Depletion of OSBP impairs VSVG transport. HeLa cells infected with OSBP shRNA were transiently transfected with myc-WT.OSBP, S242A, or S242D. After 24 h, cells were transfected with tsO45-VSVG-GFP. Cells were fixed and immunostained with anti-VSVG (8G5F11) to detect surface VSVG after the indicated times (min). Results are representative of at least three independent experiments.

Ser242 phosphorylation affects CERT Golgi localization. CHO-K1 cells stably expressing CERT-GFP were transiently transfected with myc-WT OSBP, S242A, or S242D. Cells were treated with 2.5 μg/ml 25-OH for 2 h. Cells were fixed and immunostained with anti-myc. Results are representative of at least three independent experiments.

Model of PKD regulation of Golgi lipid homeostasis. (1) PKD phosphorylation of PI4KIIIβ leads to 14-3-3 binding and stabilization of its lipid kinase activity, resulting in increased PI(4)P production at the TGN. (2) CERT and OSBP localize to the TGN via binding of their PH domains to PI(4)P. (3) PKD phosphorylation of CERT decreases its affinity for PI(4)P, releasing it from the TGN and attenuating SM and DAG production. (4) PKD phosphorylation of OSBP inhibits localization to the TGN. This impairs CERT Golgi localization and activation, and therefore SM and DAG production.

Ser242 phosphorylation inhibits 25-OH–induced TGN localization. HeLa cells infected with OSBP shRNA were transiently transfected with myc-WT OSBP, S242A, or S242D. Cells were serum-starved and treated with 2.5 μg/ml 25-OH for 2 h. Cells were then fixed and immunostained with anti-myc and (A) anti-GM130, (B) anti-Giantin, or (C) anti-TGN46. Results are representative of at least three independent experiments.

PKD phosphorylates OSBP at the Golgi. (A) Hela cells infected with OSBP shRNA were transfected with myc-OSBP or OSBP.RR109/110EE. Cells were serum-starved and treated with 2.5 μg/ml 25-OH for 2 h, fixed, and immunostained with anti-myc or anti-TGN46. (B) HEK293T cells were cotransfected with HA-PKD.SS/EE and myc-WT.OSBP or OSBP.RE109/110EE. OSBP was immunoprecipitated with anti-myc and immunoblotted with anti-pMOTIF. Total lysates were immunoblotted with anti-actin and anti-HA. Results are representative of at least three independent experiments.

PKD phosphorylates OSBP. (A) LnCap cells were lentivirally infected with PKD1 shRNA, selected with puromycin, serum-starved, and stimulated with 1 μM PdBu for 30 min. Endogenous OSBP was immunoprecipitated with anti-OSBP and immunoblotted with anti-pMOTIF or anti-OSBP. Total lysates were immunoblotted with anti-pSS738/742, anti-PKD (C20), and anti-actin. (B) HEK293T cells were lentivirally infected with PKD1 and PKD2 shRNAs and then transfected with myc-OSBP and HA-PKD.SS/EE or PKD.SS/EE* (nonsilenceable PKD). OSBP was immunoprecipitated with anti-myc and immunoblotted with anti-pMOTIF and anti-myc. Total lysates were immunoblotted with anti-HA. (C) HEK293T cells were lentivirally infected with PKD1, PKD2, or PKD1, and PKD2 shRNAs then were transfected with myc-OSBP. Cells were serum-starved and stimulated with 1 μM PdBu for 30 min. OSBP was immunoprecipitated with anti-myc and immunoblotted with anti-pMOTIF. Total lysates were immunoblotted with anti-myc or anti-pSS738/742. Results are representative of at least three independent experiments.

OSBP is recognized by the PKD substrate antibody. (A) Domain structure of OSBP revealing the alignment of PKD phosphorylation sites Ser 240 and Ser 269 from different species. (B and C) HEK293T cells transfected with myc-OSBP stimulated with (B) 1 μM PdBu or (C) 50 ng/μL PDGF for 10 or 30 min. OSBP was immunoprecipitated with anti-myc and immunoblotted with anti-pMOTIF or anti-myc. Control immunoblotting of total lysate was with anti-actin and anti-pSS738/742. (D) HEK293T cells were transfected with myc-OSBP and HA-PKD.SS/EE or PKD.KW. OSBP was immunoprecipitated with anti-myc and immunoblotted with anti-pMOTIF or anti-myc. Total lysates were immunoblotted with anti-actin and anti-HA. (E) HEK293T cells transfected with myc-OSBP were treated with or without 1 μM Gö6976 for 1 h and then stimulated with 1 μM PdBu for 30 min. OSBP was immunoprecipitated with anti-myc and immunoblotted with anti-pMOTIF or anti-myc. Total lysates were immunoblotted with anti-pSS738/742 and anti-actin. Results are representative of at least three independent experiments.

Ser242 phosphorylation affects cholesterol-regulated Golgi localization. (A) HeLa cells infected with OSBP shRNA were transiently transfected with myc-WT OSBP, S242A, or S242D. Cells were grown in 5% lipoprotein-deficient serum (LPDS) for 24 h and treated with 50 μg human LDL/ml for 18 h. Cells were then fixed and immunostained with anti-myc or anti-TGN46. Results are representative of at least three independent experiments. (B) Quantitation of Golgi fragmentation was determined by counting the number of cells with dispersed TGN46 staining (to total number of cells with fragmented and nonfragmented Golgi). The percentage of cells with Golgi fragmentation was determined for OSBP-depleted cells expressing the indicated mutants −/+ LDL. Statistical evaluations were made between the following: WT and S242A, +LDL; WT and S242D, −LDL; and WT and S242D, +LDL. The data shown are mean values ± SD of four independent experiments, with n = 100 for each condition. *p < 0.05; **p < 0.01; ***p < 0.001.

PKD phosphorylates OSBP at Ser242. (A) HEK293T cells were transfected with HA-PKD.SS/EE and myc-WT.OSBP, OSBP.S242A, or OSBP.S271A. OSBP was immunoprecipitated with anti-myc and immunoblotted with anti-pMOTIF. Total lysates were immunoblotted with anti-myc and anti-HA. (B) HEK293T cells expressing myc-WT.OSBP, OSBP.S242A, or OSBP.S271A were serum-starved, and OSBP was immunoprecipitated with anti-myc. Immune complexes were incubated in kinase buffer containing ATP in the absence or presence of recombinant PKD1. PKD phosphorylation was analyzed by immunoblotting with anti-pMOTIF and control anti-myc. (C) The MS/MS spectrum of the phosphorylated tryptic peptide SLpSELESLK acquired via collision induced dissociation with an Orbitrap XL mass spectrometer. Notice that the mass of the y₃¹⁺ ion does not contain a phosphate group while a prominent y₇¹⁺ shows the addition of 79.97 Da (phosphate), suggesting the phosphate group is present on Ser3 in the above peptide sequence. Similarly, the b-ion series is consistent with the phosphate group at Ser3. (D) Quantification of peptide SLpSELESLK using a TIC quantification method. Quantification of peptide from HEK293T cells coexpressing OSBP and constitutively active PKD (SS/EE PKD) or OSBP and SS/EE PKD with PKD1/2 shRNA is relative to cells expressing only OSBP. (E) Expression of OSBP and knockdown of SS/EE PKD were confirmed by immunoblotting with anti-myc and anti-HA. Results are representative of at least three independent experiments.