Proteolysis of AKAP121 regulates mitochondrial activity during cellular hypoxia and brain ischaemia

Abstract

A-kinase anchor protein 121 (AKAP121) assembles a multivalent signalling complex on the outer mitochondrial membrane that controls persistence and amplitude of cAMP and src signalling to mitochondria, and plays an essential role in oxidative metabolism and cell survival. Here, we show that AKAP121 levels are regulated post-translationally by the ubiquitin/proteasome pathway. Seven In-Absentia Homolog 2 (Siah2), an E3-ubiquitin ligase whose expression is induced in hypoxic conditions, formed a complex and degraded AKAP121. In addition, we show that overexpression of Siah2 or oxygen and glucose deprivation (OGD) promotes Siah2-mediated ubiquitination and proteolysis of AKAP121. Upregulation of Siah2, by modulation of the cellular levels of AKAP121, significantly affects mitochondrial activity assessed as mitochondrial membrane potential and oxidative capacity. Also during cerebral ischaemia, AKAP121 is degraded in a Siah2-dependent manner. These findings reveal a novel mechanism of attenuation of cAMP/PKA signaling, which occurs at the distal sites of signal generation mediated by proteolysis of an AKAP scaffold protein. By regulating the stability of AKAP121-signalling complex at mitochondria, cells efficiently and rapidly adapt oxidative metabolism to fluctuations in oxygen availability.

Figure 1

Siah2 binds to AKAP121. (A) Schematic representation of mouse AKAP121 and its human homologue AKAP149. Mitochondrial targeting motif (MT), PTPD1 (PTP), PKA R subunits and mRNA binding motifs are boxed. The AKAP121 bait (residues 329–573) was fused to the C-terminus of the DNA-binding domain of GAL4 (GAL4-BD). (B) Schematic representation of the clone (clone M) isolated by yeast two-hybrid analysis and its sequence homology with the C-terminus of mouse Siah2. C/H-rich, cysteine-rich region (zinc fingers); RING domain is also shown. (C) MBP-tagged full-length AKAP121 was incubated with purified GST-Siah2 or GST polypeptide. The bound and input fractions were immunoblotted (IB) with anti-AKAP121 (upper panel) and anti-GST (lower panel) antibodies. (D) In vitro translated, ³⁵S-labeled AKAP121 and Siah2 proteins were immunoprecipitated with anti-HA or control immune IgG. The input (1/5) and bound fractions were resolved on 10% SDS–PAGE gels. Gel was fixed, dried and exposed to X-ray film. ^*, small translation product. (E) HEK293 were transiently co-transfected with Flag–Siah2 RING mutant (Flag–Siah2_rm) and AKAP121 vectors. Twenty-four hours after transfection, cells were harvested and lysed. Lysates were subjected to immunoprecipitation with anti-Flag or control immune IgG and immunoblotted with the indicated antibodies. (F) Mitochondrial and supernatant fractions were isolated from HEK293 cells transiently co-transfected with AKAP121 and Siah2_rm and immunoblotted with the indicated antibodies. A representative set of gels is shown. (G) Mouse fibroblasts (NIH3T3) were transiently transfected with HA-tagged Siah2 and subjected to double immunostaining for AKAP121 and HA. Images were obtained by confocal microscopy.

Figure 2

Siah2 promotes degradation of AKAP121. (A, B) HEK293 cells were transiently transfected with CMV, AKAP121 and HA–Siah2 expression vectors. Forty-eight hours after transfection, cells were harvested and lysed (A). Where indicated, cells were treated with MG132 (8 h), a proteasome-specific inhibitor (B). Lysates were size-fractionated on SDS–PAGE and immunoblotted with anti-AKAP121, anti-ERK2 and anti-HA antibodies. (C) Quantitative analysis of the experiment shown in (B). The data are indicated as mean±s.e.m. of four independent experiments that gave similar results, and are expressed as fold decrease relative to control (AKAP121 and endogenous AKAP149, untreated cells), which was set as 1. ^*P<0.05 versus control. (D) Lysates from HEK293 cells transiently transfected with AKAP121 and Siah2 or Siah2_rm were immunoblotted with the indicated antibodies. (E) HEK293 cells were transiently transfected with AKAP121 or AKAP121_Δ336−550 vectors. Where indicated, the Siah2 vector was included in the transfection mixture. Cell lysates were immunoblotted with the indicated antibodies.

