Identification of phosphorylation sites in the recombinant catalytic subunit of cAMP-dependent protein kinase.
The catalytic subunit of cAMP-dependent protein kinase expressed in Escherichia coli is a phosphoprotein. By in vivo labeling with [32Pi]orthophosphate, the sites of phosphorylation were identified as Ser-10, Ser-139, Thr-197, and Ser-338. Two of these sites, Thr-197 and Ser-338, are found in the mammalian enzyme (Shoji, S., Titani, K., Demaille, J. G., and Fischer, E. H. (1979) J. Biol. Chem. 254, 6211-6214). The predominant isoform is phosphorylated at Ser-10, Ser-338, and Thr-197. The isoforms cannot be readily interconverted by in vitro autophosphorylation, suggesting that the phosphates are relatively stable once the mature protein is assembled. Unlike the mammalian enzyme, the recombinant enzyme is not myristylated at its animo terminus. By coexpressing the catalytic subunit and N-myristyl transferase, the recombinant catalytic subunit is myristylated, and, under these conditions, phosphorylation at Ser-10 is reduced. The fact that recombinant catalytic subunit mutants that are enzymatically impaired are not phosphorylated in vivo indicates that the phosphorylation of the catalytic subunit observed in E. coli is due to autophosphorylation. Whether this process is intramolecular or intermolecular cannot be distinguished. Although autophosphorylation accounts for the modification of the catalytic subunit when it is expressed in E. coli, there may be heterologous protein kinases that are responsible for its in vivo phosphorylation when the enzyme is expressed in eukaryotic cells.
Department of Chemistry, University of California, San Diego La Jolla 92093-0654.
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Yonemoto
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Garrod
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Bell
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Taylor
D000595:Amino Acid Sequence; D001665:Binding Sites; D002851:Chromatography, High Pressure Liquid; D000242:Cyclic AMP / Q000494:pharmacology; D007525:Isoelectric Focusing; D007527:Isoenzymes / Q000378:metabolism; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D019814:Myristic Acid; D009227:Myristic Acids / Q000378:metabolism; D010446:Peptide Fragments / Q000737:chemistry / Q000378:metabolism; D010710:Phosphates / Q000378:metabolism; D010766:Phosphorylation; D011494:Protein Kinases / Q000737:chemistry / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D014357:Trypsin / Q000378:metabolism
D007527:Isoenzymes; D009227:Myristic Acids; D010446:Peptide Fragments; D010710:Phosphates; D011994:Recombinant Proteins; D019814:Myristic Acid; D000242:Cyclic AMP; D011494:Protein Kinases; D014357:Trypsin