Evidence for the role of the phosphorylation of M6a at tyrosine 251 in neuroplasticity.

KARINA FORMOSO; ALBERTO C C FRASCH; CAMILA SCORTICATI

Buenos Aires

Conferencia; 5th Special Conference of the International Society of Neurochemistry; 2012

IBRO

Neuronal glycoprotein M6a is involved in neuronal plasticity (e.g. neurite extension and filopodia outgrowth) through unknown mechanisms. Previously we documented that filopodium formation stimulated by M6a depends on the localization of this protein in membrane microdomains and requires the activity of Src and MAPK kinases, suggesting their participation in M6a signal propagation. Recently a phosphorylated form of M6a (Y251-M6a) has been found in the mouse brain. Besides, in silico analysis of Y251-M6a residue showed that it could be a target of the SH2 domain of Fyn kinase. Therefore, the aim of this work was to establish the role of tyrosine-phosphorylation and downstream tyrosine kinase targets that induce neurite and filopodia outgrowth. To analyze the importance of the phosphorylation state of M6a we overexpressed either Y251A-M6a (non-phosphorylatable mutant) or Y251D-M6a (constitutively phosphorylated mutant) in hippocampal neurons and we quantified neurite outgrowth. The results showed that both the number and extension of neurites were significantly reduced when Y251A-M6a was overexpressed compared with M6a-wt and Y251D-M6a. Recently, we have documented that the Src family of kinases is involved in filopodium formation induced by M6a in neurons and SYF cells (lack of Src, Fyn and Yes kinases). To further explore the involvement of the Src family of kinases in M6a signaling cascade we coexpressed M6a in neurons with the wt and mutant forms of Src, Fyn and Yes and we tested for filopodium promotion. We used Y531F-Fyn, Y527F-Src or Y535F-Yes all bearing a mutation in its SH2 domain (non-phosphorylatable mutant). The results showed that M6a ability to promote filopodium formation is greatly reduced when all mutant forms of kinases were coexpressed with M6a in neurons. These data suggest that Fyn, Yes and Src might be involved, in a redundant manner, in the signaling propagation cascade of M6a. Moreover, using confocal microscopy we have determined a colocalization pattern of M6a with the Src family in neurons. In summary, we concluded that Y251-M6a and Src family of kinases phosphorylation state affects M6a´s effect on neuroplasticity. Further studies will be needed to demonstrate conclusively if this effect is achieved by the interaction of Y251-M6a with the SH2 domain of the Src family of kinases.