Phosphorylation of the Zebrafish M6Ab at Serine 263 Contributes to Filopodium Formation in PC12 Cells and Neurite Outgrowth in Zebrafish Embryos

Abstract

BackgroundMammalian M6A, a member of the proteolipid protein (PLP/DM20) family expressed in neurons, was first isolated by expression cloning with a monoclonal antibody. Overexpression of M6A was shown to induce filopodium formation in neuronal cells; however, the underlying mechanism of is largely unknown. Possibly due to gene duplication, there are two M6A paralogs, M6Aa and M6Ab, in the zebrafish genome. In the present study, we used the zebrafish as a model system to investigate the role of zebrafish M6Ab in filopodium formation in PC12 cells and neurite outgrowth in zebrafish embryos.Methodology/Principal FindingsWe demonstrated that zebrafish M6Ab promoted extensive filopodium formation in NGF-treated PC12 cells, which is similar to the function of mammalian M6A. Phosphorylation at serine 263 of zebrafish M6Ab contributed to this induction. Transfection of the S263A mutant protein greatly reduced filopodium formation in PC12 cells. In zebrafish embryos, only S263D could induce neurite outgrowth.Conclusions/SignificanceOur results reveal that the phosphorylation status of zebrafish M6Ab at serine 263 is critical for its role in regulating filopodium formation and neurite outgrowth.

Tyrosine 251 at the C‐terminus of neuronal glycoprotein M6a is critical for neurite outgrowth

“…The phosphorylation state of M6a at Y251 seems not to be involved in the formation of filopodia-like processes in hippocampal neurons. In agreement with these findings, phosphorylation at Ser263 of the M6azebrafish ortholog M6Ab promotes filopodia formation, but not neurite extension, in PC12 cells (Huang et al, 2011). Moreover, M6a lacking intracellular phosphorylation sites for casein-kinase 2, and protein PKC-expressing neurons bore filopodia, but the motility of the protrusions was slow compared with that of M6a wt .…”

“…Accordingly, we found that the structure of EC2 is critical to induce filopodium outgrowth and synaptogenesis (Fuchsova et al, ) and that mutations in M6a intracellular sites, which are motifs for protein kinase C (PKC) and casein kinase‐2 phosphorylation, decrease the motility of filopodia induced by M6a (Brocco et al, ). The zebrafish M6a ortholog, which lacks a phosphorylation site for PKC, fails to induce neurite extension in PC12 cells (Huang et al, ). Also, M6a depends on membrane lipid microdomain association and requires the activity of Src kinases and mitogen‐activated protein kinases (MAPK) for its filopodium induction (Scorticati et al, ).…”

Filopodia formation driven by membrane glycoprotein M6a depends on the interaction of its transmembrane domains

“…M6a is a membrane glycoprotein encoded by the gene GPM6A. M6a, together with M6b, proteolipid protein (PLP) and DM20, belongs to the PLP family, and has been related to neuronal plasticity in different experimental models (Lagenaur et al 1992;Alfonso et al 2005;Michibata et al 2009;Huang et al 2011;Sato et al 2011a;Zappia et al 2012). M6a positively contributes to neuritogenesis, filopodia outgrowth and synapse formation in M6a-over-expressing neurons, but its mechanism of action remains unknown (Alfonso et al 2005;Fuchsova et al 2009;Formoso et al 2015).…”

Protein tyrosine phosphatase receptor type O (Ptpro) regulates cerebellar formation during zebrafish development through modulating Fgf signaling

“…The 293T cells were maintained at 37 °C in minimal essential medium-alpha medium, 10 % fetal bovine serum, 100 mg/ml streptomycin, and 100 U/ml penicillin. Plasmid transfection and cell collection were done with standard procedures as described previously [67]. Transfected 293T cells collected from one 6-well plate were lysed in 0.1 ml immunoprecipitation lysis buffer (150 mM NaCl, 20 mM HEPES (pH 7.2), 10 mM NaF, 1 mM EDTA, 0.5 % NP-40, 1 mM Na3VO4, 1 mM PMSF, and 1 mM DTT), then sonicated for 10 s three times using a UP50H machine at an 80 % power level (Dr. Hielscher, Teltow, Germany).…”