Photoaffinity Labeling of Cyclic AMP-Dependent and Cyclic GMP-Dependent Protein Kinases
Photoaffinity labeling is a useful technique for the study of ligand-receptor interactions. Irradiation of chemically modified ligands with light produces highly reactive intermediates which are capable of forming covalent bonds with the receptors. Photoaffinity labeling has been used for many different ligands and has been especially successful for cyclic nucleotides. Several photoaffinity analogues of cAMP and cGMP are available. 8-Azido-cAMP (8-N3-cAMP) has been the one most widely and successfully used. 8-N3-cAMP binds to the regulatory subunit of a Type II cAMP-dependent protein kinase with a slightly lower affinity than cAMP and is able to label all cAMPbinding sites under saturating conditions. The data indicate that 8-N3-cAMP is an effective analogue of cAMP and a very efficient photoaffinity label for cAMP-binding sites. Regulatory subunits of Type I and Type II cAMP-dependent protein kinases are the only proteins present in all tissues studied which are specifically labeled by 8-N3-cAMP. Other minor labeled proteins appear to be proteolytic fragments of the two types of regulatory subunits. The finding that most, if not all, specific cAMP-receptor proteins are regulatory subunits of cAMP-dependent protein kinase strongly supports the hypothesis that cAMP-dependent protein kinases and their subunits mediate all the physiological effects of cAMP. Photoaffinity labeling has been used to detect and characterize proteolytic fragments of cAMP-dependent protein kinases and to study the regulation of the amounts of cAMP-dependent protein kinases and their subunits. Similarly, photoaffinity analogues of cGMP have been used to label cGMP-dependent protein kinases. Photoaffinity analogues of cyclic nucleotides are powerful tools for the investigation of cAMP- and cGMP-receptor proteins. Studies with these analogues should contribute substantially to an understanding of the mechanism of action of cyclic nucleotides as second messengers.
KeywordsCyclic Nucleotide Regulatory Subunit Photoaffinity Label Dent Protein Kinase Pendent Protein Kinase
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- Aiba H, Krakow JS (1979) Photoaffinity labeling of the E. coli cyclic AMP receptor protein with 8-azido-adenosine 3′,5′-monophosphate. Fed Proc 38:231Google Scholar
- Bodwin JS, Clair T, Cho-Chung YS (1978) Inverse relation between estrogen receptors and cyclic adenosine 3′:5′-monophosphate-binding proteins in hormone-dependent mammary tumor regression due to dibutyryl cyclic adenosine 3′:5′-monophosphate treatment or ovariectomy. Cancer Res 38:3410–3413PubMedGoogle Scholar
- Cho-Chung YS (1979) On the mechanism of cyclic AMP-mediated growth arrest of solid tumors. Adv Cyclic Nucleotide Res 12:in pressGoogle Scholar
- Cooperman BS (1976) Photoaffinity labeling of proteins and more complex receptors. In: Smith KC (ed) Aging, carcinogenesis and radiation biology. Plenum, New York, pp 315–340Google Scholar
- DeCrombrugghe B, Chen B, Anderson W, Nissley P, Gottesman M, Perlman M, Pastan I (1971) LacDNA, RNA polymerase, cyclic AMP receptor protein, cyclic AMP, Lac repressor and inducer are the essential elements for controlled Lac transcription. Nature 231:139–142Google Scholar
- DeJonge H, Rosen OM (1977) Self-phosphorylation of cyclic guanosine 3′:5′-monophosphate-dependent protein kinase from bovine lung. J Biol Chem 252:2780–2783Google Scholar
- Kuo JF, Wyatt GR, Greengard P (1971) Cyclic nucleotide-dependent protein kinases. IX. Partial purification and some properties of guanosine 3′,5′-monophosphate-dependent and adenosine 3′,5′-monophosphate-dependent protein kinases from various tissues and species of arthropoda. J Biol Chem 246:7159–7167PubMedGoogle Scholar
- Miller JP, Boswell KH, Muneyama K, Simon LN, Robins RK, Shuman DA (1973) Synthesis and biochemical studies of various 8-substituted derivatives of guanosine 3′,5′-cyclic phosphate, inosine 3′,5′-cyclic phosphate, and xanthosine 3′,5′-cyclic phosphate. Biochemistry 12:5310–5319PubMedCrossRefGoogle Scholar
- Potter RL, Taylor SS (1979 b) The structural and functional domains of cAMP-dependent protein kinase I and II. Fed Proc 38:316Google Scholar
- Prashad N, Rosenberg RN, Wischmeyer B, Ulrich C, Sparkman D (1979) Induction of adenosine 3′,5′-monophosphate-binding proteins by N6,O2′-dibutyryladenosine 3′,5′-monophosphate in mouse neuroblastoma cells. Analysis by two-dimensional gel electrophoresis. Biochemistry 18:2717–2725PubMedCrossRefGoogle Scholar