Endoplasmic reticulum is a main localization site of mTORC2 - PubMed (original) (raw)

Endoplasmic reticulum is a main localization site of mTORC2

Delphine R Boulbés et al. Biochem Biophys Res Commun. 2011.

Abstract

The Akt kinase is a critical effector in growth factor signaling. Activation of Akt driven by the growth factor dependent PI3K (phosphatidylinositol-3-OH kinase) is coupled to the plasma membrane translocation and phosphorylation of Akt on two sites by PDK1 (phosphoinositide-dependent protein kinase-1) on Thr-308 and by mTORC2 (mammalian Target of Rapamycin Complex 2) on Ser-473. In our study we examined the sub-cellular localization of mTORC2 and identified that this kinase complex predominantly resides on endoplasmic reticulum (ER). Our immunostaining analysis did not show a substantial co-localization of the mTORC2 component rictor with Golgi, lysosome, clathrin-coated vesicles, early endosomes, or plasma membrane but indicated a strong co-localization of rictor with ribosomal protein S6 and ER marker. Our biochemical study also identified the mTORC2 components rictor, SIN1, and mTOR as the highly abundant proteins in the ER fraction, whereas only small amount of these proteins are detected in the plasma membrane and cytosolic fractions. We found that growth factor signaling does not alter the ER localization of mTORC2 and also does not induce its translocation to the plasma membrane. Based on our study we suggest that the mTORC2-dependent phosphorylation of Akt on Ser-473 takes place on the surface of ER.

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Figures

Fig. 1

Detection of mTORC2 by co-localization of rictor and mTOR. MDA-MB-435 cells growing in 10% serum were stained with the rictor and mTOR antibodies. The images of rictor, mTOR, and merged images are presented.

Fig. 2

Bulk of rictor does not reside in endosomes, Golgi, and lysosomes. The immunofluorescence study has been performed to stain rictor with the specific markers of different organelles in MDA-MB-435. Clathrin was used as a specific marker for clathrin-coated vesicules, EEA1 was used as a specific marker for early endosomes, GM 130 was used as a specific marker for Golgi apparatus, and LAMP2 was used as a specific marker for lysosomes.

Fig. 3

Rictor is co-localized with the ribosomal protein S6 and ER markers. The immunofluorescence study has been performed in MDA-MB-435 cells to stain rictor with the ribosomal protein S6 (left panel) and the ER markers - calreticulin (middle panel) and ER-GFP (right panel).

Fig. 4

The biochemical sub-cellular fractionation identified mTORC2 in ER. MDA-MB-435 cells incubated in the medium containing 10% serum (GM, growth medium), serum starved for 24 hrs (SS), and serum starved for 24 hrs and stimulated with IGFI (50 ng/ml) for 20 min (IGF) were analyzed by fractionation and isolation the cytosolic, ER (endoplasmic reticulum) and PM (plasma membrane) fractions. Each fraction representing different cell culture condition were lysed and analyzed by loading the equal protein amount on a gel by immunoblotting with the indicated antibodies.

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Endoplasmic reticulum is a main localization site of mTORC2 - PubMed (original) (raw)