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Oxysterol-binding protein-related protein (ORP) 9 is a PDK-2 substrate and regulates Akt phosphorylation

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Lessmann,  Eva
Research Group and Chair of Molecular Immunology of the University of Freiburg, Max Planck Institute of Immunobiology and Epigenetics, Max Planck Society;

Minguet,  Susana
Max Planck Society;

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Huber,  Michael
Research Group and Chair of Molecular Immunology of the University of Freiburg, Max Planck Institute of Immunobiology and Epigenetics, Max Planck Society;

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Citation

Lessmann, E., Ngo, M., Leitges, M., Minguet, S., Ridgway, N. D., & Huber, M. (2007). Oxysterol-binding protein-related protein (ORP) 9 is a PDK-2 substrate and regulates Akt phosphorylation. Cellular Signalling, 19, 384-392.


Cite as: https://hdl.handle.net/11858/00-001M-0000-002B-91CB-A
Abstract
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 (VPEFS287Y) 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-β dependence was confirmed by lack of ORP9S phosphorylation at S287 in PKC-β-deficient, but not PKC-α-deficient, mast cells. Moreover, co-immunoprecipitation of PKC-β and ORP9S, and in vitro phosphorylation of ORP9S in this complex, argued for direct phosphorylation of ORP9S by PKC-β, introducing ORP9S as a novel PKC-β substrate. Akt was also detected in a PKC-β/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.