a d h ΔSH2 Δ(SH3SH2) Myr SH3 SH2 Kinase domain 85 5 249 55 5 csrc ΔSH3 FlagHK HAcSrc HAcSrcΔSH2 HAcSrcΔSH3 HAcSrcΔ(SH3SH2) WB: FlagHK HAcSrc HAcSrcKD HAcSrcY529F WB: HisHK2HK2 Input GSTcSrc GST : : GST pull down.4.6 25 WB:HK2 GST e b HK Nhalf: 454 Chalf: 497 2 Cox 8aGFP HKRFP FlagcSrc DAPI Merge GST pull down GSTcSrc Truncated GSTcSrc i Regulatory domain : FlagHK FlagHKNhalf FlagHKChalf HAcSrc WB: PP2 FlagHK2 HAcSrc WB: Catalytic 454 domain 97 : j f c PP2 FlagHK HAcSrc WB: : : HAcSrc FlagHK2 WB: g HAcSrcKD HAcSrcY529F Anti: HA HAcSrc FlagHK2 WB: FlagHK2 HAcSrc WB: : :.45.5
Supplementary Figure. csrc activity is essential for its interaction with HK and HK2. (a) SH2 (Src homology) domain is indispensable for csrc to interact with HK. FlagHK was cotransfected into HEK 293T cells separately with HAcSrc and its deletion mutants ΔSH3 (with aa 865 deleted), ΔSH2 (with aa 5249 deleted) and Δ(SH3SH2) (with aaa 86249 deleted). s were performed with M 2 beads (antiflag). Immunoprecipitates and total cell lysates were analyzed by WB with antiha for csrc and antiflag for HK. Myr, myristylation domain. (b) Nhalf (aa 454) of HK is mainly responsible for its interaction with csrc. (c) PP2, a csrc inhibitor, impaired the interaction between csrc and HK. HEK 293T cells were transfected with the combinations of plasmids as indicated. 24 hours posttransfection, cells were treated with μm PP2 for 4 hours. The cell lysates were immunoprecipitated with M 2 beads. The immunoprecipitates were detected for proteins as depicted. (d) The activity of csrc is essential for its interaction with HK. csrc, csrckd and csrcy529f was transfected alone, or together with FlagHK into HEK 293T cells. Lysates were immunoprecipitated with M 2 beads, followed by detection with antiflag for HK and antiha for coimmunoprecipitated csrc. (e) In HeLa cells, HK and csrc complex showed less colocalization in mitochondria than in cytoplasm. Cox 8a was a mitochondrial marker. scale bar, 3μm (f, g) csrc can interact with HK2. HEK 293T cells were transfected with HAcSrc and FlagHK2 in combinations as indicated. Reciprocal s were performed to precipitate HAcSrc (f) and FlagHK2 (g). (h) HK2 interacts with csrc in vitro. GST pull down was carried out with bacterially expressed HisHK2 and GSTcSrc. GST protein was used as a negative control. Large amounts of HisHK2 were precipitated by GSTcSrc, but not by GST. Left panel, coomassie brilliant blue staining of precipitates; right panel, WB detection of the same precipitates. (i) The interaction between csrc and HK2 was abrogated by treatment of HEK 293T cells with PP2. (j) csrc kinase activity is required for its interaction with HK2 in HEK 293T cells.
