Hsp70 associates with Rictor and is required for mTORC2 formation and activity - PubMed (original) (raw)

Hsp70 associates with Rictor and is required for mTORC2 formation and activity

Jheralyn Martin et al. Biochem Biophys Res Commun. 2008.

Abstract

mTORC2 is a multiprotein kinase composed of mTOR, mLST8, PRR5, mSIN1 and Rictor. The complex is insensitive to rapamycin and has demonstrated functions controlling cell growth, motility, invasion and cytoskeletal assembly. mTORC2 is the major hydrophobic domain kinase which renders Akt fully active via phosphorylation on serine 473. We isolated Hsp70 as a putative Rictor interacting protein in a yeast two-hybrid assay and confirmed this interaction via co-immunoprecipitation and colocalization experiments. In cells expressing an antisense RNA targeting Hsp70, mTORC2 formation and activity were impaired. Moreover, in cells lacking Hsp70 expression, mTORC2 activity was inhibited following heat shock while controls demonstrated increased mTORC2 activity. These differential effects on mTORC2 activity were specific, in that mTORC1 did not demonstrate Hsp70-dependent alterations under these conditions. These data suggest that Hsp70 is a component of mTORC2 and is required for proper assembly and activity of the kinase both constitutively and following heat shock.

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Figures

Figure 1

Figure 1

Yeast two-hybrid assay mapping of interacting regions of Rictor and Hsp70. The indicated deletion mutants of Gal4DBD-Rictor or Gal4AD-Hsp70 were cotransfected into AH109 cells and plated onto selective media to determine whether an interaction between the proteins was detectable via activation of the HIS3 reporter (+++, strong growth; ++, moderate growth; -, no growth). Colonies which grew were assayed for _β_-gal activity.

Figure 2

Figure 2

Rictor interacts with Hsp70 in mammalian cells. (A) Rictor, Raptor or Hsp70 was immunoprecipitated from HeLa cells and precipitates subjected to Western analysis for the indicated proteins. Lane 1, beads, no antibody; Lane 2, immunoprecipitation with an irrelevant antibody (control IgG); Lane 3, input cell lysate; Lane 4, indicated immunoprecipitate probed with antibodies for the indicated proteins. (B) Hsp70 is liberated from Rictor in the presence of ATP. Rictor immunoprecipitates were subjected to successive washes in the absence or presence of 20 mM ATP and immunoblotted for Hsp70 and mTOR. (C) Co-localization of endogenous Hsp70 (green) and Rictor (red) in HeLa cells at 37°C and (D), following heat shock. Panels on the right show the merged composite images. Results in A-D are representative of three independent experiments.

Figure 3

Figure 3

Hsp70 is required for TORC2 formation and activity. (A) TORCs were immunoprecipitated from P-19 cells stably expressing an antisense RNA targeting Hsp70. These cells have previously been shown to have markedly reduced Hsp70 levels [12], in which the antisense RNA-mediated downregulation of Hsp70 was demonstrated to be specific. (B) mTORC2 in vitro kinase assay. TORC complexes from P-19neo#3 or P-19AS-Hsp70#1 cells were obtained using mTOR antibodies and subjected to an in vitro kinase assay using inactive 6HIS-tagged Akt1 as a substrate. TORC2 activity was monitored by assessing Akt1 serine 473 phosphorylation. Results in A, B were performed three times with similar results.

Figure 4

Figure 4

mTORC2 is activated in an Hsp70-dependent manner following heat shock. (A) mTORC2 complexes were immunoprecipitated using Rictor antibodies from P-19neo#3 or P-19AS-Hsp70#1 cells under control (C, 37°C) or following heat shock (H) conditions and immunoprecipitates subjected to Western analysis for the indicated TORC2 components. (B) mTORC2 activity is increased in control P-19neo#3 cells and ablated in P-19AS-Hsp70#1 cells following heat shock. mTOR immunoprecipitates from the indicated cell lines were subjected to an in vitro kinase assay utilizing inactive (6HIS)-tagged Akt1 as a substrate. mTORC2 activity was monitored by assessing Akt1 serine 473 phosphorylation. (C) Hsp70-dependent TORC activity is specific for TORC2. Extracts from P-19neo#3 or P-19AS-Hsp70#1 cells under control or following heat shock conditions were immunoblotted for endogenous phosphorylated and total p70/S6K and Akt as indicated. Results in A-C are representative of three or four independent experiments.

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