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• W2898767623 abstract "•ADP generated from CDC7-mediated phosphorylation inhibits CDC7-ASK activity•EGFR- and ERK1/2-dependent activation of CK2α phosphorylates PGK1 at S256•Phosphorylated PGK1 binds to CDC7 and converts ADP to ATP•PGK1 releases ADP’s inhibition on CDC7 to promote DNA replication and tumorigenesis DNA replication is initiated by assembly of the kinase cell division cycle 7 (CDC7) with its regulatory activation subunit, activator of S-phase kinase (ASK), to activate DNA helicase. However, the mechanism underlying regulation of CDC7-ASK complex is unclear. Here, we show that ADP generated from CDC7-mediated MCM phosphorylation binds to an allosteric region of CDC7, disrupts CDC7-ASK interaction, and inhibits CDC7-ASK activity in a feedback way. EGFR- and ERK-activated casein kinase 2α (CK2α) phosphorylates nuclear phosphoglycerate kinase (PGK) 1 at S256, resulting in interaction of PGK1 with CDC7. CDC7-bound PGK1 converts ADP to ATP, thereby abrogating the inhibitory effect of ADP on CDC7-ASK activity, promoting the recruitment of DNA helicase to replication origins, DNA replication, cell proliferation, and brain tumorigenesis. These findings reveal an instrumental self-regulatory mechanism of CDC7-ASK activity by its kinase reaction product ADP and a nonglycolytic role for PGK1 in abrogating this negative feedback in promoting tumor development. DNA replication is initiated by assembly of the kinase cell division cycle 7 (CDC7) with its regulatory activation subunit, activator of S-phase kinase (ASK), to activate DNA helicase. However, the mechanism underlying regulation of CDC7-ASK complex is unclear. Here, we show that ADP generated from CDC7-mediated MCM phosphorylation binds to an allosteric region of CDC7, disrupts CDC7-ASK interaction, and inhibits CDC7-ASK activity in a feedback way. EGFR- and ERK-activated casein kinase 2α (CK2α) phosphorylates nuclear phosphoglycerate kinase (PGK) 1 at S256, resulting in interaction of PGK1 with CDC7. CDC7-bound PGK1 converts ADP to ATP, thereby abrogating the inhibitory effect of ADP on CDC7-ASK activity, promoting the recruitment of DNA helicase to replication origins, DNA replication, cell proliferation, and brain tumorigenesis. These findings reveal an instrumental self-regulatory mechanism of CDC7-ASK activity by its kinase reaction product ADP and a nonglycolytic role for PGK1 in abrogating this negative feedback in promoting tumor development. The serine/threonine protein kinase cell division cycle 7 (CDC7) is conserved from yeast to humans and is essential for chromosomal DNA replication (Yamada et al., 2014Yamada M. Masai H. Bartek J. Regulation and roles of Cdc7 kinase under replication stress.Cell Cycle. 2014; 13: 1859-1866Crossref PubMed Scopus (30) Google Scholar). The activity of the catalytic subunit of CDC7 is positively regulated by formation of a complex with its regulatory activation subunit, which is called Dbf4 in yeast and activator of S-phase kinase (ASK) in humans (Jiang et al., 1999Jiang W. McDonald D. Hope T.J. Hunter T. Mammalian Cdc7-Dbf4 protein kinase complex is essential for initiation of DNA replication.EMBO J. 1999; 18: 5703-5713Crossref PubMed Scopus (170) Google Scholar). Formation of a complex of CDC7 and ASK/Dbf4 (commonly referred to as Dbf4-dependent kinase or DDK) facilitates ATP binding and substrate recognition by CDC7 (Remus et al., 2009Remus D. Beuron F. Tolun G. Griffith J.D. Morris E.P. Diffley J.F. Concerted loading of Mcm2–7 double hexamers around DNA during DNA replication origin licensing.Cell. 2009; 139: 719-730Abstract Full Text Full Text PDF PubMed Scopus (475) Google Scholar, Yeeles et al., 2015Yeeles J.T. Deegan T.D. Janska A. Early A. Diffley J.F. Regulated eukaryotic DNA replication origin firing with purified proteins.Nature. 