Figure 2. A conserved phosphodegron in REST is required for regulation by βTRCP.

a, 293T cells were transfected with GST, GST-βTRCP -or Flag-REST expression plasmids. Flag-REST lysates were treated with buffer, λ-phosphatase, or λ-phosphatase + phosphatase-inhibitor as indicated. Flag-REST lysates were then mixed with GST- or GST-βTRCP lysates, precipitated with glutathione beads and immunoblotted with αFlag antibodies. b, Phosphorylation of the conserved REST degron in vivo. Sequence alignments of REST proteins (Hs REST residues 1024−1032) from several species and the phosphodegron from Hs CDC25A. Phospho-serines within the REST degron identified by MS/MS are shown in upper sequence. c, 293T cells expressing the indicated combinations of GST, GST-βTRCP1 (denoted on bottom), and Flag-REST mutants (denoted at top). GST-bound complexes were immunoblotted with αFlag (upper and lower panels) or αGST (middle). d, ³⁵S-βTRCP1 was transcribed/translated in vitro and incubated with biotin-conjugated peptides spanning the REST degron (unphosphorylated or phosphorylated) or the IκB degron (phosphorylated). Peptide-associated-proteins were precipitated with streptavidin-conjugated beads, analyzed by SDS-PAGE, and quantified using a phosphoimager. Peptide sequence spanning the REST degron is shown in the top panel. e, 293T cells expressing wild-type or degron-mutant Flag-REST cDNAs were examined for Flag-REST protein half-life in a cycloheximide (Chx) timecourse.