A potent synthetic peptide inhibitor of the cAMP-dependent protein kinase.

Abstract

As an important new reagent for studying the CAMPdependent protein kinase, a 20-residue peptide has been synthesized that corresponds to the active site of the skeletal muscle inhibitor protein.This synthetic peptide inhibits the protein kinase competitively with a Ki = 2.3 nM; its sequence, Thr-Thr-Tyr-Ala-Asp-Phe-Ile-Ala-Ser-Gly-Arg-Thr-Gly-Arg-Arg-Asn-Ala-Ile-His-Asp, is that of a peptide previously reported by us which was derived from the native inhibitor protein by Ve protease digestion (Cheng, H. C., Van Patten, S. M., Smith, A. J., and Walsh, D. A. (1985) Biochem.J. 231,655-661).Studies with analogues of this peptide show that its high affinity binding to the protein kinase (as also of the inhibitor protein) appears to be due to it mimicking the protein substrate by binding to the catalytic site via the arginine-cluster basic subsite (Arg-Arg), and also to a critical contribution from one or more of the 6 N-terminal residues (Thr-Phe).The availability of this high affinity synthetic peptide should open up a variety of avenues to probe the cellular actions of CAMP.11 15 1 6The heat stable inhibitor protein of the CAMP-dependent protein kinase was co-discovered with the kinase in early work exploring the mechanism of CAMP-mediated control of glycogen metabolism.It was found to act by binding to the catalytic subunit of the protein kinase but not the holoenzyme (l), to be competitive with respect to the protein substrate (2, 3), and to bind with high affinity in an ATP-dependent manner (2-4).'For these reasons it was proposed that the inhibitor protein contained a region corresponding to a substrate phosphorylation site, even though it is not itself phosphorylated (5).Recently, we have shown that Staphylococcus aureus Vs protease digestion of the inhibitor protein yielded a fragment (IPzo) that retained -30% of the aotivity of the native inhibitor (6).IPzo inhibited the protein kinase competively uers'sus protein substrate with a Ki = 0.3 nM.The sequence of IPzo * The costs of publication of this article were defrayed in part by the payment of page charges.This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C.