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 subunit of cyclic AMP-dependent protein kinase (cAPK) whose fluorescence emission signal has allowed the synergistic binding between nucleotides and physiological inhibitors of cAPK to be examined (Whitehouse, S., and Walsh, D. A. (1983) J. Biol. Chem. 258, 3682-3692). In the presence of the regulatory subunit, RI, the affinity of cAPK for adenosine, ADP, AMPPNP (adenosine 5'-(beta, gamma-imino)triphosphate), or ATP was 5-, 50-, 120-, and 15,000-fold enhanced, while in the presence of the heat-stable inhibitor protein of cAPK (PKI), there was a 3-, 20-, 33-, and 2000-fold enhancement in the binding of these nucleotides, respectively. 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 contrast, did not significantly enhance the binding of ATP or AMPPNP. The full binding synergism between PKI and either ATP (2000-fold) or AMPPNP (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 most likely critical. Since this region is remote from the ATP gamma-phosphate, the binding synergism must arise through an extended network communication mechanism between the PKI NH2 terminus and the ATP binding site.
Lew
J
J
Department of Chemistry and Biochemistry, University of California, San Diego, La Jolla, California 92093-0654, USA.
Coruh
N
N
Tsigelny
I
I
Garrod
S
S
Taylor
S S
SS
D015081:2-Naphthylamine / Q000031:analogs & derivatives / Q000737:chemistry; D000227:Adenine Nucleotides / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D018360:Crystallography, X-Ray; D017868:Cyclic AMP-Dependent Protein Kinases / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D004791:Enzyme Inhibitors / Q000737:chemistry / Q000378:metabolism*; D051379:Mice; D008969:Molecular Sequence Data; D009711:Nucleotides / Q000378:metabolism*; D011485:Protein Binding; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D013050:Spectrometry, Fluorescence
D000227:Adenine Nucleotides; D004791:Enzyme Inhibitors; D009711:Nucleotides; D011994:Recombinant Proteins; C038518:acrylodan; D015081:2-Naphthylamine; D017868:Cyclic AMP-Dependent Protein Kinases