okadaic-acid and 25-hydroxycholesterol

Oxysterol binding protein (OSBP), a high affinity receptor for 25-hydroxycholesterol that localizes to a Golgi/vesicular compartment, migrated on SDS-PAGE as a doublet of 96 and 101 kDa. The reduced mobility of the upper band of this doublet is the result of phosphorylation on multiple serine residues. Phosphorylation of rabbit OSBP stably overexpressed in CHO-K1 cells was altered by staurosporine and okadaic acid, while other protein kinase activators and inhibitors such as TPA, sphingosine and bis-indolylmaleimide were without affect. Treatment of overexpressing and control cells with brefeldin A (BFA) caused dephosphorylation of OSBP that coincided with disruption of the Golgi apparatus. [32P]Phosphate pulse-chase and immunoprecipitation experiments showed that BFA inhibited phosphorylation of OSBP, but not its rate of dephosphorylation. Phosphopeptide maps of OSBP from overexpressing and control CHO-K1 cells were similar, and BFA promoted dephosphorylation of all five peptides. Compared to overexpressing cells, one tryptic phosphopeptide was more abundant in control CHO-K1 cells and was preferentially dephosphorylated by BFA treatment. OSBP was phosphorylated in vitro by the Golgi enriched fraction of CHO-K1 cells or rat liver by a staurosporine- and BFA-insensitive kinase. The phosphorylation status of OSBP was not affected by 25-hydroxycholesterol and did not alter in vitro 25-[3H]hydroxycholesterol binding. Furthermore, dephosphorylation of OSBP by staurosporine did not affect 25-hydroxycholesterol-mediated localization to the Golgi apparatus. Rapid phosphorylation/dephosphorylation of OSBP requires interaction with the Golgi apparatus and an associated kinase. (c) 1998 Elsevier Science B.V.

Topics: Animals; Brefeldin A; CHO Cells; Cricetinae; Cyclopentanes; Electrophoresis, Polyacrylamide Gel; Enzyme Inhibitors; Fluorescent Antibody Technique, Indirect; Golgi Apparatus; Hydroxycholesterols; Molecular Weight; Okadaic Acid; Phosphorylation; Protein Kinases; Protein Synthesis Inhibitors; Rabbits; Rats; Receptors, Steroid; Serine; Staurosporine