2015; 519: 431-435Crossref PubMed Scopus (327) Google Scholar). CDC7 expression levels appear to remain constant throughout the cell cycle. However, the level of ASK/Dbf4 expression is cell-cycle regulated, and CDC7 is activated during S phase in eukaryotes and plays an essential role in initiating DNA replication at replication origins by phosphorylating the MCM2 and MCM4 subunits of the MCM2–MCM7 helicase complex (Araki, 2016Araki H. Elucidating the DDK-dependent step in replication initiation.EMBO J. 2016; 35: 907-908Crossref PubMed Scopus (11) Google Scholar, Labib, 2010Labib K. How do Cdc7 and cyclin-dependent kinases trigger the initiation of chromosome replication in eukaryotic cells?.Genes Dev. 2010; 24: 1208-1219Crossref PubMed Scopus (277) Google Scholar). Subsequently, CDC45 and GINS (a protein complex composed of the proteins SLD5, PSF1, PSF2, and PSF3) assemble onto each MCM2–MCM7 complex, thereby forming two active CDC45-MCM-GINS helicases that surround each strand of parental DNA for bidirectional replication (Botchan and Berger, 2010Botchan M. Berger J. DNA replication: making two forks from one prereplication complex.Mol. Cell. 2010; 40: 860-861Abstract Full Text Full Text PDF PubMed Scopus (19) Google Scholar, Evrin et al., 2009Evrin C. Clarke P. Zech J. Lurz R. Sun J. Uhle S. Li H. Stillman B. Speck C. A double-hexameric MCM2–7 complex is loaded onto origin DNA during licensing of eukaryotic DNA replication.Proc. Natl. Acad. Sci. USA. 2009; 106: 20240-20245Crossref PubMed Scopus (387) Google Scholar, Ilves et al., 2010Ilves I. Petojevic T. Pesavento J.J. Botchan M.R. Activation of the MCM2–7 helicase by association with Cdc45 and GINS proteins.Mol. Cell. 2010; 37: 247-258Abstract Full Text Full Text PDF PubMed Scopus (420) Google Scholar, Moyer et al., 2006Moyer S.E. Lewis P.W. Botchan M.R. Isolation of the Cdc45/Mcm2–7/GINS (CMG) complex, a candidate for the eukaryotic DNA replication fork helicase.Proc. Natl. Acad. Sci. USA. 2006; 103: 10236-10241Crossref PubMed Scopus (544) Google Scholar). CDC7 and ASK are overexpressed in human cancer cell lines and expressed at higher levels in many primary tumors than in matched normal tissue, and that increased CDC7 activity is associated with poor clinical outcome (Bonte et al., 2008Bonte D. Lindvall C. Liu H. Dykema K. Furge K. Weinreich M. Cdc7-Dbf4 kinase overexpression in multiple cancers and tumor cell lines is correlated with p53 inactivation.Neoplasia. 2008; 10: 920-931Abstract Full Text PDF PubMed Scopus (100) Google Scholar, Montagnoli et al., 2010Montagnoli A. Moll J. Colotta F. Targeting cell division cycle 7 kinase: a new approach for cancer therapy.Clin. Cancer Res. 2010; 16: 4503-4508Crossref PubMed Scopus (85) Google Scholar). Therefore, CDC7 is a target for cancer treatment (Montagnoli et al., 2010Montagnoli A. Moll J. Colotta F. Targeting cell division cycle 7 kinase: a new approach for cancer therapy.Clin. Cancer Res. 2010; 16: 4503-4508Crossref PubMed Scopus (85) Google Scholar). However, the mechanism of dynamic regulation of CDC7 activity mediated by assembly and disassembly of the CDC7/ASK complex in response to oncogenic signals during tumor growth remains largely unclear. In addition to their originally identified metabolic activities, metabolic enzymes can possess nonmetabolic functions and play critical roles in regulation of G1-S phase transition, mitosis, cytokinesis, and many other instrumental cellular activities (Li et al., 2018Li X. Egervari G. Wang Y. Berger S.L. Lu Z. Regulation of chromatin and gene expression by metabolic enzymes and metabolites.Nat. Rev. Mol. Cell Biol. 2018; 19: 563-578Crossref PubMed Scopus (194) Google Scholar, Lu and Hunter, 2018Lu Z. Hunter T. Metabolic kinases moonlighting as protein kinases.Trends Biochem. Sci. 2018; 43: 301-310Abstract Full Text Full Text PDF PubMed Scopus (126) Google Scholar, Wang and Lei, 2018Wang Y.P. Lei Q.Y. Metabolic recoding of epigenetics in cancer.Cancer Commun (Lond). 