Heat shock protein 70-mediated sensitization of cells to apoptosis by Carboxyl-Terminal Modulator Protein
- Longzhen Piao†1, 9,
- Yuwen Li†1,
- Keum-Jin Yang1,
- Kyeong Ah Park1,
- Hee Sun Byun1,
- Minho Won1,
- Janghee Hong1,
- Jeong-Lan Kim2,
- Gi Ryang Kweon3,
- Gang Min Hur1,
- Jeong Ho Seok1,
- Jae Youl Cho4,
- Taehoon Chun5,
- Daniel Hess6,
- Ragna Sack6,
- Sauveur-Michel Maira7,
- Derek P Brazil8,
- Brian A Hemmings6 and
- Jongsun Park1Email author
© Piao et al; licensee BioMed Central Ltd. 2009
Received: 31 March 2009
Accepted: 15 July 2009
Published: 15 July 2009
The serine/threonine protein kinase B (PKB/Akt) is involved in insulin signaling, cellular survival, and transformation. Carboxyl-terminal modulator protein (CTMP) has been identified as a novel PKB binding partner in a yeast two-hybrid screen, and appears to be a negative PKB regulator with tumor suppressor-like properties. In the present study we investigate novel mechanisms by which CTMP plays a role in apoptosis process.
CTMP is localized to mitochondria. Furthermore, CTMP becomes phosphorylated following the treatment of cells with pervanadate, an insulin-mimetic. Two serine residues (Ser37 and Ser38) were identified as novel in vivo phosphorylation sites of CTMP. Association of CTMP and heat shock protein 70 (Hsp70) inhibits the formation of complexes containing apoptotic protease activating factor 1 and Hsp70. Overexpression of CTMP increased the sensitivity of cells to apoptosis, most likely due to the inhibition of Hsp70 function.
Our data suggest that phosphorylation on Ser37/Ser38 of CTMP is important for the prevention of mitochondrial localization of CTMP, eventually leading to cell death by binding to Hsp70. In addition to its role in PKB inhibition, CTMP may therefore play a key role in mitochondria-mediated apoptosis by localizing to mitochondria.
Protein kinase B (PKB/Akt) is activated by receptor tyrosine kinases and regulates cell proliferation, survival, and motility [1, 2]. PKB activation occurs when PtdIns[3–5]P3 (a product of PI3K) binds to the pleckstrin homology domain of PKB. Phosphorylation of two amino acids (Thr308 and Ser473) is then required for full PKB activation [3, 4]. Unphosphorylated PKB is inactive, but PKB phosphorylation on Thr308 (by PDK1) stimulates PKB activity by approximately 100-fold . Phosphorylation on a second regulatory site at the carboxyl terminus (Ser473; termed the hydrophobic motif) by rictor-mTOR and DNA-PK can further activate PKB seven- to ten-fold ([5–7]. The crystal structure of PKB suggests that Ser473 phosphorylation is important for kinase activation and stabilization [8, 9].
Recently, C-terminal modulator protein (CTMP) was identified as a PKB binding partner . CTMP overexpression inactivates PKB in v-Akt-transformed cells transplanted into mice , in cultured cells , and in a K-ras-induced lung cancer model . The tumor suppressor-like properties of CTMP are supported by a report demonstrating inhibition of CTMP expression by hypermethylation of its promoter in malignant glioblastomas, where PKB activity is frequently altered .
Mitochondria regulate cellular energy supplies, apoptosis, and signaling pathways. Alterations in mitochondrial function are responsible for a range of inherited and acquired human diseases and are implicated in the aging process . Cytosolic mitochondrial protein precursors contain information that is necessary to direct them to the mitochondria. Mitochondrial precursor proteins in the cytosol are present as complexes with factors that stabilize them, as they are prone to degradation and aggregation. Several such factors are implicated as cytosolic chaperones; however, convincing data exist only for heat shock protein 70 (Hsp70) and Hsp90 [15, 16].
Here, we describe CTMP phosphorylation on two sites following treatment with pervanadate, an insulin-mimetic. Surprisingly, CTMP C-terminally tagged with GFP was localized to mitochondria, whereas CTMP N-terminally tagged with GFP was mainly found in the cytoplasm. Consistent with this observation, mitochondrial localization of endogenous and exogenous CTMP has recently been reported  while this study was in review. Mitochondrial localization of CTMP was dependent on an N-terminal mitochondrial targeting sequence (MTS) and was inhibited by phosphorylation on Ser37/Ser38. Finally, CTMP overexpression sensitizes the cell to apoptosis by sequestering Hsp70 away from apoptotic protease activating factor 1 (Apaf-1), suggesting that CTMP is involved in apoptotic processes through its mitochondrial localization and binding to Hsp70.
