SYNERGISTIC BINDING OF NUCLEOTIDES AND INHIBITORS TO CAMP-DEPENDENT PROTEIN-KINASE EXAMINED BY ACRYLODAN FLUORESCENCE SPECTROSCOPY

We have engineered an acrylodan-modified derivative of the catalytic s ubunit of cyclic AMP-dependent protein kinase (cAPK) whose fluorescenc e emission signal has allowed the synergistic binding between nucleoti des and physiological inhibitors of cAPK to be examined (Whitehouse, S ., and Walsh, D. A. (1983) J. Biol. Chem. 258, 3682-3692). In the pres ence of the regulatory subunit, R(I), the affinity of cAPK for adenosi ne, ADP, AMPPNP (adenosine 5'-(beta,gamma-imino)triphosphate), or ATP was 5-, 50, 120-, and 15,000-fold enhanced, while in the presence of t he heat-stable inhibitor protein of cAPK (PKI), there was a 3-, 20, 33 -, and 2000-fold enhancement in the binding of these nucleotides, resp ectively. A short inhibitor peptide, PKI-(14-22), enhanced the binding of ADP to the same degree as did full-length PKI (20-fold) but, in co ntrast, did not significantly enhance the binding of ATP or AMPPNP. Th e full binding synergism between PKI and either ATP (2000 fold) or AMP PNP (33-fold) to cAPK could, however, be mimicked by a longer peptide, PKI-(5-24), suggesting that the PKI NH2 terminus (residues 5-13) is m ost likely critical. Since this region is remote from the ATP gamma-ph osphate, the binding synergism must arise through an extended network communication mechanism between the PKI NH2 terminus and the ATP bindi ng site.