2018; 38: 25Crossref Scopus (48) Google Scholar). In the glycolytic pathway, PGK1 catalyzes 1,3-biphosphoglycerate (1,3-BPG) and ADP to produce 3-phosphoglycerate (3-PG) and ATP (Li et al., 2016dLi X. Zheng Y. Lu Z. PGK1 is a new member of the protein kinome.Cell Cycle. 2016; 15: 1803-1804Crossref PubMed Scopus (44) Google Scholar). In response to oncogenic signaling, activated ERK1/2 phosphorylates PGK1 and promotes the translocation of a small portion of PGK1 to the mitochondria, where PGK1 phosphorylates and activates pyruvate dehydrogenase kinase isozyme 1 (PDHK1) to inhibit pyruvate metabolism in the mitochondria, thereby enhancing aerobic glycolysis (Li et al., 2016aLi X. Jiang Y. Meisenhelder J. Yang W. Hawke D.H. Zheng Y. Xia Y. Aldape K. He J. Hunter T. et al.Mitochondria-translocated PGK1 functions as a protein kinase to coordinate glycolysis and the TCA cycle in tumorigenesis.Mol. Cell. 2016; 61: 705-719Abstract Full Text Full Text PDF PubMed Scopus (238) Google Scholar). The nonmetabolic function of PGK1 is also exhibited in its regulation of autophagy, during which PGK1 phosphorylates Beclin 1 and significantly increases VPS34 activity and PI(3)P production to initiate autophagy (Qian et al., 2017aQian X. Li X. Cai Q. Zhang C. Yu Q. Jiang Y. Lee J.H. Hawke D. Wang Y. Xia Y. et al.Phosphoglycerate kinase 1 phosphorylates beclin1 to induce autophagy.Mol. Cell. 2017; 65: 917-931.e6Abstract Full Text Full Text PDF PubMed Scopus (151) Google Scholar, Qian et al., 2017bQian X. Li X. Lu Z. Protein kinase activity of the glycolytic enzyme PGK1 regulates autophagy to promote tumorigenesis.Autophagy. 2017; 13: 1246-1247Crossref PubMed Scopus (68) Google Scholar). In this study, we demonstrated that CDC7-ASK activity is inhibited by CDC7 protein kinase reaction product ADP. Casein kinase 2α (CK2α) activation induced by epidermal growth factor (EGF) receptor (EGFR) activation phosphorylates PGK1, leading to interaction between PGK1 and CDC7 to alleviate the inhibitory effect of ADP on CDC7-ASK. EGFR mutation and overexpression are detected in many types of cancer, and EGFR activation promotes DNA replication and cell-cycle progression (Kuan et al., 2001Kuan C.T. Wikstrand C.J. Bigner D.D. EGF mutant receptor vIII as a molecular target in cancer therapy.Endocr. Relat. Cancer. 2001; 8: 83-96Crossref PubMed Scopus (277) Google Scholar, Yang et al., 2012aYang W. Xia Y. Cao Y. Zheng Y. Bu W. Zhang L. You M.J. Koh M.Y. Cote G. Aldape K. et al.EGFR-induced and PKCε monoubiquitylation-dependent NF-κB activation upregulates PKM2 expression and promotes tumorigenesis.Mol. Cell. 2012; 48: 771-784Abstract Full Text Full Text PDF PubMed Scopus (182) Google Scholar). However, the mechanism underlying EGFR activation-regulated CDC7 activation and subsequent DNA replication is largely unknown. To this end, we immunoprecipitated CDC7 in EGFR-overexpressing U87 (U87/EGFR) glioblastoma (GBM) cells and performed liquid chromatography (LC)-tandem mass spectrometry (MS) analyses, which revealed that PGK1 and previously known CDC7-interacting proteins, such as ASK, were associated with CDC7 (Figures 1A and S1A). A co-immunoprecipitation assay demonstrated that EGF-induced interaction between PGK1 and CDC7 in U87 and T98G GBM cells was blocked by treatment with the EGFR inhibitor AG1478 (Figures 1B and S1B), demonstrating that EGFR activation is required for the association of CDC7 with PGK1. In line with this finding, expression of constitutively active