Figure 5.

Amph phosphorylation and dephosphorylation. (A) Time course of Amph2 dephosphorylation is more rapid and extensive than Amph1 or DynI. Seconds refer to the time after addition of KCl. Data are averaged from four separate experiments (means ± SEM). (B) Repetitive depolarization/repolarization of synaptosomes induces cyclical changes in the phosphorylation state of the Amphs. After the first stimulation (Stim1), synaptosomes were pelleted for 5 s at 13,000 × g and resuspended in repolarization buffer (HBM containing 4.5 mM KCl), allowed to equilibrate for 1 min at 37°C, before addition of KCl back to 35 mM (Stim2). Dynamin, Amph1, and Amph2 were immunoprecipitated with their respective antiserum. (C) Effect of PKC activator (PMA) and inhibitor (Ro31–8220) on the phosphorylation state of Amph1 and Amph2. Synaptosomes were labeled as described in A and 10 μM of either PMA or Ro31–8220 were added just before harvesting.