Figure 3

Oxygen and glucose deprivation (OGD) induces degradation of AKAP121 via Siah2. (A) HEK293 cells were transiently transfected with CMV or AKAP121 vectors. Where indicated, Flag–Siah2_rm vector was included in the transfection mixture. Forty-eight hours after transfection, cells were either left untreated (0) or exposed to OGD. Cells were harvested at 2 and 4 h post-treatment and immunoblotted with anti-AKAP121, anti-AKAP-KL, anti-ERK2 and anti-Flag antibodies. (B) Quantitative analysis of the experiment shown in panel A. The data are indicated as mean of three independent experiments that gave similar results, and are expressed as fold variations±s.e.m. relative to control (AKAP121 and endogenous AKAP149, 0 time point), which was set as 1. ^*, P<0.05 vs AKAP121 (0 time point). (C, D) Hippocampal neurons (C) or NIH3T3 mouse fibroblasts (D) were left untreated (0 time point) or subjected to OGD for 2 or 4 h. Cells were re-oxygenated (ReOX) following OGD and the medium was supplemented with glucose for additional 15 h (D) or treated during OGD with MG132 (C). Cells were harvested and protein lysates immunoblotted with anti-AKAP121 and anti-ERK2 antibodies. Cumulative data are expressed as fold variations±s.e.m. relative to control (0 time point), which was set as 1. (E) HEK293 cells were transiently transfected with siRNA_c or SMARTpool siRNA_Siah2 (siRNA_Siah2). Twenty-four hours following transfection, cells were either left untreated (0) or subjected to OGD (4 h). Lysates were immunoblotted with anti-AKAP121, anti-β-tubulin and anti-Siah2 antibodies. (F) NIH3T3 were transiently transfected with siRNA_c or siRNA_Siah2 #1 or siRNA_Siah2 #2 (see Materials and methods). Twenty-four hours following transfection, cells were either left untreated (0) or subjected to OGD (4 h). Lysates were immunoblotted with anti-AKAP121, anti-β-tubulin and anti-Siah2 antibodies. (G) Pulse–chase experiment. Mouse fibroblasts where transfected with siRNA_c or siRNAs for Siah2 (SMARTpool). Twenty-four hours later, cells were pulse-labeled for 3 h with [³⁵S]Met/Cys, chased with excess cold methionine and subjected to OGD for the indicated time points. Cell lysates were immunoprecipitated with anti-AKAP121 antibody or preimmune serum (PI) and the precipitates were subjected to autoradiography. (H) The data are indicated as the mean of two independent experiments shown in panel G and are expressed as fold variations relative to control (0 time point), which was set as 100.

Figure 4

Hypoxia–Siah2 signalling promotes ubiquitination of AKAP121. (A) HEK293 cells were co-transfected with AKAP121 and HA-tagged ubiquitin. Where indicated, vectors encoding Siah2 or Siah2_rm were included in the transfection mix. Twenty-four hours after transfection, cells were treated with MG132 (20 μM) for an additional 8 h. Lysates were then prepared, immunoprecipitated with anti-AKAP121 and immunoblotted with anti-HA antibodies. (B) HEK293 cells were co-transfected with AKAP121 and HA-tagged ubiquitin. Where indicated, control (siRNA_c) or SMARTpool siRNA_Siah2 (siRNA_siah2) was included in the transfection mix. Twenty-four hours after transfection, cells were subjected to OGD for 4 h in the presence or absence of MG132, and lysed. Lysates were immunoprecipitated with anti-AKAP121 and immunoblotted with anti-HA and anti-AKAP121 antibodies. (C) In vitro translated and ³⁵S-labeled AKAP121 was incubated with purified GST–Siah2 and His₆-tagged ubiquitin in the presence or absence of E1, UbcH5c (E2) and at 37°C for 45 min. The reaction mix was denatured, size-fractionated by 7% SDS–PAGE and analysed by autoradiography.