EGFRvIII induced the binding of PGK1 to CDC7 (Figure S1C), and EGF stimulation induced the co-localization of PGK1 and CDC7 in the nuclei of U87 cells (Figure S1D) without obviously altering the subcellular distribution of PGK1 and CDC7 (Figure S1E). To understand the nuclear function of PGK1 in response to EGFR activation, we next analyzed the mechanism of EGFR activation-induced binding of PGK1 to CDC7. We pretreated U87/EGFR cells with LY294002, SU6566, SP600125, and TBB, which inhibited EGF-induced activation of phosphoinositide 3-kinase, c-Src, c-Jun N-terminal kinase (JNK), and CK2, respectively (Figure S1F). We found that only CK2 inhibition blocked EGF-induced binding of PGK1 to CDC7 (Figure 1C). Similar results were obtained with expression of the catalytically inactive CK2α K68M mutant (Ji et al., 2009Ji H. Wang J. Nika H. Hawke D. Keezer S. Ge Q. Fang B. Fang X. Fang D. Litchfield D.W. et al.EGF-induced ERK activation promotes CK2-mediated disassociation of alpha-Catenin from beta-Catenin and transactivation of beta-Catenin.Mol. Cell. 2009; 36: 547-559Abstract Full Text Full Text PDF PubMed Scopus (206) Google Scholar) (Figure 1D). A co-immunoprecipitation assay demonstrated that CK2α bound to PGK1 in response to EGF stimulation (Figure 1E). An in vitro glutathione S-transferase (GST) pull-down assay revealed that purified GST-PGK1 interacted with purified His-CK2α (Figure S1G), indicating that these two proteins can directly interact with each other. Analyses of PGK1 amino acid sequences suggested that PGK1 S256 is a potential CK2α-mediated phosphorylation residue. An in vitro phosphorylation assay demonstrated that purified wild-type (WT) His-CK2α, but not His-CK2α K68M, phosphorylated WT GST-PGK1, but not the GST-PGK1 S256A mutant, in the presence of [γ32P]-ATP, and this phosphorylation was detected using a validated, specific anti-PGK1 pS256 antibody (Figures 1F and S1H). Consistent with this in vitro result, EGF induced PGK1 S256 phosphorylation (Figures 1G and S1H), a portion of which was distributed in the nucleus (Figure S1H), and this phosphorylation was blocked by pretreatment of U87/EGFR cells with a CK2α inhibitor (Figure 1G). Streptavidin pull-down of S protein, Flag epitope, and streptavidin-binding peptide (SFB)-tagged PGK1 protein in U87/EGFR cells demonstrated that WT PGK1, but not PGK1 S256A, bound to CDC7 in response to EGF stimulation, and this interaction was abrogated by treatment with calf intestinal alkaline phosphatase (CIP) (Figure 1H). Consistent with this result, purified active His-CK2α enabled purified WT GST-PGK1, but not purified GST-PGK1 S256A, to bind to purified His-CDC7, and this interaction was abrogated by CIP treatment (Figure 1I). These results indicated that CK2α-mediated PGK1 phosphorylation promotes the binding of PGK1 to CDC7. It was reported that EGF-induced activation of extracellular signal-regulated kinase (ERK) results in phosphorylation and activation of CK2α (Ji et al., 2009Ji H. Wang J. Nika H. Hawke D. Keezer S. Ge Q. Fang B. Fang X. Fang D. Litchfield D.W. et al.EGF-induced ERK activation promotes CK2-mediated disassociation of alpha-Catenin from beta-Catenin and transactivation of beta-Catenin.Mol. Cell. 2009; 36: 547-559Abstract Full Text Full Text PDF PubMed Scopus (206) Google Scholar). As expected, U0126 ERK inhibitor treatment blocked EGF-induced binding of CK2α to PGK1 in both U87/EGFR and T98G cells (Figures S1I and S1J). In contrast with WT CK2α, CK2α T360/S362A, with mutation of ERK phosphorylation residues, lost its association with PGK1 (Figure S1K). An in vitro GST pull-down assay demonstrated that inclusion of purified active His-ERK2 dramatically increased the binding of purified WT GST-CK2α, but not purified GST-CK2α T360/S362A, to purified