a WB: commercial HK antibody b HeLa shgfp shhk c WB: AntiHK HeLa A549 IgG HK IgG HK HisHK(364aa) antibody GSTHK(364aa) antibody WB: Commercial HK antibody HisHK(364aa) antibody AntiHK Antiβactin GSTHK(364aa) antibody Antiβactin d shgfp shsrc e : IgG HK IgG HK IgG HK HK WB: AntipTyr PP2 WB: AntipTyr ptyr/actin..5 AntiHK AntiHK AntiHK AnticSrc csrc/actin.9.43.4 Antiβactin AntiHK AnticSrc Antiβactin f : HAcSrc HAcSrcKD FlagHK2 WB: AntipTyr g CIAP HAcSrc FlagHK2 WB: AntipTyr : h PP2 FlagHK2 HAcSrc WB: AntipTyr : i HisHK2 GSTcSrc GSTcSrcKD WB: AntipTyr AnticSrc AntiHK2 j FlagHK2 HAcSrc WB: AntipTyr : k GSTcSrc HisHK2 HisHK2Y686F WB: AntipTyr AntiHK2 AnticSrc Supplementary Figure 2. Both HK and HK2 are phosphorylated by csrc. (a) HK antibody raised with GSTHK (aa 364) as immunogen showed high valency towards HK. Bacterially expressed GST or Histagged fragments (aa 364) of HK were employed to immunize rabbits, followed by affinity purification of corresponding antibodies. Purified antibodies were subjected to detect endogenous HK in HCT6, HeLa and U2OS cells. A commercially available HK antibody acts as a positive control. (b) High specificity of homemade antihk antibody is indicated by knock down of endogenous HK in HeLa. (c) Homemade HK antibody is suitable for immunoprecipitation. Endogenous HK in HeLa and A549 cells were successfully ed by employing homemade antihk antibody. (d) Partial knockdown of csrc in A549 cells diminishes tyrosine phosphorylation of endogenous HK. (e) Tyrosine phosphorylation of endogenous HK was effectively abolished by treating A549 cells with PP2. (f) It is csrc, but not csrckd that strongly phosphorylates HK2 in HEK 293T cells. (g) Treatment of cell lysates with CIAP thoroughly abolished the tyrosine phosphorylation of HK2 mediated by csrc in HEK 293T cells. (h) Phosphorylation level of HK2 induced by csrc in HE EK 293T cells was eliminated by the treatment of cells with PP2. (i) Recombinant HK2 can be effectively phosphorylated by bacterially purified csrc rather than csrckd. (j) The mutation of HK2 Y686F could abolish the tyrosine phosphorylation induced by csrc in HEK 293T cells. (k) In vitro kinase assay showed that csrc could phosphorylate HK2 WT but not HK2 Y686F.
a WB: AntipY732 c AntipY732 2 3 4 5 6, 2, 3 (unphosphotyr732 p peptide) p 4, 5, 6 (phosphotyr732 peptide) AntipY732 preincubated with HKpY732 peptides AntipY732 Preincubated with HK peptides b FlagHK FlagHKY732F HAcSrc WB: : : WB: AntipY732 AntipY732 preincubated with HKpY732 peptides AntipY732 Preincubated with HK peptides d h FlagHK2 FlagHK2Y686F HAcSrc WB: AntipTyr AntipHK2Y686 2AntipHK2Y686 HK2 csrc HK2 FlagHK FlagHK2 HAcSrc PDGF PP2 WB: AntipcSrc : : e PP2 WB: AntipY732 i AntiHK AntipcSrc AnticSrc Antiβactin WB: AntipY732 AntiHK2 AntiHK AntipcSrc AnticSrc Antiβactin k Flag: vector csrc Hck Lck Blk Yes HAHKHK HAHK Y732F WB: AntiHKpY732 : HA 5 5 f g : Pervanadate FlagHK WB: AntipY732 HAPDGFR py732/actin. 2. PP2 AntiHK AntipcSrc pcsrc/actin. 3. AnticSrc Antiβactin WB: AntipY732 j HFF WB: AntipY732 AntiHK AnticSrc Antiβactin AntipcSrc l Flag: vector csrc Hck Lck Blk Yes HAHK2 HAHK Y686F WB: AntipTyr : HA
Supplementary Figure 3. csrc phosphorylates HK at Tyr732 and HK2 at Tyr686. (a) The antipy732 antibody specifically recognized phosphorylated oligopeptides containing phosphorylated Y732 residue were used to immunize rabbits, followed by purification of antihkphosphoy732 antibody (designated as antipy732) with CNBr beads. The specificity of antipy732 was determined by detecting KLHtagged peptides with or without Y732 phosphorylated by immunodot blotting. (b) The specificity of our selfmade antipy732 antibody was confirmed by WB with this antibody preincubated with HKpY732 or HK peptides. The expression plasmids encoding HK and HK Y732F were transfected into HEK 293T cells with or without HAcSrc. Cell lysates were used to immunoprecipitation by M 2 beads and followed by WB with indicated antibodies. (c) The antibody against to HK Y732 phosphorylation is specific for immunohistochemistry. Colorectal tissues were immunostainned with the corresponding antibodies. (d) Two antibodies prepared by using synthesized HK2 