In order to examine whether ERK2 could utilize the canonical D-site for a docking interaction with Staufen2, we deleted the Lys290-Gly304 region to produce the Δ15 mutant, which we subsequently used in a co-IP assay. Deletion of the D-site dramatically reduced the interaction of ERK2 (Fig. 1D), but it IC87114 did not completely block the interaction, suggesting that the other D-site might be compensating for the loss.
To examine whether deleting the D-site hampered colocalization of Staufen2 with MAPKs, we expressed GFP-tagged full-length (wild type, WT) or Δ15 (deletion of Lys290-Gly304) Staufen2 in cultured hippocampal neurons using a Sindbis viral expression system. After a 12-h infection, we stained the neurons with MAPK antibody, and look for colocalization in the somatic regions between the GFP-tagged Staufen2 proteins and MAPKs. Deleting the canonical D-site decreased the Staufen2/MAPK colocalization by 40% compare to the wild type control [WT, 69.65 ± 2.973% (N = 20); Δ15, 41.84 ± 2.941% (N = 15); ???p
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Identification of an association of ALADIN and
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