PKD phosphorylates CERT on serine 132. (A) Alignment of the peptide sequences used to raise the pMOTIF antibody and two potential PKD motifs in CERT. (B) HEK293T cells transiently expressing Flag-tagged CERT-WT, -S132A, and -S272A were lysed, and CERT phosphorylation was analyzed as described in Fig. 1 A. (C and D) Recombinant GST-Flag-CERT-WT and -S132A proteins were incubated in kinase buffer containing γ-[³²P]ATP (C) or cold ATP (D) in the absence (−) and presence (+) of purified PKD1. Proteins were separated by SDS-PAGE and transferred to membrane. (C) Incorporation of radioactive phosphate was analyzed using a phosphorimager (top) followed by immunoblotting with Flag-specific antibody to verify equal loading of the CERT proteins. (D) Immunoblotting was performed with the pMOTIF antibody and, after stripping, with Flag-specific antibody to verify equal loading of the CERT proteins. PKD1 and CERT proteins are marked with arrows; the bands with asterisks are the results of nonspecific binding.

CERT regulates PKD activation and secretory transport. (A) HEK293T cells transiently expressing CERT-WT and -S132A were lysed, and PKD activation was analyzed by immunoblotting with pS910 PKD antibody (top). Equal loading was verified by reprobing with PKD1-specific antibody (middle). The expression of CERT proteins was verified by immunoblotting with GFP-specific antibody (bottom). (B and D) HEK293T cells were transfected with the indicated expression plasmids (B), and COS7 cells were transfected with the indicated siRNAs (D) together with ssHRP-Flag plasmid as described in Materials and methods. The medium was analyzed for HRP activity after 0, 1, 3, and 6 h by chemiluminescence. Values correspond to the mean of triplicate samples, and error bars represent SEM. RLU, relative light units. (C) COS7 cells were transfected with the siRNAs indicated, and CERT expression was analyzed after 72 h by immunoprecipitation and Western blotting using a CERT-specific antibody (top). Tubulin levels were not affected (bottom). CERT is marked with an arrow.

CERT is detected by a PKD substrate antibody. (A) HEK293T cells were transfected with expression plasmids encoding Flag-tagged CERT_L and CERT. Cells were lysed, and CERT isoforms were immunoprecipitated with anti-Flag antibody. Immunoprecipitated proteins were subjected to SDS-PAGE followed by immunoblotting with PKD substrate antibody (pMOTIF; top) and, after stripping, with anti-Flag antibody (bottom). (B) HEK293T cells were transfected with Flag-CERT expression plasmid along with GFP-PKD1-KD or empty vector. CERT was analyzed by Western blotting as described in A. The expression of PKD1-KD was verified by immunoblotting with a PKD1-specific antibody (bottom). (C) HEK293T cells were either mock transfected or transfected with PKD1- and PKD2-specific siRNAs followed by transfection with Flag-CERT expression plasmid 48 h later. After 24 h, CERT phosphorylation was analyzed as described in A (top). Silencing of PKD1 and PKD2 was verified by immunoblotting of lysates with specific antibodies (bottom). The band marked with an asterisk is the result of nonspecific binding. PKD1 is marked with an arrow. (D) HEK293T cells were transfected with Flag-CERT expression plasmid. Cells were left untreated (con) or were serum starved overnight followed by stimulation with either 10% serum for 2 and 6 h or 2.5 μg/ml 25-hydroxycholesterol for 1 h. CERT phosphorylation was analyzed as described in A. (E) COS7 cells expressing Flag-CERT and PKD1-GFP (top) or GFP-CERT (bottom) were fixed and stained with Flag- and TGN46-specific antibodies (red), respectively. Bars, 10 μm.

CERT phosphorylation on serine 132 modulates PI(4)P binding and ceramide transfer activity. HEK293T cells transiently expressing the indicated GFP-tagged CERT variants were harvested by hypotonic lysis, and the cytosol fraction was recovered after 100,000 g centrifugation. Samples containing equal amounts of GFP fluorescence were used for protein–lipid overlay (A), flotation (B), and in vitro ceramide transfer assays (D). (A) Phosphatidylinositol phosphate arrays were incubated with cytosol from cells transiently expressing GFP-tagged CERT-WT and -S132A, and bound proteins were detected with GFP-specific primary followed by HRP-labeled secondary antibody. (B) MLVs consisting of PC or PC + 5% PI(4)P were incubated with cytosol and separated by centrifugation. The amount of CERT protein in the top (MLV) and bottom fractions was quantified by measuring GFP fluorescence and set as 100%. Results are plotted as percentages of protein recovered in the MLV fraction. (C) Cytosol (C) and the 100,000 g pellet (P) containing cellular membranes were analyzed by immunoblotting with GFP-specific antibody. The purity of the individual fractions was confirmed by detection of the transferrin receptor in the membrane and tubulin in the cytosolic fraction. (D) Donor liposomes containing TNP-PE and pyrene-ceramide were mixed with unlabeled acceptor liposomes. After 60 s, cytosol from cells transiently expressing GFP-tagged CERT-WT, -S132A, or GFP alone (con) was added, and pyrene fluorescence at 395 nm was recorded.

CERT-S132A localizes to PI(4)P-positive secretory vesicles. (A and B) COS7 cells were transiently transfected with the indicated expression plasmids. Cells were fixed and stained with GS28- (red; A, top), TGN46- (red; A, middle), and Flag-specific antibodies (red; A, bottom; and B). The boxed areas are shown in the enlargement. Double-positive vesicles are marked with arrows. Bars, 20 μm; (enlargement) 5 μm.