Figure 5

Regulation of mitochondrial membrane potential (ΔΨ_m) and dehydrogenase activity (MTT) by AKAP121 and Siah2. (A) TMRE analysis of HEK293 cells transiently co-transfected with GFP and CMV or AKAP121 expression vectors (AKAP121, AKAP121_Δ45−110, AKAP121_{L313, 319P}). Cumulative data are expressed as mean±s.e. of changes in TMRE fluorescence and represent fold increase over control (CMV) cells, which was set as 100. The intensity of fluorescence was evaluated in single cells with Meta Morph software analysis. ^*P<0.05 versus CMV-transfected (GFP positive) cells; ^**P<0.05 versus AKAP121-transfected (GFP positive) cells; ^P<0.05 versus CMV-transfected (GFP positive) cells. Statistical analysis was performed by ANOVA and Newman–Keuls methodology. (B) TMRE analysis of HEK293 cells transiently co-transfected with GFP and the indicated vectors, left untreated or exposed to OGD for 4 h before harvesting. Cumulative data are expressed as mean±s.e.m. of changes in TMRE fluorescence and represent fold increase over control (CMV, untreated cells), which was set as 100. ^*P<0.05 versus CMV-transfected (GFP positive) cells; ^**P<0.05 versus CMV-, CMV/OGD- and Siah2-transfected (GFP positive) cells; ^P<0.05 versus AKAP121- and AKAP121/OGD-transfected (GFP positive) cells. (C) MTT assay in HEK293 cells transiently transfected with AKAP121. Twenty-four hours after transfection, cells were harvested and mitochondrial dehydrogenase activity was assayed as described under Materials and methods. ^*P<0.02 versus each preceding time point. (D) MTT assay in HEK293 cells transiently transfected with the indicated vectors. ^*P<0.05 versus control; ^**P<0.05 versus AKAP121-transfected cells; ^P<0.05 versus siRNA_c-transfected cells. (E) Same as in panel D, except that cells were subjected to OGD for 4 h before harvesting. ^*P<0.05 versus control, ^**P<0.05 versus control and AKAP121-transfected cells; ^P<0.05 versus control and siRNA_c-transfected cells. (F, G) MTT assay in HEK293 cells transiently transfected with the following vectors: empty vector (control), AKAP121, Siah2, control siRNA (siRNA_c), siRNA_AKAP121 and SMARTpool siRNA_Siah2 (siRNA_Siah2). siRNA_AKAP121 vector was described previously (Livigni et al, 2006). Twenty-four hours following transfection, cells were left untreated (F) or subjected to OGD (G) for 4 h before harvesting. ^*P<0.02 versus control; ^**P<0.05 versus control.

Figure 6

AKAP121 disappears during cerebral ischaemia. (A) Brain sections from rats subjected to pMCAO include parietal cortical (PC1 and PC2) and hippocampal (CA1 and CA3) areas isolated from the ischaemic ispilateral hemisphere and from the non-ischaemic contralateral hemisphere. Sections from sham-operated rats were used as controls. Brain sections were immunostained with anti-AKAP121 antibody and analysed by confocal microscopy. (B) Quantitative analysis of the AKAP121 immunoreactivity from rat brain subjected to pMCAO. Fluorescence intensity was measured by using ImageJ 1.38 software. The data are expressed as percent of fluorescence intensity±s.e.m. compared with sham-operated controls. Statistical analysis was performed using separated one-way ANOVAs for each region and post hoc repeated-measure comparisons (LSD test). Rejection level was set at P<0.01. ^*P<0.01 versus contralateral and sham-operated groups.

Figure 7

Sections from control or ischaemic parietal cortex include ischaemic core (PC1) and non-ischaemic parietal cortex (PC2) from the ispilateral hemisphere, PC1 from the contralateral hemisphere and PC1 from sham-operated rats. (A) Sections were doubly immunostained with anti-AKAP121 and anti-NeuN antibodies, and analysed by confocal microscopy. (B) Quantitative analysis of NeuN-positive cells from the ischaemic core (PC1), the area surrounding the ischaemic core (PC2) and the hippocampus (CA1 and CA3) of the ipsilateral and contralateral hemispheres. (C) LSD multiple-comparison test between AKAP121-positive and NeuN-positive cells. Values are expressed as percentage of AKAP121-positive cells calculated as follows: % AKAP⁺ cells=(number of AKAP⁺ NeuN⁺ cells)/(number of NeuN⁺ cells) × 100).

Figure 8

Siah2 is required for AKAP121 disappearance in ischaemic rat brain. (A) Immunoblot analysis of lysates from PC1, PC2 and hippocampus (Hip) of ischaemic, contralateral and sham-operated hemispheres from the rat brain. (B) Cumulative data are expressed as mean±s.e.m. of three independent experiments that gave similar results. The values are relative to the same areas of sham animals that were set as 1. (C) Rat brains were perfused with control (siRNA_c) or SMARTpool siRNA_Siah2 (siRNA_Siah2), and 12 h later were subjected to pMCAO. Lysates were immunoblotted with the indicated antibody. A representative experiment is shown. In this set of experiments, the antibody directed against residues 200–450 of mouse AKAP121 (see Materials and methods) detected an AKAP121 degradation product (^*) that specifically accumulated in ischaemic PC1.

Figure 9

AKAP121 assembles a PKA and PTPD1/src-signalling complex on mitochondria that is required for oxidative metabolism and ATP synthesis. Hypoxia induces Siah2 accumulation. The E3 Ub–ligase binds to and ubiquitinates AKAP121, leading to proteasomal degradation of the anchor protein and consequent decrease of oxidative metabolism.