peptides containing phosphorylated Y686 residue as immunogen failed to specifically recognize phosphorylated HK2. and 2 refer to serums from two immunized rabbits. (e) Inhibition of csrc activity by PP2 resulted in decreasing HKY732 phosphorylation. (f) HKY732 phosphorylation was significantly increased by exposure of A549 cells to. mm pervanadate for minutes. (g) Treatment with PP2 could block the HK Y732 phosphorylation induced by overexpression of PDGFR in HEK 293T cells. (h) The associations between HK /HK2 and csrc were enhanced by stimulating cells with PDGF, but this effect could be reversed by cotreatment with PP2. After 24 hours of transfection, HEK 293T cells were exposed to PP2 4 hours and PDGF for minutes alone or in combination. (i) Y732 phosphorylation levels of HK strongly correlate with Y49 phosphorylation levels of csrc in various cancer cell lines. (j) Both PDGF(2ngml ) and EGF ( ng ml ) effective ely stimulated HKY732 phosphorylation in human foreskin fibroblast (HFF) cells. (k, l) Among four Src family members examined other than csrc, Yes showed much weak effect on HK Y732 phosphorylation (k) and HK2 tyrosine phosphorylation (l) in HEK 293T cells compared to csrc. HK. Keyhole limpet hemocyanin (KLH)tagged
a Relative HK activity 2.5 * * 2.5.5 FlagHK2 FlagHK2Y686F e WB: Antiactin Relative HK activity 2.5 2.5.5 * FlagHK2 PP2 SU6656 WB: AntipcSrc AnticSrc Antiβactin f b Relative HK activity 4 3 2 FlagHK2 FlagHK2Y686F HAcSrc WB: Re elative HK activity Antiactin 2.5 2.5.5 shgfp shsrc FlagHK2 WB: AnticSrc Antiactin * N.S. * g HK activity relative to blank vector c WB: AntiHK Relative HK activity 2.5 2 5.5.5 FlagHK PP2 WB: 2.5.5 shhk rhk rhky732f rhkkd AntipcSrc Antiactin Antiactin h d Relative HK activity 2.5 2.5.5 shgfp shsrc FlagHK WB: lative HK activity AnticSrc Antiactin *.5 *.25 *.75 N.S..5.25 shhk2 rhk2 rhk2y686f Re WB: AntiHK2 Antiactin i Trypsin (min) WB: AntiHK HisHK HisHK Y732F 5 2 6 5 2 6 25 j Trypsin (min) WB: AntiHK2 HisHK2 HisHK2 Y686F 5 2 6 5 2 6 25 k HK activity relative to vector 4 3 2 * FlagHK2 PDGF PP2 WB: AntipcSrc Antiactin l HK activity relative to vector.5.5 WB: AntiHK2 AntiHK Antiactin *
Supplementary Figure 4. csrc promotes catalytic activities of both HK and HK2. (a) In HEK 293T cells, HK2Y686F showed greatly decreased enzymatic activity as compared to WT HK2. (b) Cotransfection of csrc enhanced the activity of HK2, but not HK2Y686F in HEK 293T cells. (c) PP2 effectively inhibited HK activity. A549 was overexpressed with FlagHK followed by treatment with or without PP2. Hexokinase activity was determined as described in Methods. (d) Knockdown of csrc diminished catalytic ability of HK. FlagHK was expressed in A549 cells with csrc already knockeddown. HK activity was measured accordingly. (e) Treatment with PP2 or SU6656 disrupted the increase of hexokinase activity induced by overexpression of HK2 in HeLa cells. (f) Knock down of csrc abolished the augment of hexokinase activity caused by expression of FlagHK2 in HeLa cells. (g) Reexpression expression of HK, but not HKY732F or HKKD KD in HK knockeddowndown A549 cells rescued hexokinase activity. (h) Reexpressed HK2, but not its mutation, rescued the HK activity of HeLa cells deprived of endogenous HK2. (i, j) The digestion patterns of HK mutants were the same as their wild type controls. Equal amounts of HK and their mutant protein were digested with trypsin (2 μm) at 37 for indicated times, followed by SDSPADE and Western blot to determine the digestion patterns. (k) PDGF functioned to stimulate HK2 enzyme activity, and such effect was neutralized by treatment of cells with PP2 in HeLa cells. (l) HK and HK2 were knocked down with corresponding shrnas, followed by measurement of total HK activity accordingly. After normalization of enzyme activities to corresponding knockdown efficiencies, two isozymes contribute almost equally to total HK activity in HeLa cells. HK activities in each panel were normalized to the control cells (the first bar) and shown as means±s.d. (three experimental replicates). Unpaired student s t test was used to analyze the significance. *p<.5, p<., *p<., N.S. represents no significant difference.
