Rapid accumulation of Akt in mitochondria following phosphatidylinositol 3-kinase activation

Abstract

We describe here a new component of the phosphatidylinositol 3-kinase/Akt signaling pathway that directly impacts mitochondria. Akt (protein kinase B) was shown for the first time to be localized in mitochondria, where it was found to reside in the matrix and the inner and outer membranes, and the level of mitochondrial Akt was very dynamically regulated. Stimulation of a variety of cell types with insulin-like growth factor-1, insulin, or stress (induced by heat shock), induced translocation of Akt to the mitochondria within only several minutes of stimulation, causing increases of nearly eight- to 12-fold, and the mitochondrial Akt was in its phosphorylated, active state. Two mitochondrial proteins were identified to be phosphorylated following stimulation of mitochondrial Akt, the beta-subunit of ATP synthase and glycogen synthase kinase-3beta. The finding that mitochondrial glycogen synthase kinase-3beta was rapidly and substantially modified by Ser9 phosphorylation, which inhibits its activity, following translocation of Akt to the mitochondria is the first evidence for a regulatory mechanism affecting mitochondrial glycogen synthase kinase-3beta. These results demonstrate that signals emanating from plasma membrane receptors or generated by stress rapidly modulate Akt and glycogen synthase kinase-3beta in mitochondria.

Fig. 1

Distribution of Akt in the cytosol, nucleus and mitochondria. SH-SY5Y cells maintained in serum-containing media were fractionated into cytosolic (C), nuclear (N), and mitochondrial (M) fractions as described in ‘Materials and methods’. (a) Fractions (5 µg protein) were immunoblotted for Akt. (b) To verify separation of the cytosolic, nuclear, and mitochondrial fractions, extracts from each fraction were immunoblotted for tubulin, histone, and subunit IV of cytochrome oxidase (COX IV) and over-exposed immunoblots are shown to verify separation of each fraction. (c) Mitochondria were subfractionated into the outer membrane (OM), intermembrane space (IMS), inner membrane (IM) and matrix (Mx) fractions, and these were immunoblotted for the compartment-specific proteins porin, cytochrome c, COX IV, and Mn-superoxide dismutase (MnSOD), respectively, and for Akt. The non-specific band in the porin immunoblot provides verification of equal loading of protein in each lane and immunoblots of marker proteins were overexposed to verify fractionation procedures. (d) Purified intact mitochondria or cytosolic preparations were subjected to trypsin digestion as described in ‘Materials and methods’. Mitochondrial extracts were immunoblotted for Akt and Tom20, then stripped and reprobed with COX IV antibody. The cytosol fraction was immunoblotted for Akt to verify that this procedure was adequate to completely proteolyze Akt.

Fig. 2

Stimulation of PI3K signaling induces accumulation of Akt in mitochondria. Cells in serum-free media were treated with IGF-1 (50 ng/mL) for 0, 3, 7.5, 15, 30, and 45 min, and cytosolic and mitochondrial fractions were prepared. (a) Cytosolic fractions from SH-SY5Y cells were immunoblotted for phospho-Ser473-Akt, phospho-Thr308-Akt, and total Akt. (b) Mitochondrial fractions from SH-SY5Y cells and HEK293 cells were immunoblotted for total Akt, phospho-Ser473-Akt, phospho-Thr308-Akt, and cytochrome oxidase (COX IV). Akt bands were analyzed by densitometry. Means ± SE, n = 3 experiments; *p < 0.05 (anova) compared to values from untreated cells. (c) Mitochondrial outer membrane, inner membrane, and matrix fractions from control and IGF-1 treated SH-SY5Y cells were immunoblotted for Akt. (d) Cells were pre-incubated for 30 min without and with wortmannin (40 nm) or LY294002 (20 µm) prior to treatment with IGF-1 (50 ng/mL for 15 min). Mitochondrial and cytosolic extracts were immunoblotted for Akt. (e) 3T3L1 adipocytes were treated with insulin (100 nm for 15 min) and SH-SY5Y cells were subjected to an elevated temperature (45°C) ‘heat shock’ (HS) for 15 min. Mitochondrial extracts were immunoblotted for total Akt, phospho-Ser473-Akt, phospho-Thr308-Akt, and cytochrome oxidase (COX IV).

Fig. 3

CCCP treatment blocks IGF-1-induced accumulation of Akt in mitochondria. SH-SY5Y cells were pre-incubated with CCCP (50 µm for 30 min) prior to treatment with IGF-1 (50 ng/mL, 15 min). Mitochondrial extracts were immunoblotted for Akt, phospho-Ser473-Akt, phospho-Thr308-Akt and cytochrome c. Cytosolic extracts were immunoblotted for Akt, phospho-Ser473-Akt, and phospho-Thr308-Akt.

Fig. 4

PAS antibody-immunoreactive proteins in mitochondria. SH-SY5Y cells were pre-incubated without and with wortmannin (40 nm, 30 min) prior to IGF-1 treatment (50 ng/mL, 15 min). (a) Purified cytosolic and mitochondrial proteins were separated in a 4–15% gradient SDS-polyacrylamide gel and immunoblotted with a phospho-Akt substrate (PAS) antibody. (b) Mitochondrial extracts from control, IGF-1, and wortmannin plus IGF-1 treated cells were immunoprecipitated with the PAS antibody and immunoblotted with ATP synthase β-subunit antibody and mitochondrial extracts were also immunoblotted for total levels of ATP synthase β-subunit.

Fig. 5

IGF-1 treatment induces serine-9 phosphorylation of GSK3β in mitochondria. (a) Mitochondrial subfractions from SH-SY5Y cells were immunoblotted for GSK3β. (b) Cytosol and purified intact mitochondria were treated with trypsin (200 µg/mL for 20 min at 4°C) and immunoblotted for GSK3β. (c) SH-SY5Y cells were treated with IGF-1 (50 ng/mL) for 0, 3, 7.5, 15, 30, and 45 min and mitochondrial extracts were immunoblotted for phospho-Ser9-GSK3β and total GSK3β. (d) Purified intact mitochondria isolated from unstimulated and IGF-1 (50 ng/mL, 15 min) stimulated cells were incubated with trypsin (200 µg/mL for 20 min at 4°C) and mitochondrial extracts were immunoblotted for phospho-Ser9-GSK3β. (e) Mitochondrial outer membrane and inner membrane fractions from control and IGF-1 treated SH-SY5Y cells were immunoblotted for phospho-Ser9-GSK3β and total GSK3β. (f) Cells were pre-incubated for 30 min without or with wortmannin (40 nm) or LY294002 (20 µm) prior to treatment with IGF-1 (50 ng/mL, 15 min). Mitochondrial extracts were immunoblotted for phospho-Ser9-GSK3β and total GSK3β.