His-PGK1, and this ERK2-enhanced interaction was abrogated by CIP treatment (Figure S1L). In addition, ERK-phosphorylated CK2α induced the binding of purified GST-PGK1 to purified His-CDC7 but not to purified His-ASK (Figure S1M). These results indicated that EGF-induced and ERK-activated CK2α phosphorylates PGK1 at S256, leading to interaction between PGK1 and CDC7. To determine whether the interaction between PGK1 and CDC7 regulates CDC7 activity, we depleted PGK1 in U87/EGFR and T98G cells and expressed RNA interference-resistant (r) WT Flag-rPGK1 or Flag-rPGK1 S256A in these cells (Figure S2A). Expression of Flag-rPGK1 S256A, which had unchanged glycolytic activity (Figure S2B), did not alter the EGF-enhanced glycolytic rate as measured in a 3H-glucose labeling experiment (Figure S2C). We found that EGF stimulation resulted in phosphorylation of the CDC7 substrate MCM2 S53, and this phosphorylation was greatly reduced by PGK1 depletion (Figure 2A). Of note, this phosphorylation reduction was restored by expression of WT Flag-rPGK1, but not Flag-rPGK1 S256A (Figure 2A). Consistent results demonstrated that EGF-induced CDC7/ASK complex formation was disrupted by PGK1 depletion and reconstituted expression of rPGK1 S256A (Figure 2B), supporting that PGK1 S256 phosphorylation and the interaction between PGK1 and CDC7 are important for EGF-enhanced CDC7 activation. To measure the nuclear concentrations of relevant metabolites, we used green fluorescent protein (GFP)-tagged nuclear membrane surface protein KASH (Swiech et al., 2015Swiech L. Heidenreich M. Banerjee A. Habib N. Li Y. Trombetta J. Sur M. Zhang F. In vivo interrogation of gene function in the mammalian brain using CRISPR-Cas9.Nat. Biotechnol. 2015; 33: 102-106Crossref PubMed Scopus (548) Google Scholar) (Figures S2D and S2E) to purify nuclei of U87/EGFR cells (Figure S2F) without contamination of non-nucleus proteins (Figure S2G). Mass spectrometric analyses demonstrated that the nuclear concentrations of ADP, ATP, and BPG in these cells were about 30 μM, 3 mM, and 1 mM, respectively (Figure S2H). The measured ADP and ATP concentrations are consistent with those in previous reports (Ando et al., 2012Ando T. Imamura H. Suzuki R. Aizaki H. Watanabe T. Wakita T. Suzuki T. Visualization and measurement of ATP levels in living cells replicating hepatitis C virus genome RNA.PLoS Pathog. 2012; 8: e1002561Crossref PubMed Scopus (75) Google Scholar, Gribble et al., 2000Gribble F.M. Loussouarn G. Tucker S.J. Zhao C. Nichols C.G. Ashcroft F.M. A novel method for measurement of submembrane ATP concentration.J. Biol. Chem. 2000; 275: 30046-30049Crossref PubMed Scopus (241) Google Scholar, Huang et al., 2010Huang H. Zhang X. Li S. Liu N. Lian W. McDowell E. Zhou P. Zhao C. Guo H. Zhang C. et al.Physiological levels of ATP negatively regulate proteasome function.Cell Res. 2010; 20: 1372-1385Crossref PubMed Scopus (103) Google Scholar, Larcombe-McDouall et al., 1999Larcombe-McDouall J. Buttell N. Harrison N. Wray S. In vivo pH and metabolite changes during a single contraction in rat uterine smooth muscle.J. Physiol. 1999; 518: 783-790Crossref PubMed Scopus (52) Google Scholar). Similar nuclear ATP concentrations in intact U87/EGFR cells were also observed via real-time measurement using a fluorescent indicator of ATP, nucAT1.03 (Figure S2I) (Imamura et al., 2009Imamura H. Nhat K.P. Togawa H. Saito K. Iino R. Kato-Yamada Y. Nagai T. Noji H. Visualization of ATP levels inside single living cells with fluorescence resonance energy transfer-based genetically encoded indicators.Proc. Natl. Acad. Sci. USA. 2009; 106: 15651-15656Crossref PubMed Scopus (709) Google Scholar). We next performed an in vitro phosphorylation reaction with incubation of purified GST-CDC7, His-ASK, and