Mitochondrial PDHK1 is tyrosine phosphorylated and activated by FGFR1 in cancer cells
To better understand how tyrosine kinase signaling, commonly upregulated in tumors, regulates the Warburg effect, we examined whether oncogenic FGFR1 phosphorylates and regulates PDHK1 (). We found that active, recombinant FGFR1 (rFGFR1) effectively phosphorylates purified GST-tagged PDHK1 in an in vitro kinase assay (). Further mass spectrometric analysis identified three tyrosine residues of PDHK1, including Y136, Y243 and Y244, that are phosphorylated by FGFR1 (; numbering of PDHK1 is as per Swiss Prot entry Q15118). In addition, GST-tagged PDHK1 was tyrosine phosphorylated in 293T cells transiently co-transfected with FGFR1 wild type (WT), but not in cells co-expressing a kinase dead (KD) form of FGFR1 (). Moreover, in an in vitro PDHK1 kinase assay, tyrosine-phosphorylated GST-PDHK1 from cells co-expressing FGFR1 WT but not FGFR1 KD demonstrated enhanced kinase activity and effectively phosphorylated recombinant PDHA1 as a substrate ( upper).
FGFR1 directly phosphorylates and activates PDHK1
Overexpression of FGFR1 or its mutational activation have been implicated in various human solid tumors, including breast cancer, pancreatic adenocarcinoma, and malignant astrocytoma (Kobrin et al., 1993
; Luqmani et al., 1992
; Morrison et al., 1994
; Penault-Llorca et al., 1995
). In addition, recurrent chromosomal translocations involving the FGFR1
gene on 8p11.2-11.1 are associated with stem cell myeloproliferative disorder (MPD), which result in expression of active FGFR1 fusion tyrosine kinases. We found that inhibition of FGFR1 by a small molecule inhibitor TKI258 results in decreased tyrosine phosphorylation levels of GST-PDHK1 in cells co-expressing FGFR1 WT ( left
). Similar results were obtained in TKI258-treated cells co-expressing GST-PDHK1 and ZNF198-FGFR1 4ZF, which is an active fusion tyrosine kinase associated with the t(8;13)(p11;q12) MPD (Xiao et al., 1998
) (Figure S1
). Interestingly, immunoblotting results show that FGFR1 as a receptor tyrosine kinase is co-localized with PDHK1 and its substrate PDHA1 in mitochondria ( right
). Moreover, TKI258 treatment significantly decreased phosphorylation levels of PDHA1 at S293 in human myeloid leukemia KG1a cells harboring a FOP2-FGFR1 fusion protein (Gu et al., 2006
) (; left
) and lung cancer NCI-H1299 cells overexpressing FGFR1 (Marek et al., 2009
) (; right
). In consonance with this, targeting PDHK1 by a PDHK inhibitor, dichloroacetate (DCA), or shRNA results in decreased S293 phosphorylation levels of PDHA1 in FGFR1-expressing cancer cells ().
FGFR1 activates PDHK1 through direct phosphorylation at multiple tyrosine sites that promotes ATP and PDC binding to PDHK1
We next found that incubation with rFGFR1 results in increased tyrosine phosphorylation levels and kinase activity of GST-PDHK1 WT. However, although rFGFR1 phosphorylates all of the Y→F mutants, substitution of Y136, Y243 or Y244 attenuates the FGFR1-dependent increase of PDHK1 kinase activity assessed by S293 phosphorylation levels of PDHA1 proteins, while double mutation of both Y243 and Y244 abolishes the enhanced activation of PDHK1 by FGFR1 ().
FGFR1-dependent tyrosine phosphorylation activates PDHK1 by promoting ATP and PDC binding
As shown in , crystal structures of PDHK1 reveal that Y243 and Y244 are close to the ATP lid and ATP binding site, suggesting that phosphorylation of these residues could affect ATP binding and PDHK1 catalytic activity. In contrast, Y136 is distal to both ATP and substrate binding sites of PDHK1, but is closer to the binding site of a small molecule drug, AZD7545, which inhibits PDHK1 and 3 by aborting the kinase binding to the pyruvate dehydrogenase complex (PDC) scaffold (Kato et al., 2007
). Indeed, incubation with rFGFR1 led to increased [α-32
P]ATP binding to PDHK1 WT and Y136F mutant. In contrast, substitution of Y243 and/or Y244 abolished the FGFR1-dependent enhancement of ATP binding to PDHK1 (). To determine whether Y136 phosphorylation affects the binding of PDHK1 to PDC, we incubated active rFGFR1 with purified GST-PDHK1 WT or Y→F mutants in an in vitro
kinase assay, followed by incubation with whole cell lysates from 293T cells. Phosphorylation of PDHK1 WT by FGFR1 resulted in increased binding between PDHK1 and PDC E2 protein, as well as enhanced association between PDHK1 and its substrate PDHA1 that exists in PDC. In contrast, substitution of Y136 abolished the enhanced association of PDHK1 to PDC E2 protein or PDHA1 in the presence of rFGFR1 ().
