Negative regulation of mitochondrial VDAC channels by C-Raf kinase
© Le Mellay et al; licensee BioMed Central Ltd. 2002
Received: 13 February 2002
Accepted: 12 June 2002
Published: 12 June 2002
Growth of cancer cells results from the disturbance of positive and negative growth control mechanisms and the prolonged survival of these genetically altered cells due to the failure of cellular suicide programs. Genetic and biochemical approaches have identified Raf family serine/threonine kinases B-Raf and C-Raf as major mediators of cell survival. C-Raf cooperates with Bcl-2/Bcl-XL in suppression of apoptosis by a mechanism that involves targeting of C-Raf to the outer mitochondrial membrane and inactivation of the pro-apoptotic protein Bad. However, apoptosis suppression by C-Raf also occurs in cells lacking expression of Bad or Bcl-2.
Here we show that even in the absence of Bcl-2/Bcl-XL, mitochondria-targeted C-Raf inhibits cytochrome c release and caspase activation induced by growth factor withdrawal. To clarify the mechanism of Bcl-2 independent survival control by C-Raf at the mitochondria a search for novel mitochondrial targets was undertaken that identified voltage-dependent anion channel (VDAC), a mitochondrial protein (porin) involved in exchange of metabolites for oxidative phosphorylation. C-Raf forms a complex with VDAC in vivo and blocks reconstitution of VDAC channels in planar bilayer membranes in vitro.
We propose that this interaction may be responsible for the Raf-induced inhibition of cytochrome c release from mitochondria in growth factor starved cells. Moreover, C-Raf kinase-induced VDAC inhibition may regulate the metabolic function of mitochondria and mediate the switch to aerobic glycolysis that is common to cancer cells.
Anti-apoptotic Bcl-2 family members are overexpressed in a large number of cancers and are responsible for resistance to anticancer drugs . Oncogenic C-Raf contributes to the processes of cellular transformation and tumor development through its ability to suppress apoptotic cell death. Experimental evidence has been obtained, showing that C-Raf maintains cell survival by (i) induction of autocrine loops via the mitogenic cascade resulting in the activation of protein kinase B (PKB) [5, 6], and (ii) cooperation with Bcl-2, which targets C-Raf to mitochondria and results in the inactivation of the pro-apoptotic protein Bad [3, 7, 8]. Additionally, Raf may suppress apoptosis by activation of NF-κB [9, 10], and interaction with IAP proteins (Sendtner, Troppmair and Rapp, unpublished results). The anti-apoptotic Bcl-2 family members expressed in the outer mitochondrial membrane inhibit the release of apoptogenic cytochrome c from mitochondria [11, 12], antagonize the pore-forming activity of Bax , and directly interact with the mitochondrial permeability transition pore (PTP) that regulates mitochondrial membrane potential [14, 15]. C-Raf induces phosphorylation of Bad, which in turn leads to sequestration of Bad in a complex with 14-3-3 in the cytosol [3, 16]. However, C-Raf also suppresses apoptosis in cells that do not express Bad or are deficient for Bcl-2  suggesting the existence of other targets for C-Raf at the outer mitochondrial membrane. Here we provide evidence that C-Raf regulates the mitochondrial voltage-dependent anion channel (VDAC) that can contribute to cell survival.
Results and Discussion
C-Raf has been demonstrated previously to interact with members of the Bcl-2 family that regulate traffic of apoptogenic proteins through the outer mitochondrial membrane. Here we identify the outer mitochondrial channel protein VDAC as binding partner and effector of C-Raf. We propose that this interaction may be responsible for the Raf-induced inhibition of cytochrome c release. Moreover, C-Raf kinase-induced VDAC inhibition may regulate the metabolic function of mitochondria and mediate the switch to aerobic glycolysis that is common to cancer cells.
Materials and Methods
Human Bcl-2 cDNA encoding a protein lacking the COOH-terminal transmembrane domain (TM) (Bcl-2ΔC21: residues 219–239 in Bcl-2). cDNA coding for GST-Bcl-2ΔTm was expressed in pGEX-4T1 plasmid (gift of J. Reed). GST-C-RafYY340/341DD (active form of Raf), GST-C-RafK375W (inactive form of C-Raf), His-PAK1-T423E and His-LckY505F were cloned in pFastBac baculoviruses for expression in Sf9 insect cells.
Human VDAC1 cDNA was released from the pSK plasmid by BamHI and SalI restriction enzymes and subcloned into the BamHI/EcoRV sites of pTracer-CMV plasmid to produce a HA-tag fusion protein. cDNAs coding for Mas-BXB, BXB or C-Raf were expressed in the pcDNA-3 expression plasmid (Invitrogen).
Mouse promyelocytic 32Dcl.3 cells were stably transfected with pcDNA3 empty vector or pcDNA3-encoding active Mas-BXB or pcDNA-3-encoding inactive Mas-BXB K375W as previously described . 293 cells were transiently transfected by a calcium phosphate method with pTracer-CMV-encoding HA-VDAC1 and/or pcDNA3 encoding C-Raf.
