Tyrosine phosphorylation of mitochondrial pyruvate dehydrogenase kinase 1 is important for cancer metabolism

Abstract

Many tumor cells rely on aerobic glycolysis instead of oxidative phosphorylation for their continued proliferation and survival. Myc and HIF-1 are believed to promote such a metabolic switch by, in part, upregulating gene expression of pyruvate dehydrogenase (PDH) kinase 1 (PDHK1), which phosphorylates and inactivates mitochondrial PDH and consequently pyruvate dehydrogenase complex (PDC). Here we report that tyrosine phosphorylation enhances PDHK1 kinase activity by promoting ATP and PDC binding. Functional PDC can form in mitochondria outside of the matrix in some cancer cells and PDHK1 is commonly tyrosine phosphorylated in human cancers by diverse oncogenic tyrosine kinases localized to different mitochondrial compartments. Expression of phosphorylation-deficient, catalytic hypomorph PDHK1 mutants in cancer cells leads to decreased cell proliferation under hypoxia and increased oxidative phosphorylation with enhanced mitochondrial utilization of pyruvate and reduced tumor growth in xenograft nude mice. Together, tyrosine phosphorylation activates PDHK1 to promote the Warburg effect and tumor growth.

Figure 1. FGFR1 directly phosphorylates and activates PDHK1

(A) Schematic representation of PDHK1. Three identified FGFR1-direct tyrosine phosphorylation sites are shown, including Y136, Y243 and Y244. (B) Active recombinant FGFR1 (rFGFR1) directly phosphorylates purified GST-tagged PDHK1 at tyrosine residues in an in vitro kinase assay. Phosphorylated rFGFR1 and GST-PDHK1 were detected by a specific phospho-Tyr antibody (pY99). (C) Purified GST-PDHK1 from cells co-expressing FGFR1 wild type (WT), but not an FGFR1 kinase dead form (KD) shows increased kinase activity in an in vitro PDHK1 kinase assay using recombinant pyruvate dehydrogenase A (rPDHA) as a substrate (upper). Western blot revealed that GST-PDHK1 was tyrosine phosphorylated in cells with co-expression of FGFR1 WT but not KD mutant (lower). (D) Inhibition of FGFR1 by TKI258 (2 hours) results in decreased tyrosine phosphorylation levels of PDHK1 in cells co-expressing FGFR1 WT (left). Cells co-expressing FGFR1 KD and GST-PDHK1 were included as a negative control. Western blot result shows that FGFR1 is co-localized with PDHK1 and its substrate PDHA1 in mitochondria (right). (E) Inhibition of FGFR1 by TKI258 (2 hours) results in decreased S293 phosphorylation levels of PDHA1 in leukemia KG1a cells (FOP2-FGFR1) (left) and lung cancer H1299 cells (FGFR1) (right). (F) Left: Targeting PDHK1 by the PDHK inhibitor DCA results in decreased S293 phosphorylation levels of PDHA1 in KG1a and H1299 cells. Right: Inhibition of PDHK1 by specific shRNA decreases S293 phosphorylation levels of PDHA1 in H1299 cells. The numbers represent the relative intensity of the specific bands of PDHK1 (upper) or phospho-PDHA1 (S293; lower), which are normalized to the value of control sample without treatment with human PDHK1 (hPDHK1) shRNA.

Figure 2. FGFR1-dependent tyrosine phosphorylation activates PDHK1 by promoting ATP and PDC binding

(A) GST-PDHK1 proteins were pulled down by beads and treated with protein tyrosine phosphatase (PTP), followed by treatment with activated rFGFR1. The beads were then incubated with recombinant PDHA1 in an in vitro kinase assay. S293 phosphorylation of PDHA1 was detected by immunoblotting (left). Right panel represents the relative intensity of the specific bands of phospho-PDHA1 E1α, which are normalized to the value of control sample (GST-PDHK1 WT) without treatment with rFGFR1. (B) Cartoon representation of PDHK1 structure (PDB ID: 2Q8G) (Kato et al., 2007) made using Pymol (www.pymol.org). Y243 and Y244 are proximal to the ATP lid (in blue), and predicted location of ATP binding (modeled on the AMP-PNP-bound dimeric PDHK4 structure PDB ID: 2E0A in grey). Y136 is more proximal to AZD7545 (in green), which is a PDHK1 inhibitor that disrupts formation of functional pyruvate dehydrogenase complex (PDC). (C) Phosphorylation at Y243 or Y244, but not Y136 by FGFR1 leads to increased ATP binding to PDHK1. GST-PDHK1 variants were pulled down by beads from transfected 293T cell lysates and incubated with active rFGFR1, followed by incubation with [α-³²P]ATP. The unbound [α-³²P]ATP was washed away. Retained [α-³²P]ATP on PDHK1 was measured with a scintillation counter. (D-E) FGFR1-dependent phosphorylation at Y136 (D), Y243 and Y244 (E) promotes PDHK1 binding to PDC. GST-PDHK1 variants were pulled down by beads and incubated with active rFGFR1, followed by incubation with whole cell lysates of 293T cells. The bound PDC E2 protein and PDHA1 E1α proteins were detected by immunoblotting. Upper panel shows immunoblotting results from one representative experiment. Lower panel shows summarized results from two independent experiments that represent the relative intensity of the specific bands of PDHA1 E1α and PDC E2 that bind to PDHK1, which are normalized to the value of control sample without treatment with rFGFR1. The error bars represent mean values +/- SD.