Interestingly, substitution of Y243 and/or Y244 also led to abolishment of the FGFR1-dependent increased PDHK1/PDC E2 and PDHK1/PDHA1 associations (). These results together suggest that phosphorylation at both Y243 and Y244, but not Y136 may be required to promote ATP binding to PDHK1, which consequently facilitates PDHK1 binding to PDC scaffold to access substrate PDHA1. In contrast, Y136 phosphorylation may only function to enhance binding between PDHK1 and PDC. Figure S2
shows the results of analysis of the relative stability of proteins to limited proteolytic digestion with chymotrypsin (Kang et al., 2007
), which suggests the global structure of each mutant protein was not altered, and decreased kinase activation () or ATP/PDC binding ability of PDHK1 Y→F mutants are not due to structural alterations.
FGFR1 phosphorylates mitochondrial PDHK1 in cancer cells
Oncogenic FGFR1 is localized in mitochondria in cancer cells, where it phosphorylates PDHK1
Using an antibody that specifically recognizes PDHK1 phospho-Y243, we observed that rFGFR1 and FGFR1 WT phosphorylated GST-PDHK1 WT, Y136F and Y244F mutants, but not the Y243F and Y243/244F mutants, at Y243 in an in vitro
kinase assay using recombinant proteins () and in 293T cells co-expressing these PDHK1 variants (), respectively. We also observed that inhibiting FGFR1 by TKI258 decreased PDHK1 Y243 phosphorylation in FGFR1-expressing H1299 lung cancer cells (, left
) and KG1a leukemia cells (, right
). TKI258 does not inhibit PDHK1 kinase activity in an in vitro
kinase assay (Figure S3A
). Moreover, knockdown of FGFR1 by shRNA in H1299 cells for 48 hours results in decreased phosphorylation levels of PDHK1 Y243 and PDHA1 S293 (). Further discussion regarding decreased PDHK1 protein levels in H1299 cells with long term knockdown of FGFR1 is provided in the Discussion section.
We also detected a fraction of FOP2-FGFR1 fusion and FGFR1 in mitochondria along with mitochondrial PDHK1 and PDHA1 () in KG1a and H1299 cells, respectively, with mitochondrial COX IV and cytosolic β-actin as control markers (). Figure S3B and 3C
show no plasma membrane or nuclear contamination in the mitochondrial fractions. In consonance with these observations, immunofluorescence assay results show that FGFR1 is co-localized with the control mitochondrial marker MitoTracker® Mitochondrion-Selective dyes, but not with nuclear marker DAPI in H1299 cells ( and Figure S4A
). Figure S4B
shows no cross-reaction between FGFR1 antibody and MitoTracker® Mitochondrion-Selective dyes. In addition, knockdown of FGFR1 by shRNA leads to decreased mitochondrial localization of FGFR1 in H1299 cells (), while stimulation of cells with FGFR1 ligand bFGF does not significantly alter the FGFR1 mitochondrial localization (Figure S4C
We further investigated the ultrastructural localization of FGFR1 by immunogold transmission electron microscopy (TEM). Specific immunogold particles were found in mitochondria of H1299 cells. This immunogold particle distribution was not observed when the primary anti-FGFR1 antibody was omitted (, left) and was greatly reduced by FGFR1 shRNA (, right). To localize FGFR1 within mitochondria biochemically, we performed sub-fractionation of highly purified mitochondria from H1299 and KG1a cells. Western blot analyses of the sub-fractions show that both full length FGFR1 and FOP2-FGFR1 fusion tyrosine kinase are present predominately in the mitochondrial outer membrane (Om) (). We also observed that both PDHK1 and PDHA1 are present in the mitochondrial matrix (Ma), as well as in the intermembrane space (IMS) and Om. Detailed discussion is provided in the Discussion section. To determine whether FGFR1 and FOP2-FGFR1 fusion are integrated in Om, we performed a proteinase K protection assay using purified mitochondria from H1299 and KG1a cells, respectively. Outer membrane marker Tom 40 and inner membrane marker COX IV were included as controls. As shown in , Tom 40 was completely digested by proteinase K treatment in both the presence and absence of Triton X-100, whereas complete digestion of COX IV by proteinase K only occurred after solubilization of mitochondria with Triton X-100 treatment. We found that, similar to Tom 40, full length FGFR1 was completely digested by proteinase K in both presence and absence of Triton X-100. In contrast, FOP2-FGFR1 was only partially digested by proteinase K in the absence of Triton X-100 and complete digestion of FOP2-FGFR1 was observed after solubilization of mitochondria with Triton X-100. Together, mitochondrial FGFR1 and a portion of FOP2-FGFR1 may be integrated in the mitochondrial Om, whereas the rest of mitochondrial FOP2-FGFR1 may be located inside the mitochondria, probably in the IMS, but associated with certain unknown Om protein.
