Limited proteolysis alters the photoaffinity labeling of adenosine 3',5'-monophosphate dependent protein kinase II with 8-azidoadenosine 3',5'-monophosphate.
Photoaffinity labeling of the regulatory subunits of cAMP-dependent protein kinase with 8-azidoadenosine 3',5'-monophosphate (8-N3cAMP) has proved to be a very specific method for identifying amino acid residues that are in close proximity to the cAMP-binding sites. Each regulatory subunit contains two tandem cAMP-binding sites. The type II regulatory subunit (RII) from porcine heart was modified at a single site, Tyr-381 [Kerlavage, A., & Taylor, S.S. (1980) J. Biol. Chem. 255, 8483-8488]. When a proteolytic fragment of this RII subunit was photolabeled with 8-N3cAMP, two sites were covalently modified. One site corresponded to Tyr-381 and, thus, was analogous to the native RII. The other site of modification was identified as Tyr-196, which is not labeled in the native protein. Photoaffinity labeling was carried out in the presence of various analogues of cAMP that show a preference for one of the two tandem cAMP-binding sites. These studies established that the covalent modification of Tyr-381 was derived from 8-N3cAMP that was bound to the second cAMP-binding site (domain B) and that covalent modification to Tyr-196 was due to 8-N3cAMP that was bound to the first cAMP-binding site (domain A). These sites of covalent modification have been correlated with a model of each cAMP-binding site on the basis of the crystal structure of the catabolite gene activator protein (CAP), which is the major cAMP-binding protein in Escherichia coli.
Bubis
J
J
Department of Chemistry, University of California, San Diego, La Jolla 92093.
Taylor
S S
SS
D000255:Adenosine Triphosphate / Q000031:analogs & derivatives* / Q000378:metabolism; D000345:Affinity Labels / Q000378:metabolism*; D000595:Amino Acid Sequence; D000818:Animals; D001386:Azides / Q000378:metabolism*; D007700:Kinetics; D046911:Macromolecular Substances; D009206:Myocardium / Q000201:enzymology; D010446:Peptide Fragments / Q000032:analysis; D011485:Protein Binding; D011487:Protein Conformation; D011494:Protein Kinases / Q000378:metabolism*; D013552:Swine; D014357:Trypsin
D000345:Affinity Labels; D001386:Azides; D046911:Macromolecular Substances; D010446:Peptide Fragments; C017040:8-azidoadenosine 5'-triphosphate; D000255:Adenosine Triphosphate; D011494:Protein Kinases; D014357:Trypsin