Identification of phosphorylated residues of CTMP in pervanadate-stimulated CCL64 cells
CTMP is localized to the mitochondrial intermembrane space and/or matrix
Mitochondrial targeting sequence-mediated mitochondrial localization of CTMP is inhibited by phosphorylation event
CTMP overexpression sensitizes the cell to apoptosis induced by staurosporine
To evaluate the apoptotic role of CTMP, we overexpressed CTMP in HeLa cells for 24 h and subsequently treated the cells with 1 μM staurosporine for the indicated times. Stauroporine-mediated apoptosis (assessed by PARP cleavage and Caspase 3/9 activation) was detected at 3 h of treatment (Figure 3D–3F, data not shown). Apoptosis was more pronounced in CTMP-transduced compared to controls, suggesting that CTMP overexpression increases the sensitivity of cells to programmed cell death.
CTMP-mediated Hsp70 sequestration leads to the dissociation of Hsp70 and Apaf-1
Mitochondria are essential organelles in most eukaryotic cells and function in the maintenance of cellular energy supplies. Mitochondria are responsible for thermoregulation and synthesis of essential molecules . In addition, mitochondria also participate in key signaling events regulating cell proliferation and death . PKB has a critical role in regulating apoptosis [28–30] and directly phosphorylates and interacts with key factors involved apoptosis signaling [30, 31]. PKB also indirectly regulates apoptotic transcription factors such as FOXO3a and NFκB . Additionally, activated PKB is localized to various subcellular compartments including the mitochondria and nucleus . Therefore, there is a complex interplay between PKB signaling and mitochondria-mediated apoptosis.
We provide the first evidence that CTMP, a negative regulator of PKB, localizes to the mitochondria in a MTS-dependent manner (Figure 3A and 3B). We found CTMP is located at the mitochondrial intermembrane space and/or matrix (Figure 2D–2F). In addition, we discovered CTMP is phosphorylated on Ser37/Ser38 (Figure 1) and phosphorylation on these residues inhibited CMTP mitochondrial localization (Figure 3C). Furthermore, adenovirus-mediated CTMP overexpression sensitized cells to staurosporine-stimulated apoptosis (Figure 3D–3F). Interestingly CTMP interacts with Hsp70, resulting the sequestration of Hsp70 from Apaf-1 (Figure 4). Taken together, these data suggest CTMP is a novel mitochondrial protein and is involved in apoptosis (Figure 4D).
Evidence suggest that CTMP negatively regulates PKB activity in v-Akt transformed cells , ciliary ganglion neurons , and K-ras-induced lung cancer model . This observation is further supported by a recent report showing epigenetic down-regulation of CTMP transcription in malignant glioblastomas . As previously suggested , CTMP is phosphorylated in vivo in response to pervanadate stimulation (Figure 1). In the current study, two serine residues on CTMP that are phosphorylated in response to pervanadate in vivo were identified (Figure 1). Ser37/Ser38 is located at the amino-terminus of CTMP, close to the putative MTS, as predicted by MitoProt II 1.0a4 . These two seine residues are conserved in CTMP homologues from mouse (NP_03707.1), rat (NP_001020188.1) and dog (NP_001074103.1) but lower organisms such as D. melanogaster, S. cerevisiae, S. pombe or C. elegans do not appear to contain a gene for CTMP, suggesting CTMP is a relatively recent protein during evolution and the two identified serine residues might be important for its function. In addition, Ser37 was predicted by the motif prediction program (Prosite) to be phosphorylated by Casein kinase II. However, this possibility needs to be experimentally evaluated in the context of apoptosis and will provide detailed insight into CTMP regulation.
CTMP was detected in the membrane and cytosol fraction of LN229 cells . Furthermore, two molecular species corresponding to CTMP were observed in different cell lines, suggesting post-translational modifications. In addition, CTMP associated with intracellular structures similar to membrane ruffles . Recently, a nuclear pool of CTMP was detected in human pancreatic duct epithelial cells . Consistent with recent reports , a mitochondrial pool of CTMP from HEK 293 cells dependent on its MTS was detected in our experiments (Figure 2 and 3). Furthermore, this mitochondrial CTMP localization was inhibited by phosphorylation (Figure 3C). Therefore, CTMP may have distinct roles in various subcellular compartments.