For FACS analysis 32Dcl.3 cells lines were maintained in IL-3 deprived RPMI medium with 10% serum for 24 h. Cells were incubated with 7,5 μg/ml JC-1 for 15 min at room temperature, washed with PBS and subjected to FACS analysis.
Cell fractionation and immunoblotting
32Dcl.3 cells and transfectants were maintained in IL-3 deprived RPMI complete medium for 12 to 24 h. Cells were washed three times in phosphate-buffered saline solution and frozen at -70°C. Cell pellets were homogenized in buffer (10 mM Hepes pH 7. 4, 0,3 M mannitol, 1 mM EGTA) containing protease inhibitors and centrifuged two times at 600 g for 5 minutes at 4°C. After a further centrifugation at 12 000 × g for 15 minutes at 4°C, cell pellets contained-mitochondria enriched fraction were solubilised in the same buffer and a cytosolic fraction was obtained after centrifugation at 100000 × g for 1 h at 4°C. Proteins were resuspended in Laemmli buffer, applied to SDS-PAGE, transferred to a nitrocellulose membrane and analysed as described previously .
48 hours after transfection cells were lysed in NP-40 buffer (10 mM Hepes pH 7.4, 145 mM KCl, 5 mM MgCl2, 1 mM EGTA, 0,5 % NP-40) containing protease inhibitors. After centrifugation cell lysates were successively incubated with anti-hemagglutinin antibody (12CA5) or anti-C-Raf antibody and with protein A/G agarose beads. Immunoprecipitates were washed with NP-40 buffer, resuspended in Laemmli buffer and separated by SDS-PAGE. The proteins were transferred to a nitrocellulose membrane and analysed as described previously .
HA-VDAC was immunoprecipitated from transfected 293 cells and incubated with GST-C-Raf recombinant proteins in kinase buffer (25 mM Hepes, pH 7,4, 150 mM NaCl, 25 mM β-glycerophosphate, 1 mM DTT, 10 mM MgCl2) containing 50 μM ATP and 10 μCi [32P]-γ ATP. Alternatively, GST-C-Raf was incubated with MEK as substrate instead of HA-VDAC. Phosphorylation of HA-VDAC or MEK was analysed by autoradiography. For the in vivo kinase assay transfected 293 cells were grown in DMEM medium with 0,3% serum for 24 h. Cells were cultured in phosphate free medium in the presence of 0,75 mCi [32P] for 2 h before HA-VDAC immunoprecipitation.
GST-C-RafYY340/341DD, GST-C-RafK375W, His-PAK1-T423E and His-LckY505F were produced in Sf9 insect cells and purified as described . Escherichia coli BL21 DE3(pLysS) were transformed with pGEX plasmids. After induction with isopropyl-β-D-thiogalactopyranoside (IPTG) GST-Bcl-2ΔTm protein was prepared from the bacterial soluble fraction incubated with glutathione-sepharose. To remove the GST-tag from Bcl-2ΔTm protein, beads were washed with lysis buffer (50 mM Tris, pH 8, 1 mM EDTA, 10 mM β-mercaptoethanol) containing 0,1 % Triton-X100 and 300 mM NaCl and incubated overnight at 4°C in buffer (50 mM Tris pH 8, 20 mM β-mercaptoethanol) containing thrombin (Sigma). Cleaved Bcl-2ΔTm was further purified on anion exchange Resource Q column (Pharmacia-Amersham) and the protein was eluted in a NaCl gradient. VDAC was purified from rat liver as described .
Lipid bilayer experiments
The channel-forming proteins were reconstituted into artificial lipid bilayer membranes in a teflon chamber [21, 28]. The membrane was formed from a 1% (w/v) solution of diphytanoylphosphatidylcholine (DiphPC) (Avanti Polar Lipids, Alabaster, AL) in n-decane. VDAC or Bcl-2ΔTm proteins were added to the KCl buffer in both compartments of the chamber and the single-channel conductance of the pores was measured after application of a fixed membrane potential. To test the effect of C-Raf kinase on VDAC or Bcl-2ΔTm-formed channels, GST-C-RafYY340/341DD, GST-C-RafK375W or GST alone were incubated with channel-forming proteins in kinase buffer in presence of ATP for 30 min at 30°C. Samples were applied on both sides of the DiphPC membrane in KCl buffer and single-channel formation was measured. As a control GST, GST-C-RafYY340/341DD and GST-C-RafK375W were checked to not be able to form channels in artificial bilayer membranes. In control experiments VDAC was first added to an artificial membrane. After reconstitution of several channels active and inactive C-Raf were added to the aqueous phase and their effect on VDAC was investigated.
The authors want to thank Drs. John Reed and Viktor Wixler for providing reagents. We are thankful to Ludmilla Wixler, Heike Hamm and Renate Metz for technical help and to Bruce Jordan, Rudolf Götz, Ulrike Rennefahrt and Thomas Twardzik for critical comments on the manuscript.
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