Regulation of oxysterol-binding protein Golgi localization through protein kinase D-mediated phosphorylation

Mol Biol Cell. 2010 Jul 1;21(13):2327-37. doi: 10.1091/mbc.e10-02-0090. Epub 2010 May 5.

Abstract

Protein kinase D (PKD) plays a critical role at the trans-Golgi network by regulating the fission of transport carriers destined for the plasma membrane. Two known Golgi-localized PKD substrates, PI4-kinase IIIbeta and the ceramide transfer protein CERT, mediate PKD signaling to influence vesicle trafficking to the plasma membrane and sphingomyelin synthesis, respectively. PKD is recruited and activated at the Golgi through interaction with diacylglycerol, a pool of which is generated as a by-product of sphingomyelin synthesis from ceramide. Here we identify a novel substrate of PKD at the Golgi, the oxysterol-binding protein OSBP. Using a substrate-directed phospho-specific antibody that recognizes the optimal PKD consensus motif, we show that PKD phosphorylates OSBP at Ser240 in vitro and in cells. We further show that OSBP phosphorylation occurs at the Golgi. Phosphorylation of OSBP by PKD does not modulate dimerization, sterol binding, or affinity for PI(4)P. Instead, phosphorylation attenuates OSBP Golgi localization in response to 25-hydroxycholesterol and cholesterol depletion, impairs CERT Golgi localization, and promotes Golgi fragmentation.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Sequence
  • Animals
  • Cell Line
  • Golgi Apparatus / metabolism*
  • Humans
  • Molecular Sequence Data
  • Phosphorylation
  • Protein Kinase C / genetics
  • Protein Kinase C / metabolism*
  • Protein-Serine-Threonine Kinases / genetics
  • Protein-Serine-Threonine Kinases / metabolism
  • Receptors, Steroid / genetics
  • Receptors, Steroid / metabolism*
  • Recombinant Fusion Proteins / genetics
  • Recombinant Fusion Proteins / metabolism
  • Sequence Alignment
  • Serine / metabolism

Substances

  • Receptors, Steroid
  • Recombinant Fusion Proteins
  • oxysterol binding protein
  • Serine
  • CERT1 protein, human
  • protein kinase D
  • Protein-Serine-Threonine Kinases
  • Protein Kinase C