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Functional cGMP-dependent protein kinase is phosphorylated in its catalytic domain at threonine-516
R Feil1, J Kellermann, F Hofmann
1Institut für Pharmakologie und Toxikologie, Technischen Universität München, Germany.
Insights
Phosphorylation of threonine-516 in cyclic GMP-dependent protein kinase (cGMP kinase) is essential for its enzymatic activity. This phosphothreonine-516 is crucial for forming an active protein kinase.
Area of Science:
- Biochemistry
- Molecular Biology
- Enzymology
Background:
- Threonine phosphorylation in protein kinase catalytic cores is vital for enzyme function.
- Cyclic GMP-dependent protein kinase I alpha (cGMP kinase) has threonine residues Thr-514 and Thr-516 in its catalytic core.
Purpose of the Study:
- To investigate the in vivo phosphorylation and functional significance of Thr-514 and Thr-516 in cGMP kinase.
- To determine the role of phosphothreonine-516 in cGMP kinase activity.
Main Methods:
- Overexpression and purification of active cGMP kinase in Sf9 insect cells and inactive enzyme in Escherichia coli.
- Chemical labeling of cGMP kinase cysteines and subsequent peptide analysis using amino acid sequencing and mass spectrometry.
- Site-directed mutagenesis of Thr-516 to alanine (T516A) and glutamate (T516E) to assess functional impact.
Main Results:
- Thr-516 was found to be phosphorylated in cGMP kinase purified from Sf9 cells but not from E. coli.
- The T516A mutant expressed in insect cells lacked detectable kinase activity.
- The T516E mutant retained basal phosphotransferase activity, while Thr-516 substitution with glutamate in E. coli did not yield active enzyme.
Conclusions:
- Phosphorylation of Thr-516 in cGMP kinase is critical for its catalytic activity.
- Phosphothreonine-516 is essential for the formation of an enzymatically active cGMP kinase protein.
Abstract:
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.