Multisite phosphorylation of oxysterol-binding protein regulates sterol binding and activation of sphingomyelin synthesis

Abstract

The endoplasmic reticulum (ER)-Golgi sterol transfer activity of oxysterol-binding protein (OSBP) regulates sphingomyelin (SM) synthesis, as well as post-Golgi cholesterol efflux pathways. The phosphorylation and ER-Golgi localization of OSBP are correlated, suggesting this modification regulates the directionality and/or specificity of transfer activity. In this paper, we report that phosphorylation on two serine-rich motifs, S381-S391 (site 1) and S192, S195, S200 (site 2), specifically controls OSBP activity at the ER. A phosphomimetic of the SM/cholesterol-sensitive phosphorylation site 1 (OSBP-S5E) had increased in vitro cholesterol and 25-hydroxycholesterol-binding capacity, and cholesterol extraction from liposomes, but reduced transfer activity. Phosphatidylinositol 4-phosphate (PI(4)P) and cholesterol competed for a common binding site on OSBP; however, direct binding of PI(4)P was not affected by site 1 phosphorylation. Individual site 1 and site 2 phosphomutants supported oxysterol activation of SM synthesis in OSBP-deficient CHO cells. However, a double site1/2 mutant (OSBP-S381A/S3D) was deficient in this activity and was constitutively colocalized with vesicle-associated membrane protein-associated protein A (VAP-A) in a collapsed ER network. This study identifies phosphorylation regulation of sterol and VAP-A binding by OSBP in the ER, and PI(4)P as an alternate ligand that could be exchanged for sterol in the Golgi apparatus.

FIGURE 1:

OSBP phosphorylation sites and mutations. Site 1, site 2, and PKD phosphorylation sites and mutations reported in this study are indicated in bold. PHD, PH domain; SBD, sterol-binding domain.

FIGURE 2:

Identification of an OSBP site 1 phosphomutant with increased sterol-binding activity. (A) Purified OSBP, OSBP-S381A, OSBP-S3E, and OSBP-S5E (2 μg each) were resolved by SDS–6%PAGE and stained with Coomassie Blue. (B and C) Specific binding of [³H]25OH and [³H]cholesterol by 12 pmol of OSBP (■), OSBP-S381A (▲), OSBP-S3E (●), or OSBP-S5E (♦) was assayed as described in Materials and Methods. (D) Following binding of 100 nM [³H]25OH, OSBP and OSBP-S5E were isolated on Talon resin and resuspended in binding buffer containing 100 nM 25OH at 20°C. At the indicated times, OSBP-bound 25OH was removed by centrifugation, and radioactivity in the supernatant was quantified. (E) Increasing amounts of OSBPs (see B and C for symbols) were incubated with liposomes containing 2 mol% [³H]cholesterol, and the extraction of radiolabel into the supernatant was measured. (F) Transfer of [³H]cholesterol between liposomes by OSBP, OSBP-S5E, OSBP-S381A, or OSBP-RR/EE was determined using a modified assay that involved preextraction of sterols from donor liposomes prior to addition of acceptor liposomes containing 2 mol% PI or PI(4)P. Results in all panels are the mean and SEM of three or more experiments using two to three different protein preparations.

FIGURE 3:

PI(4)P binding by OSBP is competitive with cholesterol but unaffected by site 1 phosphorylation. (A) OSBP (50 pmol) was incubated with liposomes containing [³H]cholesterol (1 mol%) and increasing amounts PI, PI(4,5)P₂, or PI(4)P (0–2 mol%). The extraction of [³H]cholesterol from liposomes by OSBP into the supernatant is expressed relative to activity in the absence of PIPs. Results are from a representative experiment. (B) The association of OSBP with liposomes during the extraction assay shown in (A) was determined by SDS–PAGE of supernatant (S) and pellet (P) fractions. (C) OSBP (50 pmol) was incubated with liposomes containing 0.5 mol% of [³H]PI or [³²P]PI(4)P, and extraction of radioactivity into the supernatant was measured after precipitation of liposomes. Results are the mean and SEM of three experiments. (D) Increasing amounts of OSBP (■), OSBP-S381A (▲), or OSBP-S5E (●) were incubated with liposomes containing 0.5 mol% [³²P]PI(4)P, and extraction of radioactivity into the supernatant was measured. The results are the mean and SEM of three experiments.

FIGURE 4:

PH-domain activity and Golgi localization is not affected by OSBP site 1 mutations. (A) Purified wild-type and OSBP mutants (200 pmol) were incubated with 0, 100, 200, and 500 pmol of PI(4)P, PI(4,5)P₂, and PC that was immobilized on nitrocellulose filters. Filters were probed with an OSBP polyclonal and goat anti-rabbit IRDye 800–conjugated secondary antibodies. (B) The association of OSBP and phosphomutants with liposomes containing 10 mol% PI, PI(4)P, or PI(4,5)P₂ was determined by quantification of distribution in the supernatant (light bars) and pellet (black bars) fractions after centrifugation. Results are from a representative experiment. (C) The indicated mCherry-OSBP fusions were transiently expressed for 24 h in OSBP-deficient CHO cells; this was followed by incubation with 25OH (6 μM) or solvent control (NA, no addition) for 2 h and immunostaining with a giantin antibody and goat anti-rabbit Alex Fluor 488. (D) The indicated amounts of recombinant OSBP (■), OSBP-S381A (▲), or OSBP-S5E (●) were incubated with GST-VAP-A (50 pmol), complexes were isolated by binding to glutathione-Sepharose, and bound OSBP was quantified by SDS–PAGE and Coomassie staining. Results are the mean and SEM of three experiments.

