Site-specific mutations in the regulatory subunit of cAMP-dependent protein kinase: selective screening for functional cAMP binding sites

An expression vector for the type I regulatory (R) subunit was constructed in pUC7, and maximum expression (20 mg/L) was achieved in E. coli 222. The expressed R was functionally indistinguishable from the wild type protein. Site-specific mutations were introduced using oligonucleotide probes. Specific targets for mutagenesis were each of the cAMP-binding sites as well as the hinge region. Deletions also were introduced. Each mutation led to the expression of stable protein which was purified to homogeneity. Although antibodies can be used to screen for protein, immunological methods do not discriminate between active and inactive proteins. Screening for functional cAMP binding sites was achieved by photolabeling with 8-N3-[32P] cAMP following transfer to nitrocellulose. The R-subunit was photolabeled after SDS-gel electrophoresis and electrotransfer and also after plaque transfers to nitrocellulose. In the absence of photolysis, bound 8N3-[32P] cAMP was removed by washing with cold 8N3cAMP. Better than 50% of the bound nucleotide was covalently incorporated following photolysis. In solution R1 is labeled at 2 sites. Trp 260 is modified by 8N3cAMP bound to site A; Tyr 381 is modified by 8N3-cAMP bound to site B. Following transfer to nitrocellulose, photolabeling of Tyr 371 is abolished. Photolabeling of site A is retained even when mutations have been introduced into site B. The type II R-subunit is labeled at a single site in solution - Tyr 381 (site B). R/sup II/ is not photolabeled following transfer to nitrocellulose. The immobilized proteins are thus photolabeled in a site-specific manner, and photolabeling can be used to screen functional binding of 8N3cAMP to site A