FGFR1 is localized to outer membrane of mitochondria and functional PDC can form in mitochondria outside of matrix in cancer cells
Functional PDC can form in mitochondria outside of matrix in some cancer cells and PDHK1 is commonly phosphorylated in these cells at Y243 by various oncogenic tyrosine kinases
Furthermore, we detected PDC activity and various PDC components in the mitochondrial Om and IMS, in addition to the matrix in both H1299 and KG1a cells (, respectively). These results suggest that oncogenic FGFR1 may inhibit PDC by phosphorylating PDHK1 in the Om in cancer cells. Detailed discussion is provided in the Discussion section.
We found that PDHK1 was phosphorylated at Y243 in diverse hematopoietic cancer cell lines associated with various constitutively activated tyrosine kinase mutants, including HEL (JAK2 Val617Phe mutant), KG1a (FOP2-FGFR1), K562 (BCR-ABL) and Mo91 (TEL-TrkC), whereas Y243 phosphorylation levels are relatively lower in FLT3-internal tandem duplication (ITD) mutant positive Molm14 and Mv4;11 cells, but not detected in EOL-1 (FIP1L1-PDGFRA) cells. Phosphorylation levels of PDHA1 at S293 in general correlated with PDHK1 Y243 phosphorylation levels in these leukemia cells (). Y243 phosphorylation of PDHK1 was also detected in various human solid tumor cell lines, including lung cancer H1299 (FGFR1; ) and A549 cells and breast cancer MCF-7 cells, but not breast cancer MDA-MB435 cells and prostate cancer PC3 and DU145 cells (). However, the phosphorylation levels of PDHA1 at S293 in these solid tumor cells did not correlate with PDHK1 Y243 phosphorylation levels (). Detailed discussion is provided below.
Y243 of PDHK1 is commonly phosphorylated by oncogenic tyrosine kinases localized to mitochondria in cancer cells
We next found that active, recombinant ABL (), JAK2 () and FLT3 () also directly phosphorylated PDHK1 at Y243 in the in vitro
kinase assays using recombinant proteins, whereas EGFR phosphorylated PDHK1 with less efficiency (Figure S5A
). Inhibition of BCR-ABL by imatinib, JAK2 by AG490 and FLT3-ITD by TKI258 resulted in decreased Y243 phosphorylation of PDHK1 in the pertinent human cancer cell lines (; left, middle
, respectively). In addition, immunoblotting results confirm the mitochondrial localization of BCR-ABL, JAK2 and FLT3 (), which are co-localized with PDHK1 and its substrate PDHA1 in mitochondria (Figure S5B
) in the pertinent human leukemia cell lines. Moreover, Western blot analyses of the sub-fractions of highly purified mitochondria from K562, HEL and Molm14 leukemia cells (; left, middle
, respectively) show that a portion of cytoplasmic BCR-ABL and JAK2 proteins are present predominately in the matrix of mitochondria, while a portion of receptor tyrosine kinase FLT3 is present predominately in the outer membrane of mitochondria. These oncogenic tyrosine kinases are also co-localized with PDHK1 and PDHA1 in the corresponding mitochondrial sub-fractions in the pertinent human leukemia cell lines, where these tyrosine kinases may phosphorylate PDHK1. Consistently, PDC activity was also detected in the mitochondrial Om and IMS, in addition to the matrix in all of these cells (Figure S5C-5E
Presence of the catalytically less active mouse PDHK1 Y134F and Y239/240F mutants in cancer cells leads to decreased cell proliferation under hypoxia and increased oxidative phosphorylation
We next generated “rescue” cell lines as previously described (Hitosugi et al., 2009
) by RNAi-mediated stable knockdown of endogenous human PDHK1 (hPDHK1) and rescue expression of Flag-tagged mouse PDHK1 (mPDHK1) WT, or the corresponding Y134F and Y239/240F mutants (). Both Flag-mPDHK1 WT and Y134F mutant, but not Y239/240F mutant, were phosphorylated at Y239 (corresponding to Y243 in human PDHK1 numbering) by FGFR1 in H1299 cells (Figure S6A
). In addition, Y134F and Y239/240F mutants showed decreased kinase activity that led to reduced phosphorylation levels of PDHA1 at S293 in Y134F and Y239/240F rescue cells, respectively, compared to cells with mPDHK1 WT (Figure S6B
). Moreover, although hypoxia results in increased PDHK1 expression and Y243 phosphorylation levels (Figure S6C
), endogenous hPDHK1 protein levels were not detected in diverse rescue cells under normoxia nor hypoxia, suggesting that the efficacy of hPDHK1 knockdown mediated by shRNA is not altered under hypoxia (Figure S6D
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
We also observed that, under normoxia, cells rescued with any of the mPDHK1 variants showed a comparable rate of proliferation that was greater than that of parental cells, in which endogenous hPDHK1 was stably knocked down. However, Y134F and Y239/240F rescue cells showed a significantly slower proliferation rate under hypoxic conditions than did cells rescued with mPDHK1 WT (). Moreover, compared to cells rescued with mPDHK1 WT, the Y134F and Y239/240F rescue cells and parental cells with stable knockdown of endogenous hPDHK1 had a higher rate of oxygen consumption (), an increased production of intracellular reactive oxygen species (ROS) () and reduced lactate production (). In addition, treatment with oligomycin, a specific inhibitor of mitochondrial ATP synthase, resulted in an increased inhibition of ATP production and a decreased proliferation rate among parental control H1299 cells with stable knockdown of endogenous hPDHK1 and rescue cells expressing Y134F and Y239/240F mutants, compared to cells with mPDHK1 WT (, respectively). These results together suggest that cells expressing catalytically less active mPDHK1 mutants, including Y134F and Y239/240F, rely more on oxidative phosphorylation for ATP production and cell proliferation compared to cells with mPDHK1 WT.