FIGURE 5:

OSBP site 1 phosphomutants restore 25OH-activated SM synthesis in OSBP-depleted CHO cells. (A and B) CHO cells deficient in endogenous OSBP were transiently transfected for 48 h with wild-type OSBP and site 1 phosphomutants. SM (A) and ceramide (B) synthesis was measured by [³H]serine incorporation after treatment with 25OH (6 μM, black bars) or solvent control (empty bars) for 6 h. Results are the mean and SEM of three experiments. (C) Total cell lysates from mock and OSBP-transfected cells were resolved by SDS–6%PAGE and immunoblotted with an OSBP polyclonal antibody.

FIGURE 6:

A site 2 phosphorylation motif is adjacent to the PH domain. (A) OSBP and the indicated site1 and site 2 mutants were transiently transfected into CHO cells for 48 h. Cells were then harvested for immunoblotting or labeled with ³²PO₄ (0.3 mCi/ml) for 4 h. ³²PO₄-labeled OSBPs were immunoprecipitated with monoclonal 11H9, resolved by SDS–PAGE, and subject to autoradiography for 12 h at −80°C. (B) ³²PO₄-labeled OSBPs were digested with trypsin and resolved by two-dimensional thin-layer electrophoresis and chromatography on cellulose-coated plates (Mohammadi et al., 2001). Arrows indicate the position of the site 2 phosphopeptide. Asterisks indicate the positions of site 1 phosphopeptides.

FIGURE 7:

Identification of a site 1/site 2 double mutant that does not restore sterol activation of SM synthesis. (A) OSBP site 2 mutants (S3A and S3D) were transiently expressed in OSBP-depleted CHO cells, and SM and ceramide synthesis was measured by [³H]serine incorporation as described in the legend to Figure 5. (B) A series of four OSBP site1/site2 double phosphomutants were transiently expressed in OSBP-depleted CHO cells, and SM and ceramide synthesis was measured as described above. The results shown in (A) and (B) are the means and SEM of three experiments. (C and D) Immunoblot analysis of site 2 (C) and site 1/site 2 double phosphomutants (D) expressed in OSBP-depleted CHO cells.

FIGURE 8:

OSBP S381A/S3D collapses and aggregates the ER. (A) OSBP-depleted CHO cells transiently expressing OSBP-S381A/S3D for 24 h were treated with 25OH (6 μg/ml) or solvent control (no addition, NA) for 2 h. Cells were immunostained with OSBP monoclonal 11H9 and Alexa Fluor 594–conjugated antibodies, which was followed by incubation with VAP-A polyclonal and Alexa Fluor 488–conjugated secondary antibodies. (B) Following 25OH treatment, CHO cells were immunostained with an OSBP polyclonal and Alexa Fluor 594–conjugated antibodies, which was followed by incubation with PI4K IIIβ monoclonal and Alexa Fluor 488–conjugated secondary antibodies. Arrowheads indicate dispersed Golgi staining in expressing cells. (C) CHO cells expressing OSBP-S381A/S3D/FF-AA, treated with or without 25OH, were coimmunostained for VAP-A or giantin, using Alexa Fluor 488–conjugated secondary antibodies. CHO cells expressing OSBP (D) or OSBP-S381A/S3D (E, F, and G) were processed for TEM, as described in Materials and Methods. The ER is indicated by arrowheads. Scale bar: 500 nm. (F) High magnification of the boxed area in (E). (H and I) OSBP-S381A/S3D was visualized in CHO cells with 11H9 and a goat anti-mouse secondary conjugated to 5-nm colloidal gold particles. Scale bar: 100 nm.

FIGURE 9:

Enhanced interaction between VAP-A and OSBP-S381A/S3D. (A) Triton X-100 extracts were prepared from OSBP-depleted CHO cells transiently expressing the indicated single OSBP and site 1 or site 2 mutants and treated without (−) or with (+) 25OH (6 μM for 2 h). VAP-A was immunoprecipitated from extracts with a polyclonal antibody and immunoblotted for OSBP and VAP-A. (B) OSBP was coimmunoprecipitated with VAP-A from Triton X-100 extracts of OSBP-depleted CHO cells expressing site 1/site 2 double phosphomutants. (C) The amount of OSBP coimmunoprecipitated with VAP-A in (B) was quantified as a percentage of total OSBP input (average and range of two experiments).