Figure 5.

Phosphorylation of huntingtin at S421 modifies the nature of the molecular motor complexes. (A) GST–HAP1 pull-down experiments of protein extracts from HEK cells transfected with htt, mutant htt-polyQ, htt-S421A or htt-S421D reveal that the binding of HAP1 with the htt 480 amino-acid fragment is not modulated by phosphorylation. (B) GST–HAP1A pull-down experiments from HEK cells transfected with htt-S421A or htt-S421D show that the interactions between HAP1A and p150^Glued and between HAP1A and KHC are not modified by phosphorylation. (C) The HAP1A–KHC interaction is unchanged whether htt S421 is phosphorylated or not. Extracts of htt-S421A- and htt-S421D-transfected mouse neuronal cells are immunoprecipitated using anti-β-galactosidase (control) or anti-HAP1A antibodies and immunoprobed with anti-KHC (H2) and anti-HAP1 (EM78) antibodies. (D–G) The p150^Glued–KHC interaction is significantly increased when S421 is phosphorylated. (D) Mouse neuronal cell extracts are immunoprecipitated using anti-His (control) or anti-p150^Glued antibodies and immunoprobed with anti-p150^Glued and anti-KHC antibodies. (F) Mouse neuronal cell extracts are immunoprecipitated using anti-β-galactosidase (control) or anti-KHC antibodies and immunoprobed as in (D). Quantitative assessments of the optical densities were performed and expressed as KHC/p150^Glued (E) or p150^Glued/KHC ratios (G) (^*P<0.05, ^**P<0.01; see Supplementary data for detailed statistical analyses and number of measures).