Phosphorylation of M6a at Serine-267 induces filopodia formation in rat hippocampal neurons

MICAELA GARCIA; KARINA FORMOSO; ALBERTO CC FRASCH; CAMILA SCORTICATI

Huerta Grande, Cordoba

Congreso; XXIX Congreso Anual de la Sociedad Argentina de Investigaciones en Neurociencias; 2014

The neuronal membrane glycoprotein M6a, together with PLP/DM20 and M6b, is a member of the proteolipid protein family. M6a is composed of 278 amino acids that form four transmembrane domains, two external loops, and the N- and C-terminal regions facing the cell cytoplasm. M6a induces neurite outgrowth, increases filopodium/spine density and participates in synaptogenesis, but the mechanism of action remains unknown. It has been suggested that M6a acts as a Ca2+ channel gated by selectivy phosphorylation of protein kinase C (PKC) in the downstream pathway of the nerve growth factor in PC12 cells. We previously demonstrated that the lack of both PKC and CK phosphorylation sites of M6a at the C-terminal domain impaired filopodium mobility in neurons. Recently, a phosphorylated form of M6a at Ser-267 has been found in postnatal and in 21-days old mouses brains. In silico analysis of residue 267 of M6a showed that it could be a target of PKC. To analyze the importance of the phosphorylation state of M6a we overexpressed S267A-M6a (non-phosphorylatable mutant) or S267D-M6a (constitutively phosphorylated mutant) in hippocampal neurons and we quantified filopodium formation at 5 DIV. The results showed that both the wild type and the phosphorylated form of M6a significantly increased the number of processes in 20 um of neurite length compared with control group. However, in S267A expressing neurons the number remains at control levels. By treating M6a expressing neurons with different doses of Gö6976 inhibitor we determined that the calcium dependent PKCs are involved in M6a-induced filopodia. In summary, we concluded that phosphorylation of S267 is implicated in M6a filopodia formation pathway and might act driven by PKC calcium dependent isoforms.