Functional cGMP-dependent protein kinase is phosphorylated in its catalytic domain at threonine-516.
The phosphorylation of threonine residues in the catalytic core of several protein kinases is important for the functional integrity of these enzymes. The corresponding residues of cGMP-dependent protein kinase I alpha (cGMP kinase) are Thr-514 and/or Thr-516. The in vivo phosphorylation and functional role of these residues was studied. cGMP kinase was overexpressed and purified as a catalytically active and inactive enzyme in Sf9 insect cells and in Escherichia coli, respectively. The enzymological and physicochemical properties of the Sf9 cGMP kinase were indistinguishable from that of the purified bovine lung enzyme. The cysteines of cGMP kinase including Cys-518 were labeled with vinylpyridine. Amino acid sequencing and mass spectroscopy of the labeled peptides showed that Thr-516 was phosphorylated in the enzyme purified from Sf9 cells but not in that from E. coli. The functional importance of phosphothreonine-516 was investigated by substitution of Thr-516 by alanine (T516A) or by glutamate (T516E). Expression in insect cells of the T516A mutant resulted in a protein lacking detectable kinase activity, whereas the T516E mutant retained basal phosphotransferase activity. In E. coli, the exchange of Thr-516 by glutamate did not lead to the synthesis of a catalytically active enzyme. These results demonstrate that phosphothreonine-516 of cGMP kinase is crucial for the formation of an enzymatically active protein kinase.
Feil
R
R
Institut für Pharmakologie und Toxikologie, Technischen Universität München, Germany.
Kellermann
J
J
Hofmann
F
F
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D002417:Cattle; D006152:Cyclic GMP / Q000378:metabolism; D017869:Cyclic GMP-Dependent Protein Kinases / Q000737:chemistry*; D017931:DNA Primers / Q000737:chemistry; D004926:Escherichia coli; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010446:Peptide Fragments / Q000737:chemistry; D010766:Phosphorylation; D010769:Phosphothreonine / Q000737:chemistry; D018411:Spodoptera; D013329:Structure-Activity Relationship; D013912:Threonine
D017931:DNA Primers; D010446:Peptide Fragments; D010769:Phosphothreonine; D013912:Threonine; D017869:Cyclic GMP-Dependent Protein Kinases; D006152:Cyclic GMP