a d 2Deoxyglucose upta ake Cpm μg min 48 36 2 2 * FlagHK FlagHKY732F 2Deoxyglucose upta ake Cpm μg min HAcSrc WB: Antiactin 3 25 2 5 5 shgfp shsrc FlagHK WB: AnticSrc Antiactin e 2Deoxyglucose uptak ke Cpm μg min 3 25 2 5 5 b ke 2Deoxyglucose uptak Cpm μg min 3 25 2 5 5 shhk rhk WB: AntiHK FlagHK2 PP2 WB: AntipcSrc Antiactin Antiactin 5 * rhky732f f 2Deoxyglucose uptak ke Cpm μg min * 3 25 2 5 5 shgfp shsrc FlagHK2 WB: AnticSrc Antiactin c 3 25 2 5 5 FlagHK PP2 WB: g 2Deoxyglucose uptak ke Cpm μg min AntipcSrc Antiactin 2Deoxyglucose uptak ke Cpm μg min 3 25 2 5 5 * PP2 PDGF WB: pcsrc Antiactin h 2Deoxyglucose uptak ke Cpm μg min 3 25 2 5 5 PDGF shgfp shhk2 WB: AntiHK2 AntipcSrc Antiactin i 2Deoxyglucose upta ake Cpm μg min 3 25 2 5 5 Flag HAHK WB: Antiβactin j 3 25 ke 2Deoxyglucose uptak Cpm μg min 2 5 5 Flag HAHK2 WB: Antiβactin
Supplementary Figure 5. HK/HK2catalyzed glycolysis is stimulated by csrc. (a) HKpromoted glucose uptake was enhanced by overexpression of csrc. A549 cells were infected with lentiviruses expressing the proteins indicated. HKY732F was used as a negative control. (b) Introduction of WT HK, but not its Y732F mutant, rescued the attenuationn of hexokinase activity caused by knockdown of endogenous HK in A549 cells. (c) HKstimulated glucose uptake was abolished by exposure of A549 cells to PP2. (d) Glucose uptake stimulated by overexpression of HK was abrogated by knockdown of csrc in A549 cells. (e) HK2mediated glucose uptake was suppressed by PP2 in HeLa cells. (f) The stimulation of glycolysis gy y by HK2 in HeLa cells was attenuated by knockdown of csrc. (g) PDGFpromoted glucose uptake was inhibited by exposure of A549 cells to PP2. (h) PDGF stimulated glucose uptake in HeLa cells was diminished by knockdown of HK2. (i, j) Other four Src family members examined showed much weaker effect on HK (i) and HK2 (j) promoted glucose uptake compared to csrc. Results of each panel are means±s.d. (three experimental replicates). Unpaired student s t test was used to analyze the significance. p<., *p<..