Figure 3. FGFR1 phosphorylates mitochondrial PDHK1 in cancer cells

(A-B) PDHK1 Y243 is specifically phosphorylated by rFGFR1 in an in vitro kinase assay (A) and in cells expressing FGFR1 (B), detected by an antibody that specifically recognizes phospho-Y243 of PDHK1. (C) Immunoblotting shows that targeting FGFR1 by TKI258 in lung cancer H1299 cells (left) or leukemia KG1a cells (right) decreases phosphorylation of PDHK1 Y243. (D) Knockdown of FGFR1 by lentiviral shRNA for 48 hours in H1299 cells results in decreased phosphorylation levels of PDHK1 Y243 and PDHA1 S293. (E) Western blot result shows that FGFR1 is co-localized with PDHK1 and its substrate PDHA1 in mitochondria in KG1a (left) and H1299 (right) cells. (F) Immunoblotting result shows that FGFR1 is localized in mitochondria, with mitochondrial COX IV and cytosolic β-actin as control markers in KG1a (left) and H1299 (right) cells. (G) Immunofluorescence assay reveals that FGFR1 is co-localized with mitochondrial marker MitoTracker® Mitochondrion-Selective dyes, but not with nuclear marker DAPI in H1299 cells (upper left). Knockdown of FGFR1 by shRNA (right panel) leads to decreased mitochondrial localization of FGFR1 (lower left).

Figure 4. FGFR1 is localized to outer membrane of mitochondria and functional PDC can form in mitochondria outside of matrix in cancer cells

(A) Mitochondrial localization of FGFR1 in H1299 cells under transmission electron microscopy (TEM). Left panel shows the control without primary antibody. Middle panel shows the localization of FGFR1 in mitochondria of H1299 cells, with arrows indicating representative staining of mitochondrial FGFR1. Right panel shows the control with FGFR1 knockdown by shRNA. Scale bars: 0.5 μm. (B) Localization of full length FGFR1 and FOP2-FGFR1 fusion tyrosine kinase in the outer membrane of mitochondria in lung cancer H1299 and leukemia KG1a cells, respectively. Tom40, Cyto C, complex I 39-kDa protein, and MnSOD are markers for mitochondrial outer membrane (Om), inter-membrane space (IMS), inner membrane (Im), and matrix (Ma), respectively. (C) Proteinase K protection experiment where isolated mitochondria were treated with proteinase K in the presence and absence of Triton X-100, followed by Western blot. (D-E) Left: Activity of PDC-mediated conversion of ¹⁴C-labeled pyruvate into ¹⁴C-labeled CO₂ was detected in the mitochondrial subfractions including Om, IMS and Ma of H1299 cells (D) and KG1a cells (E). The error bars represent mean values +/- SD. Right: Western blotting results show expression levels of PDHA1 (E1), E2, E3 and E3BP in indicated mitochondrial subfractions. Tom40, Cyto C and MnSOD were included as markers.

