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.

[1]  T. Steitz,et al.  Predicted structures of cAMP binding domains of type I and II regulatory subunits of cAMP-dependent protein kinase. , 1990, Biochemistry.

[2]  R. Kunisawa,et al.  Complete nucleotide sequence of the gene encoding the regulatory subunit of 3', 5'-cyclic AMP-dependent protein kinase from the yeast Saccharomyces cerevisiae , 1987, Nucleic Acids Res..

[3]  U. Walter,et al.  Molecular cloning, cDNA structure, and regulation of the regulatory subunit of type II cAMP-dependent protein kinase from rat ovarian granulosa cells. , 1986, The Journal of biological chemistry.

[4]  S. Chirala.  The nucleotide sequence of the lac operon and phage junction in lambda gt11. , 1986, Nucleic Acids Research.

[5]  S. Taylor,et al.  Expression of the type I regulatory subunit of cAMP-dependent protein kinase in Escherichia coli. , 1986, The Journal of biological chemistry.

[6]  J. de Gunzburg,et al.  Comparison of the regulatory and catalytic subunits of cAMP dependent protein kinase from Dictyostelium discoideum and bovine heart using polyclonal antibodies. , 1986, Biochemical and biophysical research communications.

[7]  N. Pace,et al.  Rapid determination of 16S ribosomal RNA sequences for phylogenetic analyses. , 1985, Proceedings of the National Academy of Sciences of the United States of America.

[8]  E. Krebs,et al.  Identification of an inhibitory region of the heat-stable protein inhibitor of the cAMP-dependent protein kinase. , 1985, Proceedings of the National Academy of Sciences of the United States of America.

[9]  S. Taylor,et al.  Monoclonal antibodies as probes for functional domains in cAMP-dependent protein kinase II. , 1985, The Journal of biological chemistry.

[10]  J. Kyhse-Andersen.  Electroblotting of multiple gels: a simple apparatus without buffer tank for rapid transfer of proteins from polyacrylamide to nitrocellulose. , 1984, Journal of biochemical and biophysical methods.

[11]  E. Krebs,et al.  Amino acid sequence of the regulatory subunit of bovine type II adenosine cyclic 3',5'-phosphate dependent protein kinase. , 1984, Biochemistry.

[12]  R. D. Wade,et al.  Guanosine cyclic 3',5'-phosphate dependent protein kinase, a chimeric protein homologous with two separate protein families. , 1984, Biochemistry.

[13]  J. de Gunzburg,et al.  An unusual adenosine cyclic 3',5'-phosphate-dependent protein kinase from Dictyostelium discoideum. , 1984, Biochemistry.

[14]  I. Majerfeld,et al.  A cytosolic cyclic AMP-dependent protein kinase in Dictyostelium discoideum. I. Properties. , 1984, The Journal of biological chemistry.

[15]  R. W. Davis,et al.  Yeast RNA polymerase II genes: isolation with antibody probes. , 1983, Science.

[16]  E. Krebs,et al.  Primary structure of the regulatory subunit of type II cAMP-dependent protein kinase from bovine cardiac muscle. , 1982, Proceedings of the National Academy of Sciences of the United States of America.

[17]  J. Corbin,et al.  Regulatory mechanisms in the control of protein kinases. , 1982, CRC critical reviews in biochemistry.

[18]  S. Taylor,et al.  Correlation of the cAMP binding domain with a site of autophosphorylation on the regulatory subunit of cAMP-dependent protein kinase II from porcine skeletal muscle. , 1979, The Journal of biological chemistry.

[19]  H. Towbin,et al.  Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. , 1979, Proceedings of the National Academy of Sciences of the United States of America.

[20]  F. Sanger,et al.  DNA sequencing with chain-terminating inhibitors. , 1977, Proceedings of the National Academy of Sciences of the United States of America.

[21]  M. M. Bradford.  A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. , 1976, Analytical biochemistry.

[22]  U. K. Laemmli,et al.  Cleavage of Structural Proteins during the Assembly of the Head of Bacteriophage T4 , 1970, Nature.

[23]  D. Ladant,et al.  Gene isolation by direct in situ cAMP binding. , 1987, Gene.

[24]  J. de Gunzburg,et al.  Cloning and cDNA sequence of the regulatory subunit of cAMP-dependent protein kinase from Dictyostelium discoideum. , 1987, Proceedings of the National Academy of Sciences of the United States of America.

[25]  Henri Buc,et al.  Activation of Transcription by the Cyclic AMP Receptor Protein , 1984 .

[26]  J. Messing.  [2] New M13 vectors for cloning , 1983 .

[27]  G. Olsen,et al.  Nucleotide sequence of the Dictyostelium discoideum small-subunit ribosomal ribonucleic acid inferred from the gene sequence: evolutionary implications , 1983 .