Pseudosubstrate peptide inhibitors of beta-cell protein kinases: altered selectivity after myristoylation.

Inhibitors of protein kinases are widely used to study stimulus-response pathways in pancreatic beta-cells. Synthetic peptides modelled on the pseudosubstrate sites of protein kinases, or of their endogenous inhibitor proteins, offer potentially specific inhibitors of individual protein kinases or kinase isoforms. However, the use of these inhibitors in studies of beta-cell physiology has been limited, since such peptide sequences are usually poorly membrane permeant. Myristoylation of these peptides enhances their ability to cross intact plasma membranes and thus inhibit intracellular protein kinases, and this approach is becoming increasingly common in identifying the cellular role(s) of particular protein kinases. In this study, using insulin-secreting beta-cells, we demonstrate that myristoylation alters the specificity of pseudosubstrate peptides such that all myristoylated peptides tested, even those lacking pseudosubstrate domains, acted as protein kinase C (PKC) inhibitors. This effect of myristoylation was limited to the inhibition of PKC, since the specificity of peptide inhibitors towards beta-cell protein kinase A activity was not affected by myristoylation. These results demonstrate that myristoylated pseudosubstrate peptides have value as protein kinase inhibitors in intact beta-cells, but emphasise that studies using them to ascribe role(s) for protein kinases in beta-cells must be interpreted with caution.