Figure 5. Y243 of PDHK1 is commonly phosphorylated by oncogenic tyrosine kinases localized to mitochondria in cancer cells

(A) Immunoblotting detected Y243 phosphorylation of PDHK1 in diverse cancer cells. Western blot results of phosphorylation levels of PDHA1 at S293 are shown. (B-D) Active, recombinant ABL (B), JAK2 (C) and FLT3 (D) directly phosphorylate PDHK1 at Y243 in corresponding in vitro kinase assays. (E) Immunoblotting shows that targeting BCR-ABL by imatinib in K562 cells (left), JAK2 by AG490 in HEL cells (middle) or FLT3-ITD by TKI258 in Mv4;11 cells (right) decreases phosphorylation of PDHK1 Y243. (F) Immunoblotting results show that BCR-ABL (left), JAK2 (middle) and FLT3 (right) are localized in mitochondria with mitochondrial COX IV and cytosolic MEK1 as control markers in leukemia K562 (BCR-ABL), HEL (JAK2), and Molm14 (FLT3) cells, respectively. BCR-ABL and JAK2 but not FLT3 were also detected in the cytoplasm. Integrin β1 and PARP are markers for plasma membrane and nucleus, respectively. (G) Sub-fractionation results show that BCR-ABL and JAK2 are localized in the mitochondrial matrix in K562 and HEL cells (left and middle, respectively), while FLT3 is localized in the mitochondrial outer membrane in Molm14 cells (right). Tom40, Cyto C, complex I 39-kDa protein, and MnSOD are markers for mitochondrial outer membrane (Om), inter-membrane space (IMS), inner membrane (Im), and matrix (Ma), respectively.

Figure 6. Expression of catalytically less active mouse PDHK1 mutants, including Y134F and Y239/240F, in H1299 cells leads to decreased proliferation under hypoxia and increased oxidative phosphorylation

(A) Immunoblotting shows shRNA-mediated stable knockdown of endogenous human PDHK1 (hPDHK1) in H1299 cells using lentiviral transduction, and rescue expression of Flag-tagged mouse PDHK1 (mPDHK1) proteins including WT, Y134F mutant and Y239/240F double mutant. Y134, Y239 and Y240 of mPDHK1 are corresponding to Y136, Y243 and Y244 of hPDHK1, respectively. (B) Rescue expression of mPDHK1 Y134F or Y239/240F mutants in H1299 cells results in reduced cell proliferation rate under hypoxic conditions (1% oxygen) but not at normal oxygen tension (normoxic; 17% oxygen) compared to cells expressing mPDHK1 WT. Cell proliferation was determined by the cell number of each cell line 48 and 96 hours after seeding (n=3; *: 0.01<p<0.05). The error bars represent mean values +/- SD. (C-E) Y134F and Y239/240F rescue cells have a significantly elevated oxygen consumption rate (C), an increased intracellular ROS production under normoxia (D), and a significantly reduced lactate production under normoxia (E), compared to cells expressing mPDHK1 WT. Media were incubated with cells for 1 hour prior to lactate assay. The error bars represent mean values +/- SD. (F-G) Treatment with oligomycin resulted in increased inhibition of ATP production (F) and decreased proliferation (G) in Y134F and Y239/240F rescue cells, compared to cells expressing mPDHK1 WT. The error bars represent mean values +/- SD. (H-I) Isolated mitochondria from rescue cells expressing mPDHK1 Y239/240F show significantly increased rates of pyruvate consumption (H) and PDC-mediated conversion of ¹⁴C-labeled pyruvate into ¹⁴C-labeled CO₂ (I), compared to mitochondria isolated from WT rescue cells. The error bars represent mean values +/- SD.

Figure 7. Phosphorylation-dependent activation of PDHK1 is mediated through phosphorylation and inactivation of PDHA1, which is important for tumor growth in xenograft nude mice

(A) Expression of PDHA1 S293D mutant, but not PDHA1 WT or S293A (lower), resulted in a significant decrease in O₂ consumption in H1299 rescue cells of mPDHK1 Y239/240F but not in WT rescue cells (upper). (B-E) Expression of PDHA1 WT, S293A or S293D mutant in rescue cells of mPDHK1 Y239/240F did not affect cell proliferation under normoxia (B; left); however, expression of PDHA1 S293D mutant, but not WT or S293A mutant resulted in decreased sensitivity of mPDHK1 Y239/240F rescue cells to hypoxia in regard to cell proliferation (B; right), increased lactate production (C) and decreased sensitivity to the treatment with oligomycin in regard to ATP levels (D) and cell proliferation (E). The error bars represent mean values +/-SD. (F) Left: Dissected tumors (indicated by red arrows) in a representative nude mouse (#1743) injected with mPDHK1 WT H1299 cells on the left flank and Y239/240F H1299 cells on the right flank are shown. Right: Expression of Flag-mPDHK1 WT and Y239/240F, as well as phosphorylation levels of PDHK1 and PDHA1 in tumor lysates. (G) Y239/240F rescue cells show significantly reduced tumor formation in xenograft nude mice compared to cells with mPDHK1 WT (p value was determined by the paired Student’s t test).