To further address this possibility, we turned to enhancer-supressor genetic assays dependent on Sema-1a/PlexA repulsive axon guidance. Ectopic expression of Sema-1a in muscles leads to reduced muscle innervation (Yu et al., 1998). These effects are due to the repulsive action of Sema-1a (Yu et al., 1998) and are suppressed by decreasing the levels

of the Sema-1a receptor, PlexA (Winberg et al., 1998b). In contrast, decreasing Palbociclib solubility dmso the levels of 14-3-3ε enhanced Sema-1a repulsion (Figures 3B–3D); suggesting that similar to PKA RII and Nervy (Figures 3C and 3D; Terman and Kolodkin, 2004), 14-3-3ε opposes Sema-1a repulsion. To further investigate these antagonistic interactions, we turned to genetic assays dependent on the repulsive effects of PlexA. Increasing the levels of neuronal PlexA generates abnormally defasciculated axons that result in discontinuous CNS longitudinal connectives and axons crossing the midline or projecting abnormally into the periphery ( Figures 4A and S3A; Metformin price Winberg et al., 1998b and Ayoob et al.,

2004). Strikingly, decreasing the levels of 14-3-3ε significantly increased these PlexA-dependent guidance defects ( Figures 4A–4C), while increasing neuronal 14-3-3ε significantly decreased these PlexA-dependent guidance defects ( Figures 4B and 4C). Together, these results along with other in vivo Sema1a/PlexA-dependent CNS and motor axon guidance assays ( Figure S3) indicate that 14-3-3ε antagonizes Sema1a/PlexA-mediated repulsive axon guidance. To begin to investigate the mechanism only by which 14-3-3ε antagonizes Sema-1a/PlexA-mediated repulsive axon guidance, we sought to determine the site of interaction between PlexA and 14-3-3ε. We found that the portion of PlexA that was necessary and sufficient for the interaction with 14-3-3ε contains a consensus 14-3-3 binding sequence (Figures 5A and 5B).

In particular, 14-3-3 proteins typically bind to single phosphorylated serine or threonine residues on target proteins (Yaffe and Elia, 2001) and Drosophila PlexA contains a mode I 14-3-3 consensus binding motif, R/KSXpSXP, where p represents the phosphorylated serine (Ser1794) residue predicted to mediate the interaction with 14-3-3 proteins ( Figure 5B; Yaffe et al., 1997 and Rittinger et al., 1999). To test this possibility, we substituted alanine (Ala) for serine (Ser) and threonine (Thr) residues within this consensus 14-3-3ε binding motif. We found that the predicted Ser1794 residue was necessary for the observed PlexA interaction with 14-3-3ε ( Figures 5C and S4A). Next, we generated phospho-mimetic forms of Ser1794 (Ser1794 to Glu1794 or Asp1794), but found that as with other 14-3-3 interacting proteins adding one negative charge was not sufficient for the interaction between PlexA and 14-3-3ε ( Figures 5C and S4A).