Post-translational lipid modification and nucleotide binding of Myelin 2',3'-Cyclic Nucleotide 3'-Phosphodiesterase (CNP)

Abstract

The myelin protein CNP $(2 sp prime,3 sp prime$-Cyclic Nucleotide 3$sp prime$-Phosphodiesterase) is thio-palmitoylated. Since acylation plays an important role in the protein-membrane interaction, CNP palmitoylation was further investigated. Seven cysteine residues in CNP were individually converted into serines and the palmitoylation was analyzed in either COS-7 cells or an in vitro acylation reaction. No single Cys to Ser mutation could reduce substantially the level of palmitoylation, which may indicate that the turnover of palmitate on CNP is high and that there are multiple palmitoylation sites. Immunostaining and subcellular fractionation showed that isoprenylation is the major factor to control the membrane association of CNP while palmitoylation may serve as a fine tuning mechanism. A double mutation of Cys 231 to Ser and Thr 374 to Pro greatly reduced CNPase activity and the level of palmitoylation. CNP was expressed in Sf9 cells and the mutant C397S was purified to near homogeneity. Since CNP contains several ATPase consensus motifs, we investigated in a preliminary way its ATPase/ATP-binding properties. CNP was affinity-photolabeled by $lbrack alpha- sp{32}$P) 8-azido ATP in a specific and saturable way, although no apparent ATPase activity was detected. The binding of 8N3 ATP could be competed by ATP, GTP and CTP at different concentrations