Abstract
Photoaffinity labeling with 8-azidoadenosine 3':5'-monophosphate is a highly selective method for probing the cAMP-binding sites of the regulatory subunits of cAMP-dependent protein kinase and for identifying specific residues that are in close proximity to the cAMP-binding sites. The cAMP-binding site of a mutant RI-subunit has been characterized here and contrasted to the native RI-subunit. This mutant RI-subunit was generated by oligonucleotide-directed muta-genesis and lacks the entire second cAMP-binding domain which includes both of the residues, Trp260 and Tyr371, that are photolabeled in the native RI-subunit. The mutant RI-subunit, nevertheless, is photoaffinity-labeled with high efficiency, and the residue covalently modified was identified as Tyr244. The position of Tyr244 based on a computer graphic model of cAMP-binding site A is proposed and correlated with the presumed locations of Tyr371 and Trp260 in the native R-subunit. Photoaffinity labeling also can be used to detect functional cAMP-binding sites following electrophoretic transfer of the denatured protein to nitrocellulose. Labeling of the immobilized protein on nitrocellulose required a functional cAMP-binding site A that can be photoaffinity-labeled in solution based on the following criteria. 1) The type I R-subunit is photolabeled, whereas the type II R-subunit is not. A primary feature which distinguishes these two R-subunits is that the RI-subunit is photolabeled at both sites A and B, whereas covalent modification of the RII-subunit occurs only at site B. 2) The truncated mutant of the RI-subunit which lacks the entire second cAMP-binding domain can be photolabeled on nitrocellulose. 3) A mutant RI-subunit which can no longer be photolabeled in site B is still photolabeled on nitrocellulose. 4) A mutation which abolished cAMP binding to site A also abolished photoaffinity labeling after transfer to nitrocellulose.
Highlights
Deletion of CAMP-binding Site B in the Regulatory Subunoift CAMPdependent Protein KinaseAlters the Photoaffinity Labelingof Site A*
When cell extracts containing equivalent amounts of the native R'-subunit and the truncated R'-subunit (W260/ St) were denatured with sodium dodecyl sulfate, electrophoresed on polyacylamide gels, electrophoretically transferred to nitrocellulose, and photoaffinity-labeled with ~ - N s [ ~ ' P ] CAMP,the results shown in Fig. 1were observed.The mutant
Photoaffinity labeling has proven to be a very specificprobe for mapping regions of the CAMP-binding sitesin the Rsubunits of CAMP-dependentprotein kinase
Summary
R'-subunit, it is possible to generate specific mutant forms of the protein that have altered CAMP-bindingproperties. The mutant R'-subunit retains a single high affinity CAMP-binding site and still forms a complex with the C-subunit [11]. Of Biochemistry, Bran- this truncated R'-subunit lacks both of the sites deis University, Waltham, MA 02154. That are typically covalently modified by 8-NScAMP in the II Supported in part by the "Consejo Nacional de Investigaciones native full-length R'-subunit. Deletion of CAMP-binding Site B subunit was photoaffinity-labeled verywell with 8-N3[32P]
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