Irreversible Inactivation of Protein Kinase C Isozymes by Thiol-Reactive Peptide Substrate Analogs

Abstract

Protein kinases play diverse and essential roles in the regulation of cell growth, survival, and differentiation, and in other critical biological functions, for example, neurotransmission (1,2). Substrate selectivity is a major factor in the division of labor among the members of the protein kinase superfamily (1). Typically, local sequences surrounding phospho-acceptor residues are highly important in the recognition of protein substrates by protein kinase C (PKC), cAMP-dependent protein kinase (PKA), and a number of other Ser/Thr protein kinases (1). This has served as the basis for the use of synthetic peptide substrates of defined sequence to identify structural determinants that govern the substrate selectivities of these kinases (1,3,4). This approach was first developed in the laboratory of Edwin G. Krebs, with the use of extensive series of synthetic peptides that corresponded to known phospho-acceptor sites in protein substrates of PKA (1,5). The synthetic peptide Leu-Arg-Arg-Ala-Ser-Leu-Gly (kemptide) was identified in those studies as an excellent artificial PKA substrate (K _m=16 μM) (5). By applying the same approach, the synthetic peptide substrate Arg-Arg-Lys-Ala-Ser-Gly-Pro-Pro-Val, which corresponds to the sequence of a major PKC phosphorylation site in lysine-rich histone, was later identified as an artificial substrate suitable for analysis of PKC catalysis, based on its K _m (130 μM) and on the lipid cofactor dependence of its phosphorylation by purified rat brain PKC (>10-fold stimulation by phorbol 12-myristate 13-acetate/phosphatidyl serine or Ca²⁺/phosphatidyl serine) (6).