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. 2013 Jan 10;49(1):121-32.
doi: 10.1016/j.molcel.2012.10.023. Epub 2012 Nov 29.

Phosphorylation of human TFAM in mitochondria impairs DNA binding and promotes degradation by the AAA+ Lon protease

Bin Lu  1 , Jae Lee, Xiaobo Nie, Min Li, Yaroslav I Morozov, Sundararajan Venkatesh, Daniel F Bogenhagen, Dmitry Temiakov, Carolyn K Suzuki
Affiliations

Phosphorylation of human TFAM in mitochondria impairs DNA binding and promotes degradation by the AAA+ Lon protease

Bin Lu et al. Mol Cell. .

Abstract

Human mitochondrial transcription factor A (TFAM) is a high-mobility group (HMG) protein at the nexus of mitochondrial DNA (mtDNA) replication, transcription, and inheritance. Little is known about the mechanisms underlying its posttranslational regulation. Here, we demonstrate that TFAM is phosphorylated within its HMG box 1 (HMG1) by cAMP-dependent protein kinase in mitochondria. HMG1 phosphorylation impairs the ability of TFAM to bind DNA and to activate transcription. We show that only DNA-free TFAM is degraded by the Lon protease, which is inhibited by the anticancer drug bortezomib. In cells with normal mtDNA levels, HMG1-phosphorylated TFAM is degraded by Lon. However, in cells with severe mtDNA deficits, nonphosphorylated TFAM is also degraded, as it is DNA free. Depleting Lon in these cells increases levels of TFAM and upregulates mtDNA content, albeit transiently. Phosphorylation and proteolysis thus provide mechanisms for rapid fine-tuning of TFAM function and abundance in mitochondria, which are crucial for maintaining and expressing mtDNA.

