Identification of phosphorylation sites in the recombinant catalytic subunit of cAMP-dependent protein kinase
- PMID: 8395513
Identification of phosphorylation sites in the recombinant catalytic subunit of cAMP-dependent protein kinase
Abstract
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.
Similar articles
-
Autophosphorylation of the catalytic subunit of cAMP-dependent protein kinase.J Biol Chem. 1992 Dec 15;267(35):25174-80. J Biol Chem. 1992. PMID: 1460017
-
Crystal structures of the myristylated catalytic subunit of cAMP-dependent protein kinase reveal open and closed conformations.Protein Sci. 1993 Oct;2(10):1559-73. doi: 10.1002/pro.5560021003. Protein Sci. 1993. PMID: 8251932 Free PMC article.
-
Expression of the catalytic subunit of cAMP-dependent protein kinase in Escherichia coli: multiple isozymes reflect different phosphorylation states.Protein Eng. 1993 Sep;6(7):771-7. doi: 10.1093/protein/6.7.771. Protein Eng. 1993. PMID: 8248101
-
[Mode of action of cyclic amp in prokaryotes and eukaryotes, CAP and cAMP-dependent protein kinases].Biochimie. 1985 Jun;67(6):563-82. doi: 10.1016/s0300-9084(85)80196-6. Biochimie. 1985. PMID: 2413906 Review. French.
-
cAMP-dependent protein kinase defines a family of enzymes.Philos Trans R Soc Lond B Biol Sci. 1993 Jun 29;340(1293):315-24. doi: 10.1098/rstb.1993.0073. Philos Trans R Soc Lond B Biol Sci. 1993. PMID: 8103934 Review.
Cited by
-
In vivo activation of protein kinase A in Schizosaccharomyces pombe requires threonine phosphorylation at its activation loop and is dependent on PDK1.Genetics. 2004 Dec;168(4):1843-53. doi: 10.1534/genetics.104.032466. Genetics. 2004. PMID: 15611161 Free PMC article.
-
Direct modulation of the protein kinase A catalytic subunit α by growth factor receptor tyrosine kinases.J Cell Biochem. 2012 Jan;113(1):39-48. doi: 10.1002/jcb.23325. J Cell Biochem. 2012. PMID: 21866565 Free PMC article.
-
Tyrosine Phosphorylation of the BRI1 Receptor Kinase Occurs via a Post-Translational Modification and is Activated by the Juxtamembrane Domain.Front Plant Sci. 2012 Aug 8;3:175. doi: 10.3389/fpls.2012.00175. eCollection 2012. Front Plant Sci. 2012. PMID: 22891071 Free PMC article.
-
An Isoform-Specific Myristylation Switch Targets Type II PKA Holoenzymes to Membranes.Structure. 2015 Sep 1;23(9):1563-1572. doi: 10.1016/j.str.2015.07.007. Epub 2015 Aug 13. Structure. 2015. PMID: 26278174 Free PMC article.
-
Aurora-A site specificity: a study with synthetic peptide substrates.Biochem J. 2005 Aug 15;390(Pt 1):293-302. doi: 10.1042/BJ20050343. Biochem J. 2005. PMID: 16083426 Free PMC article.
Publication types
MeSH terms
Substances
LinkOut - more resources
Full Text Sources
Other Literature Sources
Molecular Biology Databases