Figure 3. The serine in the FFAT core of... | Rockefeller University Press

$The serine in the FFAT core of ACBD5 is phosphorylated and inhibits VAPB binding.(A) ACBD5 constructs with nonphosphorylatable (A) and phosphomimetic (E) residues upstream (T252) or within the FFAT core (S269) were expressed in COS-7 cells. The proteins were immunoprecipitated, and endogenous bound VAPB was detected by immunoblotting using Myc/VAPB antibodies. Data were analyzed by one-way ANOVA with Dunnett’s multiple comparison test (n = 3). Total VAPB (IP fraction) was normalized against total VAPB (input) and Myc-ACBD5 (IP fraction). (B) The serine residue within the core of the ACBD5 FFAT-like motif (position 5, S269) binds VAPB in a hydrophobic pocket (Kaiser et al., 2005; Furuita et al., 2010). Phosphorylation (P) at this position likely causes steric hindrance, inhibiting the FFAT–VAPB interaction. (C) Lysates of COS-7 cells expressing Myc-ACBD5 were treated ± λPP before IP. Phosphorylation of ACBD5 at S269 was examined by immunoblotting using ACBD5 pS269/ACBD5 control/Myc antibodies. (D) Myc-ACBD5 WT, S269A phospho mutant, or control vector (Myc) was expressed in COS-7 cells. The proteins were immunoprecipitated, and phosphorylation of ACBD5 at S269 was examined by immunoblotting using ACBD5 pS269/ACBD5 control/Myc antibodies. (E) Myc-VAPB and FLAG-ACBD5 were immunoprecipitated from COS-7 cells separately and then incubated together to allow Myc-VAPB and FLAG-ACBD5 to interact. ACBD5 S269 phosphorylation in the VAPB-bound (IP) and non–VAPB-bound fractions were examined by immunoblotting using ACBD5 pS269/FLAG/Myc antibodies. Source data are available for this figure: SourceData F3.$

Figure 3.

The serine in the FFAT core of ACBD5 is phosphorylated and inhibits VAPB binding. (A) ACBD5 constructs with nonphosphorylatable (A) and phosphomimetic (E) residues upstream (T252) or within the FFAT core (S269) were expressed in COS-7 cells. The proteins were immunoprecipitated, and endogenous bound VAPB was detected by immunoblotting using Myc/VAPB antibodies. Data were analyzed by one-way ANOVA with Dunnett’s multiple comparison test (n = 3). Total VAPB (IP fraction) was normalized against total VAPB (input) and Myc-ACBD5 (IP fraction). (B) The serine residue within the core of the ACBD5 FFAT-like motif (position 5, S269) binds VAPB in a hydrophobic pocket (Kaiser et al., 2005; Furuita et al., 2010). Phosphorylation (P) at this position likely causes steric hindrance, inhibiting the FFAT–VAPB interaction. (C) Lysates of COS-7 cells expressing Myc-ACBD5 were treated ± λPP before IP. Phosphorylation of ACBD5 at S269 was examined by immunoblotting using ACBD5 pS269/ACBD5 control/Myc antibodies. (D) Myc-ACBD5 WT, S269A phospho mutant, or control vector (Myc) was expressed in COS-7 cells. The proteins were immunoprecipitated, and phosphorylation of ACBD5 at S269 was examined by immunoblotting using ACBD5 pS269/ACBD5 control/Myc antibodies. (E) Myc-VAPB and FLAG-ACBD5 were immunoprecipitated from COS-7 cells separately and then incubated together to allow Myc-VAPB and FLAG-ACBD5 to interact. ACBD5 S269 phosphorylation in the VAPB-bound (IP) and non–VAPB-bound fractions were examined by immunoblotting using ACBD5 pS269/FLAG/Myc antibodies. Source data are available for this figure: SourceData F3.

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