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Figures

Figure 1
Figure 1. TFAM bound to DNA is resistant to Lon
(A) and (B) TFAM (80 nM) was incubated with Lon (50 nM) with or without ATP (2 mM) (A), or first preincubated with DNA oligonucleotides (4 μM) for 10 min on ice, prior to adding Lon (80 nM) and ATP (2 mM) (B); TFAM was detected by immunoblotting. (C-E) TFAM (1 pmol or as indicated) incubated with radiolabeled DNA (4 pmol) was analyzed by gel shift (C and D) or Southwestern (E) assays. Southwestern membranes were probed with radiolabeled DNA or immunoblotted for TFAM, Lon, the mitochondrial processing peptidase α subunit (MPPα), steroidogenic acute regulatory protein (StAR), or bovine serum albumin (BSA). (F) Lon (66 nM) was preincubated with or without DNA (4 μM) for 10 min before adding casein (3 μM) and ATP, and analyzed by SDS-PAGE and Coomassie Blue staining.
Figure 2
Figure 2. Lon knockdown in mtDNA-depleted cells increases TFAM and mtDNA
(A) Extracts from HeLa ρ+, ρ0 and ρlow cells were blotted for TFAM. Overexposure permits detection of TFAM in ρ0 and ρlow cells. A lower molecular weight TFAM band in ρ+ cells is likely a processed form or breakdown product. (B) Extracts from ρlow cells transfected with control, Lon or ClpP siRNAs were blotted for TFAM, Lon, ClpP and actin. (C) Total DNA was isolated from ρlow cells transduced with control or Lon shRNA lentivirus (5 MOI) and relative quantitation (RQ) of mtDNA was determined by qPCR of 7S DNA and CYTB gene using the nuclear APP gene as an endogenous control. Data represent at least three independent experiments. Standard error of the mean is shown. (D) Extracts from ρlow cells transduced as in (C) were immunoblotted for Lon, TFAM and actin.
Figure 3
Figure 3. Lon-dependent proteolysis of TFAM is blocked by bortezomib and MG262 but not epoxomicin
(A) and (B) Lon (200 nM monomer) or 20S (3 nM complex) peptidase activities were measured using the fluorescent dipeptide substrate AA2-Rh110 (6 μM) incubated in the presence or absence of bortezomib at 37°C for 3 hr. Fluorescence was normalized to percent activity of no drug control. Results represent at least 3 independent experiments. (C) ρlow cells were incubated with or without bortezomib for 18 hr and extracts were blotted for TFAM or p53. (D) and (E) ρlow, ρ0 or ρ+ cells were treated with DMSO, bortezomib (5 μM), MG262 (1.25 μM) or epoxomicin (1 μM) for 18 hr; extracts were blotted for TFAM, p53, Lon or actin. TFAM precursor (pre.) and mature (mat.) proteins are indicated.
Figure 4
Figure 4. HMG box mutation or PKA-dependent phosphorylation of TFAM leads to degradation by Lon
(A) Diagram of HMG box mutants of TFAM. (B) HeLa ρ+ cells were transfected twice with siRNAs on Day (D) = 0 and 2; on D=3, the cells were transfected with plasmids for expressing HMG1/2myc or TFAMmyc; and on D=4, cells were chased with cycloheximide (CHX, 100 μg/ml). Extracts were blotted for TFAM, Lon or actin. (C) ρ+ cells were transfected as in (B) and chased with CHX and bortezomib (5 μM) and blotted as in (B). (D) TFAM-DNA complex (PDB ID 3TMM) showing HMG box lysines 51, 52, 156 and arginine 157 (red); HMG box serines 55, 56, 61 and 160 (yellow); LSPLS (green); and LSPHS (blue). (E, F) Endogenous pTFAMser55 in ρ+ cells knocked down for Lon for 48 hr (E), or treated with or without the PKA inhibitor KT5720 or bortezomib (5μM) for 16 hr (F). (G) Fluorescent double labeling of Mitotracker Orange and overexpressed PKAα or mtsPKAα in ρ+ cells. (H) TFAMmyc coexpressed with mtsPKAα or PKAα in ρ+ cells treated with or without MG262 (1.25 μM) for 16 hr. Anti-TFAM immunoprecipitates were blotted for pTFAMser55, TFAM or PKA. (I) TFAMmyc and mtsPKAα were coexpressed in ρ+ cells and treated with MG262 (1.25 μM) in the presence or absence of H89 or KT5720 for 16 hr. (J) Trypsin-treated mitochondria coexpressing TFAM and mtsPKAα were incubated with [γ~32P]-ATP, then lysed (total lysate) and centrifuged (resulting in supernatant and pellet). The supernatant was immunoprecipitated for TFAM (bound and unbound). Samples were analyzed by SDS-PAGE and autoradiography. (*) Autophosphorylated PKAα coimmunoprecipitated with TFAM (Fig. S4A and H).
Figure 5
Figure 5. TFAM phosphorylation modulates DNA-binding, transcriptional activation and Lon-sensitivity
(A) Diagram of TFAM phosphomimic mutants at serines phosphorylated by PKA in vitro. (B) ρ+ cells transiently expressing wild type TFAM or phosphomimics were treated with DMSO, MG262 (1.25 μM), bortezomib (5 μM) or epoxomicin (1 μM); extracts were blotted with anti-myc antibodies. Actin controls are shown in Fig. S4A. (C) TFAM or TFAMSSDD were preincubated with or without biotinylated dsDNATFAM prior to incubation with streptavidin agarose. Pull-down and protein input were blotted for TFAM. (D) TFAM or TFAMSSDD (160 nM) were preincubated with or without dsDNATFAM (8 μM), before adding Lon (70 nM) and ATP (2 mM); reactions were blotted for TFAM. (E) Transcription reactions with TFAM or TFAMSSDD using the LSP promoter template with α32P-ATP. (F) TFAM (160 nM) was preincubated with a 300-fold molar excess of DNA prior to adding PKA (2500 U) and [γ~32P]-ATP (8 μCi) at 30°C for 2 hr. (G) [γ~32P]-labeled PKA phosphorylated TFAM was preincubated with or without dsDNATFAM before adding Lon as in (D), and visualized by autoradiography.
Figure 6
Figure 6. Phosphorylation potentially regulates multiple aspects of TFAM biogenesis and activity
Mitochondrial phosphorylation of TFAM- a, Phosphorylation of TFAM at HMG1 serines 55 and 56 by PKA regulates mtDNA binding and release. b, Phosphorylation of TFAM by PKA or other protein kinases may alter its interactions with other proteins. Cytosolic phosphorylation of TFAM precursor- c, Precursor phosphorylation may alter its degradation by the proteasome; or d, may alter its binding to the protein translocation machinery.
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