Expression and properties of the regulatory subunit of Dictyostelium cAMP-dependent protein kinase encoded by lambda gt11 cDNA clones.
lambda gt11 phages harboring five different cDNA fragments for the regulatory (R) subunit of Dictyostelium discoideum cAMP-dependent protein kinase (CAK) directed the synthesis of this protein in Escherichia coli cells. Crude bacterial extracts were probed with an antiserum against the Dictyostelium R subunit. The presence of specific epitopes for the R subunit in a given extract was compared with high-affinity cAMP-binding activity and with the ability to inhibit the catalytic (C) subunit through protein-protein interaction. The expression and the biochemical properties of these proteins were correlated with their cDNA nucleotide sequence. The results show that the Dictyostelium R subunit can be functionally expressed in E. coli cells either as a fusion protein with beta-galactosidase or as a nonfusion protein. In both cases, the products of cDNA clones containing the entire coding sequence retained high-affinity cAMP-binding activity and the capacity to interact with the catalytic subunit. One of the fusions, lacking the 94 N-terminal residues, failed to inhibit catalytic activity, although it bound cAMP with an affinity similar to that of the native R protein from D. discoideum.
D000595:Amino Acid Sequence; D010582:Bacteriophage lambda / Q000235:genetics; D001483:Base Sequence; D003001:Cloning, Molecular; D004271:DNA, Fungal / Q000235:genetics*; D004023:Dictyostelium / Q000201:enzymology / Q000235:genetics*; D000939:Epitopes / Q000032:analysis; D004926:Escherichia coli / Q000235:genetics; D046911:Macromolecular Substances; D008969:Molecular Sequence Data; D011494:Protein Kinases / Q000235:genetics* / Q000276:immunology
D004271:DNA, Fungal; D000939:Epitopes; D046911:Macromolecular Substances; D011494:Protein Kinases