Fig. 6.
Cdk5 phosphorylation of the serine 1144 of rat synaptojanin 1 affects its interaction with endophilin and amphiphysin. (A) Serine 1144 and/or threonine 1147 were mutated to alanine, and GST fusions of the wild-type and mutant PRDs were incubated with p35/Cdk5 and [32P]ATP. Incubation mixtures were then analyzed by SDS/PAGE followed by Coomassie blue (CB) staining and autoradiography. (B) GST fusion proteins of the wild-type PRD of synaptojanin 1, or of two mutants harboring a S → A or S → D mutation at position 1144, were used in pull-down assays from rat brain cytosol. Western blotting of the bound material revealed that the “phosphomimetic mutation” (S1144D) inhibited the recovery of both amphiphysin 1 and endophilin 1 in the bound material.