abundant His-MCM2 fragment containing the N-terminal and CDC7-phosphosphorylaton region (MCM2-N) in the presence of 3 mM ATP. A GST pull-down assay demonstrated that the interaction between CDC7 and ASK was diminished after prolonged incubation, and this diminished interaction was accompanied with increasing ADP concentration (reaching about 130 μM), attenuated increase of MCM2 phosphorylation (Figures 2C and 2D), and limited reduction of ATP concentration (to about 2.8 mM) (Figure S2J). Given the minimal changes in ATP concentration, we hypothesized that ADP production affected the interaction between CDC7 and ASK. As expected, ADP dosage-dependently reduced the association between CDC7 and ASK (Figure 2E) and inhibited CDC7-mediated MCM2 phosphorylation (Figure 2F). In addition, CDC7/ASK complex formation was not affected by the presence of a large amount of ATP (Figure S2K), and ADP still disrupted the association of an ATP-binding-deficient CDC7 K90R mutant with ASK (Jiang and Hunter, 1997Jiang W. Hunter T. Identification and characterization of a human protein kinase related to budding yeast Cdc7p.Proc. Natl. Acad. Sci. USA. 1997; 94: 14320-14325Crossref PubMed Scopus (67) Google Scholar) (Figure S2L). Of note, ADP bound to WT CDC7 but not to ASK (Figure 2G). The CDC7 K90R mutant, which reduced its binding to ADP, had intact association with ASK, suggesting that a different ADP-binding region than the catalytic domain is involved in the allosteric regulation of CDC7-ASK interaction. Consistent with these results, the fused portions of CDC7 protein required for catalytic activity (Hughes et al., 2012Hughes S. Elustondo F. Di Fonzo A. Leroux F.G. Wong A.C. Snijders A.P. Matthews S.J. Cherepanov P. Crystal structure of human CDC7 kinase in complex with its activator DBF4.Nat. Struct. Mol. Biol. 2012; 19: 1101-1107Crossref PubMed Scopus (52) Google Scholar) were able to bind to ASK in an ADP-independent manner (Figure S2M). To identify the ADP-binding region regulating the binding of CDC7 to ASK, we mixed purified bacterially expressed noncatalytic fragments of CDC7 with ADP and showed that only the kinase insert 2 region (228–359 aa) of CDC7 bound to ADP (Figure S2N). CDC7 with deletion of this region abrogated the inhibitory effect of ADP on the interaction between CDC7 and ASK (Figure S2O). These results suggested that ADP binds to a region of CDC7 that is distinct from the ATP-binding site and disrupts the CDC7/ASK complex. In line with the in vitro results, the addition of equal amounts of ADP (0.6 mM) and ATP (0.6 mM) to digitonin-permeabilized cells, which increased the nuclear ADP concentration (to about 140 μM) (Figure S2P) to comparable levels that broke up the CDC7/ASK complex in vitro (Figures 2C and 2D), disrupted the EGF-induced interaction between CDC7 and ASK (Figure S2Q). In addition, depletion of ATP by treating the nuclear lysate of U87/EGFR cells with apyrase did not affect the stability of this complex (Figure S2R). These results suggested that ADP, but not ATP, binds to an allosteric region of CDC7 and reduces the association between CDC7 and ASK. PGK1 converts 1,3-BPG to 3-PG and ADP to ATP. Given that EGF stimulation did not obviously alter nuclear concentrations of ADP, ATP, or BPG (Figure S2H), we hypothesized that CDC7-associated PGK1 locally reduced ADP levels by converting it to ATP. To test this hypothesis, we incubated purified ASK, MCM2-N, WT GST-CDC7, and catalytically inactive GST-CDC7 D196N with purified WT PGK1, PGK1 S256A, or inactive PGK1 T378P in the presence or absence of 1,3-BPG. GST pull-down analyses after the kinase reaction for 30 min, enabling accumulation of ADP (Figure 2C), demonstrated that only the presence of both WT PGK1 and 1,3-BPG greatly reduced ADP levels (Figure 3A) and enhanced the interaction between CDC7 and ASK as well as MCM2 phosphorylation (Figure 3B), whereas inactive GST-CDC7 D196N, which does not hydrolyze ATP, remained bound to ASK. In addition, expression of an MCM2-N phosphorylation-deficient (PD) mutant in U87/EGFR cells (Cho et al., 2006Cho W.H. Lee Y.J. Kong S.I. Hurwitz J. Lee J.K. CDC7 kinase phosphorylates serine residues adjacent to acidic amino acids in the minichromosome maintenance 2 protein.Proc. Natl. Acad. Sci. USA. 2006; 103: 11521-11526Crossref PubMed Scopus (74) Google Scholar), which was resistant to phosphorylation by CDC7 (Figure S3A), inhibited the binding of CDC7 to WT MCM2-N (Figure S3B) and subsequent phosphorylation of WT MCM2-N (Figure S3C). Furthermore, MCM2-N PD expression dosage-dependently rescued PGK1 S256A-mediated disruption of CDC7/ASK complex formation (Figure 3C). This rescuing effect was abrogated by the addition of ADP to the cell culture medium. These results strongly suggested that CDC7-dependent MCM phosphorylation increased the local concentration of ADP that disrupts the CDC7/ASK complex and inhibits CDC7 activity. EGF-induced binding of PGK1 to CDC7 might convert local ADP to ATP and promotes CDC7 activation. CDC7-mediated MCM protein phosphorylation results in assembly of CDC45, as well as GINS proteins SLD5, PSF1, PSF2, and PSF3, on each MCM2–MCM7 complex associated with chromatin to form CDC45-MCM-GINS helicases for DNA replication (Botchan and Berger, 2010Botchan M. Berger J. DNA replication: making two forks from one prereplication complex.Mol. Cell. 2010; 40: 860-861Abstract Full Text Full Text PDF PubMed Scopus (19) Google Scholar, Evrin et al., 2009Evrin C. Clarke P. Zech J. Lurz R. Sun J. Uhle S. Li H. Stillman B. Speck C. A double-hexameric MCM2–7 complex is loaded onto origin DNA during licensing of eukaryotic DNA replication.Proc. Natl. Acad. Sci. USA. 2009; 106: 20240-20245Crossref PubMed Scopus (387) Google Scholar, Ilves et al., 2010Ilves I. Petojevic T. Pesavento J.J. Botchan M.R. Activation of the MCM2–7 helicase by association with Cdc45 and GINS proteins.Mol. Cell. 2010; 37: 247-258Abstract Full Text Full Text PDF PubMed Scopus (420) Google Scholar, Moyer et al., 2006Moyer S.E. Lewis P.W. Botchan M.R. Isolation of the Cdc45/Mcm2–7/GINS (CMG) complex, a candidate for the eukaryotic DNA replication fork helicase.Proc. Natl. Acad. Sci. USA. 2006; 103: 10236-10241Crossref PubMed Scopus (544) Google Scholar). As expected, PGK1 depletion in U87/EGFR and T98G GBM cells (Figure S2A) reduced the EGF-induced binding of CDC45 and SLD5 to MCM2 (Figure 4A) and chromatin association of CDC45, SLD5, PSF1, and PSF3 (Figure 4B). This reduction was restored by reconstituted expression of WT rPGK1, but not rPGK1 S256A (Figures 4A, 4B, and S2A). In addition, reconstituted expression of rPGK1 S256A in PGK1-depleted H1299 non-small cell lung cancer cells and MDA-MB-231 human breast cancer cells (Figure S4A) also blocked the EGF-induced chromatin association of CDC45, SLD5, PSF1, and PSF3 (Figure S4B), suggesting that the observed PGK1-dependent CDC45-MCM-GINS formation is not cell-line or tumor-type dependent. Consistent with this finding, a chromatin immunoprecipitation (ChIP) assay demonstrated that EGF stimulation induced binding of CDC45 and SLD5 to the DNA replication origins located within the LMNB2 (encoding lamin B2) and MCM4 loci (Salsi et al., 2009Salsi V. Ferrari S. Ferraresi R. Cossarizza A. Grande A. Zappavigna V. HOXD13 binds DNA replication origins to promote origin licensing and is inhibited by geminin.Mol. Cell. Biol. 2009; 29: 5775-5788Crossref PubMed Scopus (27) Google Scholar) (Figure 4C) without affecting the association of histone H3 with these loci (Figure S4C), and this increased binding of