a e level Relative lactate (fold).5.5 shgfp shhk rhk rhky732f * b Relative lactate level (fold).5.5 shgfp shhk2 rhk2 rhk2y686f * c Glc G6P F6P F,6BP G6P FBP DHAP [2 3 C] HK G6PD PFK E4P GAP GAP R5P/X5P S7P d tive Lactate oduction Relat pr 2.5 2.5 Lactate from EM pathway Lactate from PP pathway.7 2.2..3.5.5.6.24.5.5 HAcSrc FlagHK FlagHKY732F e te relative to r EM pathway Lactat vector..2..8 6.6..2. shgfp shhk rhk rhky732f. Lactate from EM pathway Lactate from PP pathway.5.56..2.7.66. Pyr Lac Lactate percentage in EMP and PPP 87 9 9 88 9 f 8 6 2 3 HAcSrc FlagHK FlagHKY732F h Oxygen con nsumption (fol ld).6.4.2.8.6.4.2 shgfp shhk rhk rhk Y732F 9 untreat oligomycin 2 rhky732f 4 C in Nucleic Acids.4.2.8.6.4 2.2 shgfp shhk rhk rhky732f DNA RNA g Re elative ATP level (fold).4.2.8.6.4 2.2 shgfp shhk rhk rhky732f untreat oligomycin N.S.
Supplementary Figure 6. csrc augments glucose flux through both EMP and PPP. (a, b) Reexpression of wildtype HK, but not its mutant, rescued the lactate production in HeLa cells. (c) A schematic diagram indicates [2 3 C] glucose metabolic fates through EMP and PPP. 3 C atom is drawn as filled circles. The carbon atoms going through the EMP are labeled as black filled circles. The circles in red represent the 3 C atoms going through PPP and then recycled back to EMP. (d) Overexpressed HK rather than HKY732F efficiently increased lactate secretion through both EMP and PPP, and such an effect was enhanced by coexpression of csrc (upper panel). The corresponding percentages of glucose metabolized dth through hemp and PPP are shown in lower panel. HK, HKY732F and csrc were expressed by infecting HeLa cells with different combinations of lentiviruses as indicated. Lactate secretion was measured by NMR. (e) In HK knock down HeLa cells, the lactate production ratio from both EMP and PPP was downregulated and this effect was reversed by reexpression of WT HK, but not its mutant. (f) Disruption of HK expression by shrna influenced DNA/RNA synthesis. HeLa cells were treated with 6 4 C glucose for 24 hours, the incorporation ratio were determined according to the protocol in Methods. (g) Oligomycin treatment resulted in enhanced inhibition of ATP synthesis in HK knock down and HKY732F rescue HeLa cells compared with shgfp cells. (h) Rescue expression of HK, but not its mutant, reversed the increase of oxygen consumption caused by interference of HK. All the results represent means±s.d. of three independent experiments. Unpaired Student s t test was used to analyze the significance. p<., *p <.,.
a b FlagrHK2 FlagrHK2Y686F shhk2 WB: WB: AntipY732 AntiHK 2 3 4 5 6 7 T N T N T N T N T N T N T N.3 Antiβactin * AntipcSrc AnticSrc Antiβactin d shhk2 rhk2 HKpY732 shhk2 rhk2y686f (g) Weight.2.. shhk2 rhk2 shhk2 rhk2y686f Colon primary adenocarcinoma and their matched normal tissue and metastatic adenocarcinoma c T T2 T3 T4 T5 T6 T7 T8 T9 T WB: AntipY732 AntiHK AntipcSrc AnticSrc Antiβactin e primary adenocarcinoma The intensity it of PY Y732 IHC staining 8 metastatic adenocarcinoma 6 2 f The intensity of PY73 2 IHC staining 8 6 2 T 3 N M * T 3 N x M y X>, y> g HK HK csrc 2 3 4 5 6 7 8 9 Growth factors RTK P csrc HK HK csrc P P glucose HK glucose6p glucose Glut ribose5p NADPH lactate Warburg Effect Nucleotide synthesis Tumorigenesis