Mitochondrial localization is mediated by the MTS. In most cases, the MTS is present as a cleavable sequence at the N terminus, also called a pre-sequence . Bioinformatic analysis of the CTMP sequence indicated that a potential MTS exists in CTMP at the N-terminus of protein. This predicted N-terminal MTS appears to be functional since an N-terminal 31-amino acid deletion-mutant of CTMP did not localize to the mitochondria (Figure 3A and 3B). N-terminal MTS of mitochondrial precursors are in most cases cleaved by the mitochondrial-processing peptidase (MPP) as soon as the cleavage sites reach the mitochondrial matrix [34, 35]. Mutational studies show the R-2 or R-3 motif are important but not sufficient to direct cleavage by MPP . Site-directed mutagenesis of the -2 or -3 arginine in different precursor molecules completely or partially inhibits processing [36, 37]. Further studies are needed to interrogate if the R-2 motif of CTMP is important in MTS-mediated mitochondria localization.
The molecular pathways that mediate apoptosis are tightly regulated by a series of positive and negative signals, the balance of which determines whether or not cells commit suicide . Recent evidence indicates that the coordinated interaction between Hsps and the components of apoptosis machinery may determine cellular susceptibility to damaging stresses . Indeed, CTMP associate specifically Hsp70, but not Hsp90, in HeLa cells (Figure 4A and 4B). Furthermore, complex formation of Hsp70 and Apaf-1 was decreased in CTMP-infected cell compared to controls (Figure 4C), indicating why CTMP overexpression sensitized the cell to apoptosis induced by staurosporine (Figure 4D). Indeed, Miyawaki and colleagues recently reported that upregulation of CTMP in hippocampal neurons is required for ischemia-induced neuronal death . Based on previous studies ([10, 12, 40], CTMP phosphorylation event occurs at the plasma membrane in response to activation of growth factor signaling, leading to the release of PKB. In the case of apoptosis, phosphorylation of the mitochondria pool of CTMP by as-yet-unidentified kinase will promote the exclusive cytoplasmic localization of CTMP, resulting the sequestration of Hsp70 from Apaf-1. One can also proposed that 14-3-3 may bind the phosphorylated CTMP and maintain it's cytoplasmic localization. In this regard, it will be of course interesting to discover how phosphorylation affects the interaction between CTMP and Hsp70. Further studies are required to elucidate mechanisms of CTMP-dependent apoptosis under pathophysiological conditions.
In summary, this work provides the first evidence for the involvement of phosphorylation in the MTS-mediated mitochondrial localization of CTMP. In addition to its role in PKB inhibition, CTMP has a key role in mitochondria-mediated apoptosis by localizing to mitochondria. Whether phosphorylation of these serine residues affects CTMP protein-protein interactions remains unknown. Further analysis is likely to elucidate additional CTMP regulatory mechanisms, which will provide a better understanding of its role in signaling and its potential in therapeutic applications.
The anti-CTMP antibody used was described previously . Antibodies were purchased from the following companies: anti-AIF and anti-COX IV (Cell Signaling), anti-GFP (Santa Cruz Biotech), anti-PARP (BD Biosciences), anti-Flag (Sigma), HRP-conjugated anti-mouse IgG (Calbiochem), HRP-conjugated anti-rabbit IgG (Calbiochem), and HRP-conjugated anti-sheep IgG antibody (Sigma). Staurosporine and sodium orthovanadate (Na3VO4) were purchased from Sigma.
Construction of expression vectors
GFP-N-terminal-tagged CTMP (GFP-NT-CTMP) was previously reported . GFP-C-terminal-tagged CTMP (GFP-CT-CTMP) was subcloned into pEGFP-N3 (Clonetech). CTMP deletion mutants were constructed in pEGFP-N1 vector (GFP-CT-CTMP-ΔN31). Ser37/Ser38 negatively charged side group-mimic mutants (S37D/S38D) were created by using the QuikChange Site-Directed Mutagenesis Kit (Stratagene), using pEGFP-N3-CTMP as a template. An adenoviral CTMP vector in pENTR-3C was prepared using the Adenoviral Expression Kit (Invitrogen). Adenoviruses were purified as described previously . Constructs were confirmed by DNA sequencing. Mutagenic and cloning oligonucleotide sequences are available upon request.
