactive peptide. These data support our hypothesis regarding the mechanism by which pseudo-RACK peptide activates PKC in heart cells and suggest that this approach is applicable to other signaling proteins with inducible protein-protein interactions.

PKCε activation promotes FGF-2 exocytosis and induces endothelial cell proliferation and sprouting

“…In the manuscript, the peptides were delivered at 1 μM. Scrambled peptides do not have these effects and do not alter PKCε activity [15]. …”

Common Mechanisms of Alzheimer's Disease and Ischemic Stroke: The Role of Protein Kinase C in the Progression of Age-Related Neurodegeneration

“…RACKs play a vital role in transporting PKC isoforms from the cytosol to the membrane [45]. Each PKC isoform has a binding site for specific RACKs in order to facilitate the appropriate translocation destination [46]. Once at the membrane, A-kinase regulating-proteins (AKAPs) and heat shock proteins (HSPs) direct PKC isoforms into close proximity with substrates [41].…”

Targeting protein kinase C subtypes in pancreatic cancer

“…Receptors for activated C-kinase (RACK) proteins serve as specific anchoring molecules to different areas of the cell, and peptides that mimic the RACK binding site on PKC can function as selective isoform-specific inhibitors of translocation and activity in vivo [25]. Based on this strategy, selective antagonists for PKCδ and PKCε have been developed and employed for investigating NF-κB signaling in pancreatic cells [5,26]. Of note, neither these peptide regulators nor other above discussed inhibitors so far have been tested in clinical trials for pancreatic cancer.…”