Oxysterol-binding protein-related protein (ORP) 9 is a PDK-2 substrate and regulates Akt phosphorylation.
The oxysterol-binding protein and oxysterol-binding protein-related protein family has been implicated in lipid transport and metabolism, vesicle trafficking and cell signaling. While investigating the phosphorylation of Akt/protein kinase B in stimulated bone marrow-derived mast cells, we observed that a monoclonal antibody directed against phospho-S473 Akt cross-reacted with oxysterol-binding protein-related protein 9 (ORP9). Further analysis revealed that mast cells exclusively express ORP9S, an N-terminal truncated version of full-length ORP9L. A PDK-2 consensus phosphorylation site in ORP9L and OPR9S at S287 (VPEFS(287)Y) was confirmed by site-directed mutagenesis. In contrast to Akt, increased phosphorylation of ORP9S S287 in stimulated mast cells was independent of phosphatidylinositol 3-kinase but sensitive to inhibition of conventional PKC isotypes. PKC-beta dependence was confirmed by lack of ORP9S phosphorylation at S287 in PKC-beta-deficient, but not PKC-alpha-deficient, mast cells. Moreover, co-immunoprecipitation of PKC-beta and ORP9S, and in vitro phosphorylation of ORP9S in this complex, argued for direct phosphorylation of ORP9S by PKC-beta, introducing ORP9S as a novel PKC-beta substrate. Akt was also detected in a PKC-beta/ORP9S immune complex and phosphorylation of Akt on S473 was delayed in PKC-deficient mast cells. In HEK293 cells, RNAi experiments showed that depletion of ORP9L increased Akt S473 phosphorylation 3-fold without affecting T308 phosphorylation in the activation loop. Furthermore, mammalian target of rapamycin was implicated in ORP9L phosphorylation in HEK293 cells. These studies identify ORP9 as a PDK-2 substrate and negative regulator of Akt phosphorylation at the PDK-2 site.
Lessmann
Eva
E
Department of Molecular Immunology, Biology III, University of Freiburg and Max Planck Institute for Immunobiology, Stübeweg 51, 79108 Freiburg, Germany.
Ngo
Mike
M
Leitges
Michael
M
Minguet
Susana
S
Ridgway
Neale D
ND
Huber
Michael
M
D064413:3-Phosphoinositide-Dependent Protein Kinases; D020816:Amino Acid Motifs; D000818:Animals; D019556:COS Cells; D002478:Cells, Cultured; D002522:Chlorocebus aethiops; D015971:Gene Expression Regulation, Enzymologic; D006801:Humans; D057927:Hydrophobic and Hydrophilic Interactions; D047468:Immunoprecipitation; D008297:Male; D051379:Mice; D010766:Phosphorylation; D011493:Protein Kinase C / Q000378:metabolism; D064546:Protein Kinase C beta; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism*; D051057:Proto-Oncogene Proteins c-akt / Q000378:metabolism*; D034622:RNA Interference; D011987:Receptors, Steroid / Q000235:genetics / Q000378:metabolism* / Q000502:physiology; D000098825:Oxysterol Binding Proteins
D011987:Receptors, Steroid; D000098825:Oxysterol Binding Proteins; D064413:3-Phosphoinositide-Dependent Protein Kinases; D017346:Protein Serine-Threonine Kinases; D051057:Proto-Oncogene Proteins c-akt; D011493:Protein Kinase C; D064546:Protein Kinase C beta