Cell culture and stimulation
U2OS, CCL64, HeLa, and HEK 293 cells were maintained in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% FBS, 2 mM glutamine, 100 unit/ml penicillin, and 100 μg/ml streptomycin (Life Technologies) and were transfected using FuGene 6 (Roche), jetPEI (Q-biogene), or Lipofectamine (Invitrogen) reagent. The transfection mixture was removed after 24 h and cells were serum-starved for 16 h before stimulation for 15 min with 100 μM pervanadate, prepared in 0.2 mM H2O2 . To induce apoptosis, cells were treated with 1 mM staurosporine for the indicated time.
In vivo labeling of stable CCL64 cells expressing wild type Flag-CTMP and phosphoamino acid analysis
Metabolic labeling of CCL64 cells was performed as described previously, with minor modifications . Briefly, cells were stimulated with buffer or 100 μM pervanadate for 15 min. The 32P-labeled band corresponding to Flag-CTMP was excised, reduced, alkylated, and cleaved with 1 μg of trypsin (Promega, sequencing grade). Phosphoamino acids were identified following hydrolysis in 6 M HCl containing 0.1 mg/ml bovine serum albumin at 110°C for 60 min. Hydrolysates were separated by thin layer electrophoresis at pH 3.5 to resolve phosphoserine (pS), phosphothreonine (pT), and phosphotyrosine (pY) . Radioactivity was detected using a PhosphoImager (Molecular Dynamics).
Phosphorylation site mapping by mass spectrometry
Extracted peptides were analyzed by high-performance liquid chromatography (HPLC) interfaced with electrospray ionization mass spectrometry (ESI-MS), using a Rheos 4000 chromatograph equipped with a 1 × 250 mm Vydac (Hesperia) C18 column and interfaced with a Sciex API 300 mass spectrometer (PE Sciex), operated in the single quadrupole mode on Q1. Analysis of mass spectra of phosphopeptides was performed as described previously . Mass spectra were acquired on a Sciex API 300 triple quadrupole mass spectrometer equipped with a NanoESI source (Protana).
Confocal imaging analysis of CTMP localization
U2OS cells were grown on glass coverslips and transfected with GFP-CTMP or pDsRed-Mito, a mitochondrial marker. After 24 h, cells were fixed in 4% paraformaldehyde at room temperature for 10 min, mounted with Vectashield (Vector Laboratories) and visualized using a OLYMPUS 510 confocal microscope. Differential localization of CTMP (Cyt: cytoplasm, PM: plasma membrane, Mit: mitochondria, Nuc: Nucleus) was examined using confocal microscopy. At least 200 cells were counted from three distinct fields for each transfected group.
Preparation of mitochondria and plasma membranes
Cells were washed with PBS and resuspended in mitochondrial fraction buffer (20 mM HEPES, pH 8.0, 10 mM KCl, 1.5 mM MgCl2, 1 mM EDTA, 250 mM sucrose, 1 mM PMSF, 10 g/ml leupeptin, 10 g/ml aprotinin, and 0.2 mM sodium orthovanadate) for 30 min on ice and then homogenized. Unbroken cells and nuclei were removed. The supernatant was centrifuged at 10,000 × g for 30 min at 4°C. The resultant supernatant, representing the cytosolic fraction, was centrifuged at 100,000 × g for 1 h at 4°C. The resultant pellet was washed with 500 μl of mitochondrial fraction buffer and solubilized in lysis buffer to generate the mitochondrial fraction. Isolated mitochondrial fractions were further treated with 2 M sodium chloride, 100 mM sodium carbonate (pH 11.2), or 1% Triton X-100 for 30 min. Samples were ultracentrifuged at 100,000 × g for 1 h to separate the supernatant (S) and precipitate (P) fractions.
We would like to thank P. Cron (FMI, Switzerland) for anti-CTMP antibodies. This work was supported by grants from the Korean government (Ministry of Education, Science and Technology: National Research Foundation of Korea grant (No. 2009-0062916); Ministry for Health, Welfare and Family Affairs: National R&D Program for Cancer Control (No. 0720560)). D. Brazil is supported by Science Foundation Ireland. FMI is part of the Novartis Research Foundation.
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