INHIBITION OF PHOSPHORYLATION OF THE OXYSTEROL BINDING-PROTEIN BY BREFELDIN-A

Oxysterol binding protein (OSBP), a high affinity receptor for 25-hydr oxycholesterol that localizes to a Golgi/vesicular compartment, migrat ed 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 mu ltiple serine residues. Phosphorylation of rabbit OSBP stably overexpr essed in CHO-K1 cells was altered by staurosporine and okadaic acid, w hile other protein kinase activators and inhibitors such as TPA, sphin gosine and bis-indolylmaleimidz were without affect. Treatment of over expressing and control cells with brefeldin A (BFA) caused dephosphory lation of OSBP that coincided with disruption of the Golgi apparatus. [P-32]Phosphate pulse-chase and immunoprecipitation experiments showed that BFA inhibited phosphorylation of OSBP, but not its rate of depho sphorylation. Phosphopeptide maps of OSBP from overexpressing and cont rol CHO-K1 cells were similar, and BFA promoted dephosphorylation of a ll five peptides. Compared to overexpressing cells, one tryptic phosph opeptide was more abundant in control CHO-KI cells and was preferentia lly dephosphorylated by BFA treatment. OSBP was phosphorylated in vitr o by the Golgi enriched fraction of CHO-K1 cells or rat liver by a sta urosporine-and BFA-insensitive kinase. The phosphorylation status of O SBP was not affected by 25-hydroxycholesterol and did not alter in vit ro 25-[H-3]hydroxycholesterol binding. Furthermore, dephosphorylation of OSBP by staurosporine did not affect 25-hydroxycholesterol-mediated localization to the Golgi apparatus. Rapid phosphorylation/dephosphor ylation of OSBP requires interaction with the Golgi apparatus and an a ssociated kinase. (C) 1998 Elsevier Science B.V.