Mitochondrial localization of CNP2 is regulated by phosphorylation of the N-terminal targeting signal by PKC: implications of a mitochondrial function for CNP2 in glial and non-glial cells.

Both 2',3'-cyclic nucleotide-3'-phosphodiesterase (CNP) isoforms are abundantly expressed in myelinating cells. CNP2 differs from CNP1 by a 20 amino acid N-terminal extension and is also expressed at much lower levels in non-myelinating tissues. The functional role of CNP2, apart from CNP1, and the significance for CNP2 expression in non-myelinating tissues are unknown. Here, we demonstrate that CNP2 is translocated to mitochondria by virtue of a mitochondrial targeting signal at the N-terminus. PKC-mediated phosphorylation of the targeting signal inhibits CNP2 translocation to mitochondria, thus retaining it in the cytoplasm. CNP2 is imported into mitochondria and the targeting signal cleaved, yielding a mature, truncated form similar in size to CNP1. CNP2 is entirely processed in adult liver and embryonic brain, indicating that it is localized specifically to mitochondria in non-myelinating cells. Our results point to a broader biological role for CNP2 in mitochondria that is likely to be different from its specific role in the cytoplasm, along with CNP1, during myelination.

Structural evidence that brain cyclic nucleotide phosphodiesterase is a member of the 2H phosphodiesterase superfamily.

2',3'-Cyclic-nucleotide 3'-phosphodiesterase (CNP) is an enzyme abundantly present in the central nervous system of mammals and some vertebrates. In vitro, CNP specifically catalyzes the hydrolysis of 2',3'-cyclic nucleotides to produce 2'-nucleotides, but the physiologically relevant in vivo substrate remains obscure. Here, we report the medium resolution NMR structure of the catalytic domain of rat CNP with phosphate bound and describe its binding to CNP inhibitors. The structure has a bilobal arrangement of two modules, each consisting of a four-stranded beta-sheet and two alpha-helices. The beta-sheets form a large cavity containing a number of positively charged and aromatic residues. The structure is similar to those of the cyclic phosphodiesterase from Arabidopsis thaliana and the 2'-5' RNA ligase from Thermus thermophilus, placing CNP in the superfamily of 2H phosphodiesterases that contain two tetrapeptide HX(T/S)X motifs. NMR titrations of the CNP catalytic domain with inhibitors and kinetic studies of site-directed mutants reveal a protein conformational change that occurs upon binding.

Oligodendrocyte-myelin glycoprotein (OMgp) is an inhibitor of neurite outgrowth.

A protein fraction purified from bovine brain myelin, previously called arretin because of its ability to inhibit neurite outgrowth, has been identified as consisting predominantly of oligodendrocyte-myelin glycoprotein (OMgp). We show that it is a potent inhibitor of neurite outgrowth from rat cerebellar granule and hippocampal cells; from dorsal root ganglion explants in which growth cone collapse was observed; from rat retinal ganglion neurons; and from NG108 and PC12 cells. OMgp purified by a different procedure from both mouse and human myelin behaves identically in all bioassays tested.