Endogenous cyclic AMP-stimulated phosphorylation of a 49700-Mr Wolfgram protein component in rabbit central nervous system was investigated by using photoaffinity labelling and 2′,3′-cyclic nucleotide 3′-phosphodiesterase activity staining after electroblotting on to nitrocellulose paper. Photoaffinity labelling with 8′-azidoadenosine 3′,5′-cyclic monophosphate showed a cyclic AMP-binding protein that appeared to be intrinsic to the myelin membrane and appeared to represent the R-subunit of a type I cyclic AMP-dependent protein kinase. This photoaffinity-labelled protein was of larger apparent Mr than the protein showing cyclic AMP-stimulated phosphorylation. Blotting of one-dimensional sodium dodecyl sulphate/polyacrylamide-gel electrophoretograms followed by staining for 2′,3′-cyclic nucleotide 3′-phosphodiesterase activity showed two activity bands corresponding to the two components of the Wolfgram protein doublet. Cyclic AMP-stimulated protein phosphorylation corresponded to the upper component of this doublet. Electroblotting of two-dimensional non-equilibrium pH-gradient electrophoretograms also showed co-migration of cyclic AMP-stimulated protein phosphorylation with enzyme activity. It is proposed that central-nervous-system myelin contains an endogenous type I cyclic-AMP dependent protein kinase that phosphorylates the larger subunit of 2′,3′-cyclic nucleotide 3′-phosphodiesterase.
Mutations that prevent cyclic nucleotide binding to binding sites A or B of type I cyclic AMP-dependent protein kinase.
