A family of polypeptide substrates and inhibitors of insulin receptor kinase

Abstract

Previous studies have shown that reduced carbamoylmethylated lysozyme (RCAM-lysozyme, MW .apprx. 14.5K) is a substrate and inhibitor (Ki .apprx. 0.6 .mu.M) of insulin receptor kinase (InsRK) autophosphorylation (Kohanski and Lane, 1986; Lane and Kohanski, 1986). In this study we have prepared a family of defined modified derivatives of RCAM-lysozyme and used them to probe the nature of the substrate and inhibitory sites of InsRK. All open-chain derivatives of lysozymes in which either the tryptophanyl, methionyl, cysteinyl, arginyl, or histidyl side chains were modified served as substrates and were potent inhibitors of InsRK autophosphorylation. This was true whether the substitutions were either hydrophilic or hydrophobic, although the hydrophilic derivatives had a higher inhibitory potency. Tryptic peptides derived from RCAM-lysozyme, however, were inactive as inhibitors, and a mixture of the three cyanogen bromide fragments (containing 12, 24, and 93 amino acids, respectively) was found to be less potent in inhibiting the receptor kinase. Derivatization of either tyrosyl or carboxyl side chains produced derivatives that were neither substrates nor capable of inhibiting receptor autophosphorylation. Derivatives with modified amino groups were substrates for InsRK but were able to inhibit InsRK autophosphorylation. The present study suggests that (a) unphosphorylated InsRK has a large hydrophilic substrate binding domain and is effectively inhibited by long-chain polypeptides but not by short sequences, (b) some of the amino, carboxyl, and hydroxyphenyl side chains are essential to the inhibitory nature of these polypeptides, and (c) derivatives that fail to inhibit autophosphorylation can still be recognized and phosphorylated by active InsRK. Thus, the two mechanistically interrelated InsRK events (e.g., autophosphorylation and substrate phosphorylation) can be dissociated from one another. The study may assist in developing more potent inhibitors of InsRK autophosphorylation and in determining the ideal sequence(s) for potential physiological inhibitors, as well as for substrates of InsRK.