Supplementary Figure 7. Phosphorylation of HKY732 is significantly correlated with csrc activity and tumor metastasis. (a) HK2Y686F impeded the xenograft tumor formation relative to WT HK2. The tumor lysates were detected with WB as indicated. The data were analyzed by employing twoway ANOVA (*p<.). (b) The activity of csrc is positively correlated to the phosphorylation of HK. The tumor lysates from patients suffered from breast cancer were detected with antibodies indicated. (c) Glioma lysates weree immunoblotted with the antibodies indicated. (d) IHC staining of a microarray containing primary colon adenocarcinomas and their matched normal tissues and metastatic adenocarcinomas with antipy73phosphorylation intensity of HK Y732 is much higher in metastatic colon adenocarcinoma tissues antibody. The scale bar is.5 mm. (e) The than in primary colon adenocarcinoma tissues. The slides quantifications were carried out with P software. Unpaired Student s t test was used to analyze the significance (p<., n=). (f) The phosphorylation intensity of HK Y732 is much higher in primary colon adenocarcinoma tissues with lymph node or distant metastasis than in primary colon adenocarcinoma tissues without any metastasis. t Unpaired Student s t t test t was used to analyze the significance ifi (*p<., n=7). This statistical result is from the IHC staining shown in Supplementary Fig. 7d. (g) A schematic diagram showing the mechanism how csrc activates HK. Upon the stimulation of growth factors, such as EGF, csrc is activated to form a transient protein complex with HK homodimer. In this complex, HK is promptly phosphorylated by csrc at Y732, which is essential for rapid liberation of HK from the complex. Librated HK exists in the form of phosphorylated monomer which shows maximum catalytic efficiency. After activation, HK increases s glucose metabolism through both EMP and PPP, which in turn provides enough ATP, ribose5p, NADPH and other intermediates required for biosynthesis of rapidly proliferating tumor cells.
Figure a Figure b Figure c 25 25 Co: Flag : Flag : HAcSrc 7 : FlagHK Co: HAcSrc : FlagHK : HAcSrc : HAcSrc Co: FlagHK : HAcSrc : FlagHK Figure d : HAcSrc Co: HK : HK 25 Figure e input input GST pull down GST pull down HisHK GSTcSrc : csrc : csrc IgG HisHKHK GSTcSrc 25 Supplementary Figure 8. Uncropped images of western blots analysis (Figure )
Figure 2a Figure 2b Figure 2c : HKpTyr : FlagHK : FlagHK : HAcSrc : HKpTyr : FlagHK : HAcSrc : FlagHK : HKpTyr : FlagHK : HAcSrc : FlagHK Figure 2d Figure 2e Figure 2f ptyr csrc HK : HKpTyr : HK : HK : csrc : βactin : HKpTyr : HK : HK : pcsrc : csrc Supplementary Figure 9. Uncropped images of western blots analysis (Figure 2) (continue)
Figure 4g Figure 4h Figure 4i : HKpTyr : FlagHK : HAcSrc : FlagHK HK/GSTcSrc HKpTyr : FlagHK : HKpY732 : HAcSrc : FlagHK Figure 4j Figure 4k Figure 4l Figure 4m HKpY732 HK GSTcSrc HKpY732 HK pcsrc csrc βactin HKpY732 HK pcsrc csrc βactin HKpY732 HK pcsrc csrc βactin Supplementary Figure 9. Uncropped images of western blots analysis (Figure 2)
Figure 4c Figure 4d Co: HAHK : FlagHK : HKpTyr : FlagHK : HAHK : HAcSrc Figure 4e : HAHK : FlagHK : HAcSrc HAHK Phostag FlagHK : FlagHK Co: HAHK : HAHK Co: HAHK : FlagHK : HAcSrc : FlagHK Figure 4g HAHK Phostag st 2nd FlagHK FlagHK MyccSrc HAHK Supplementary Figure. Uncropped images of western blots analysis (Figure 4)
Figure 8c HKpY732 HK pcsrc csrc βactin HKpY732 Figure 8d HK pcsrc csrc βactin Supplementary Figure. Uncropped images of western blots analysis (Figure 8)