Cyclic GMP-dependent protein kinase substrates in rat brain.
Cyclic GMP (cGMP)-dependent protein kinase (PKG) has a limited substrate specificity, and only cerebellar G-substrate has been demonstrated in brain. In view of the physiological importance of cGMP and PKG in the nervous system, it is important to identify endogenous PKG substrates in rat brain. We devised a combination of ion-exchange and hydrophobic chromatographies to identify potential PKG substrates. Extracts from cytosol, peripheral membrane proteins, or a fraction enriched in Ca(2+)-sensitive lipid-binding proteins were partly purified and phosphorylated with purified PKG. Using whole extracts only a single specific PKG substrate-P34-was found. However, after chromatography we detected > 40 distinct proteins that were phosphorylated by PKG to a much greater extent than by cyclic AMP-dependent protein kinase or protein kinase C. Four PKG substrates--P140, P65, P32, and P18--were detected in the cytosol. Six PKG substrates--P130, P85 (doublet), P58, P54, and P38--were enriched from the Ca(2+)-sensitive lipid-binding protein fraction. In peripheral membrane fractions > 30 relatively specific PKG substrates were enriched after chromatography, especially P130, P94, P58, P52, P45, P40, P36, P34, P28, P26, P24, and P20. These results indicate that brain is not lacking in PKG substrates and show that many are apparently quite specific substrates for this enzyme. The identification of some of these novel PKG substrates will facilitate understanding the role of cGMP signaling in the brain.
Wang
X
X
Endocrine Unit, John Hunter Hospital, Newcastle, New South Wales, Australia.
Robinson
P J
PJ
D000818:Animals; D001921:Brain / Q000378:metabolism*; D002118:Calcium / Q000494:pharmacology; D002352:Carrier Proteins / Q000378:metabolism; D017869:Cyclic GMP-Dependent Protein Kinases / Q000378:metabolism*; D003600:Cytosol / Q000378:metabolism; D050356:Lipid Metabolism; D008566:Membranes / Q000378:metabolism; D016548:Prosencephalon / Q000378:metabolism; D051381:Rats; D013379:Substrate Specificity
D002352:Carrier Proteins; D017869:Cyclic GMP-Dependent Protein Kinases; D002118:Calcium