CDC45 and SLD5 was blocked by PGK1 depletion and rPGK1 S256A expression (Figure 4C). We next incubated tumor cells with 5-bromo-2′-deoxyuridine (BrdU) and found that EGF stimulation induced BrdU incorporation into U87/EGFR and T98G GBM cells using flow cytometric (Figure 4D) analyses and enzyme-linked immunosorbent assay (ELISA) (Figure S4D) when the cells entered S phase (Figure S4E). PGK1 depletion or rPGK1 S256A expression in these cells blocked EGF-induced DNA synthesis (Figures 4D and S4D) but did not affect the percentage of BrdU-positive cells (Figure S4F). In addition, incubation of digitonin-permeabilized cells with ADP or ATP revealed that ADP, but not ATP, reduced EGF-induced BrdU incorporation (Figures 4E and S4G). These results indicated that EGF-induced binding of PGK1 to CDC7 promotes DNA replication. To determine the role of the association between PGK1 and CDC7 in tumorigenesis, we examined the proliferation of U87/EGFRvIII and T98G/EGFRvIII cells with depletion of PGK1 and reconstituted expression of WT rPGK1 or rPGK1 S256A (Figure S5A). As expected, depletion of PGK1 and rPGK1 S256A expression inhibited cell proliferation (Figure 5A). In addition, the clonogenic proliferative ability of U87/EGFRvIII cells was reduced by EGFR and CK2 inhibition, and this inhibition was partially rescued by expression of PGK1 with the S256D phosphorylation-mimic mutant of PGK1 (Figures S5B and S5C). Next, we intracranially injected U87/EGFRvIII cells into athymic nude mice. Dissection of the mouse brains 2 weeks after injection revealed that the animals injected with U87/EGFRvIII cells with reconstituted expression of WT rPGK1 had rapid tumor growth (Figure 5B). In contrast, much smaller tumor was detected in the mice injected with U87/EGFRvIII cells with reconstituted expression of rPGK1 S256A mutant (Figure 5B). In addition, immunohistochemical (IHC) staining of tumor samples with an anti-PGK1 pS256 antibody and antibodies against MCM pS53 and BrdU demonstrated that rPGK1 S256A expression greatly abrogated PGK1 pS256 staining and reduced the levels of MCM pS53 and BrdU (Figure 5C). These results indicated that interaction between PGK1 and CDC7 is required for EGF-promoted and CDC7-mediated phosphorylation of MCMs, DNA replication, cell proliferation, and brain tumorigenesis. To determine the clinical significance of the observed PGK1-regulated DNA replication, we performed IHC analyses and found that the phosphorylation levels of CK2α T360/S362, PGK1 S256, and MCM2 S53 in tumor samples obtained from 20 patients with low-grade diffuse astrocytoma (World Health Organization grade II) were markedly lower than those from 30 patients with anaplastic astrocytoma (grade III) and 50 patients with GBM (grade IV) (Figures 6A and 6B ). However, the total expression levels of CK2α, PGK1, and MCM2 did not differ in these tumor samples (Figures S6A and S6B). In addition, we revealed that the phosphorylation levels of CK2α T360/S362, PGK1 S256, and MCM2 S53 were positively correlated with each other in glioma specimens (Figure 6C). Quantification of the IHC staining on a scale of 0 to 8 demonstrated that these correlations were significant (Figure 6C). These results supported a role for CK2α-dependent PGK1 phosphorylation in the clinical aggressiveness of human glioma. EGFR mutation and overexpression are detected in many types of cancer. For instance, the constitutively active EGFRvIII mutant, which lacks 267 aa in its extracellular domain, is commonly found in GBMs and breast, ovarian, prostate, an" @default.
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• W2898767623 title "Nuclear PGK1 Alleviates ADP-Dependent Inhibition of CDC7 to Promote DNA Replication" @default.
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