Phosphorylation of RalBP1 at C-terminal Thr 645 inhibits PSD-95 binding.
(A) C-terminal aa sequence (SKDRKETPI*) of rat RalBP1 (rRalBP1). (B) Amino acid sequence of WT and mutant rRalBP1 C-termini. (C) RalBP1 is phosphorylated at Thr 645 in heterologous cells. Lysates of HEK293T cells transfected with Flag-rRalBP1 (WT or T645A) were immunoblotted with the indicated antibodies. RalBP1-pT645, phosphopeptide antibody; RalBP1-T645; non-phosphopeptide antibody. Flag antibodies were used for normalization. (D) λ-phosphatase incubation dephosphorylates phospho-Thr 645 from RalBP1 expressed in heterologous cells and in the brain. Flag-rRalBP1 (WT or T645A) expressed in HEK293T cells, or endogenous RalBP1 from rat brain (S2 and P2 fractions), on nitrocellulose membranes was incubated with λ-phosphatase, followed by immunoblotting with RalBP1-pT645, RalBP1-T645, and RalBP1-Total antibodies. RalBP1-Total was raised against the C-terminal third of RalBP1 and thus recognizes both phospho- and non-phospho-RalBP1; λ-PPase, λ-phosphatase. (E) A mutation (R642A; −5 position) at the RalBP1 C-terminus inhibits RalBP1 phosphorylation. Lysates of HEK293T cells transfected with Flag-rRalBP1 (WT or R642A) were immunoblotted with the indicated antibodies. (F) PKA directly phosphorylates RalBP1 on Thr 645 in vitro. Increasing amounts of GST-RalBP1 were incubated with the catalytic subunit of PKA, followed by the immunoblot analysis with GST and RalBP1-pT645 antibodies. (G) RalBP1 T645E, a phosphomimetic mutant, fails to interact with PDZ domains from PSD-95 family proteins (PSD-95, PSD-93, SAP97, and SAP102) in yeast two-hybrid assays. Note that the human RalBP1 C-terminus used in this experiment ends with RKETSI (human) instead of RKETPI (rat; Figure 1A). (H) RalBP1 T645E fails to form a complex with PSD-95 in heterologous cells. Lysates of HEK293T cells doubly transfected with PSD-95+Flag-rRalBP1 (WT or T645E), or singly with PSD-95, were immunoprecipitated with Flag antibodies and immunoblotted for Flag and PSD-95. (I) Phosphorylated RalBP1 shows a reduced association with PSD-95, compared to total (phosphorylated+non-phosphorylated) RalBP1. Lysates of HEK293T cells doubly transfected with PSD-95 and RalBP1 (WT), or singly with RalBP1, were immunoprecipitated with PSD-95 antibodies and immunoblotted.