Tyrosine phosphorylation of PDHK1 is important for PDC-mediated pyruvate metabolism, probably through inhibitory phosphorylation of PDHA1
The pyruvate dehydrogenase complex (PDC) contributes to pyruvate decarboxylation, in which pyruvate is converted to acetyl-CoA and carbon dioxide. To determine the role of tyrosine phosphorylation of PDHK1 in regulation of PDC activity, we performed a pyruvate consumption experiment using highly purified mitochondria. We found that mitochondria isolated from rescue cells expressing mPDHK1 Y239/240F mutant show a significantly increased pyruvate consumption rate, compared to mitochondria isolated from WT rescue cells (). Moreover, we examined the PDC activity by assessing the rate of PDC-mediated conversion of pyruvate to CO2. As shown in , isolated mitochondria from cells expressing mPDHK1 Y239/240F mutant have a significantly increased rate of transforming 14C-labeled pyruvate to 14C-labeled CO2, compared to mitochondria isolated from WT rescue cells. These data suggest an important role for tyrosine phosphorylation of PDHK1 in regulation of PDC activity, probably by phosphorylating PDHA1.
We next sought to determine the role of PDHA1 in PDHK1-regulated cancer cell metabolism. As shown in , expression of PDHA1 WT or an active, phospho-deficient mutant S293A in H1299 rescue cells of either mPDHK1 WT or Y239/240F mutant resulted in an increased O2 consumption rate. In contrast, mPDHK1 WT rescue cells expressing a phospho-mimetic, catalytically less active form of PDHA1 (S293D) showed less increase in O2 consumption compared to cells expressing PDHA1 WT or S293A mutant, whereas expression of PDHA1 S293D mutant, but not PDHA1 WT or S293A mutant, resulted in a significant decrease in O2 consumption in H1299 rescue cells of mPDHK1 Y239/240F. Furthermore, expression of PDHA1 WT, S293A or S293D mutants in rescue cells of mPDHK1 Y239/240F did not affect cell proliferation under normoxia. 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 (). Moreover, rescue cells of mPDHK1 Y239/240F transfected with PDHA1 S293D mutant showed increased lactate production () and decreased sensitivity to the treatment with oligomycin, an inhibitor of ATP synthase, in regard to ATP production and cell proliferation (, respectively), compared to cells transfected with PDHA1 WT or S293A mutant. These data together with data presented in suggest that rescue expression of mPDHK1 Y239/240F makes cells rely more on oxidative phosphorylation, whereas expression of the inactive, phospho-mimetic PDHA1 S293D mutant attenuates such a metabolic switch.
Phosphorylation-dependent activation of PDHK1 is mediated through phosphorylation and inactivation of PDHA1, which is important for tumor growth in xenograft nude mice
Expression of mPDHK1 Y239/240F mutant in cancer cells leads to reduced tumor growth in xenograft nude mice
We next performed xenograft experiments in which nude mice were injected with Flag-mPDHK1 WT and Y239/240F rescue H1299 cells (Figure S7A
). Ten million cells each were injected (Flag-mPDHK1 WT rescue cells on the left flank and Y239/240F cells on the right flank; n=10), and the mice were monitored for tumor growth over a 5-week time period. The masses of tumors derived from Y239/240F rescue cells were significantly reduced compared to those of tumors formed by Flag-mPDHK1 WT rescue cells ().