View metadata, citation and similar papers at core.ac.uk brought to you by CORE

provided by Elsevier - Publisher Connector

FEBS Letters 588 (2014) 3074–3080

journal homepage: www.FEBSLetters.org

Phosphoglucomutase1 is necessary for sustained cell growth under repetitive depletion ⇑ ⇑ Eunju Bae, Hee Eun Kim, Eunjin Koh , Kyung-Sup Kim

Department of Biochemistry and Molecular Biology, Brain Korea 21 PLUS Project for Medical Sciences, Institute of Genetic Science, Integrated Genomic Research Center for Metabolic Regulation, Yonsei University College of Medicine, Seoul 120-752, Republic of Korea

article info abstract

Article history: (PGM)1 catalyzes the reversible conversion reaction between glucose-1-phos- Received 20 March 2014 phate (G-1-P) and glucose-6- (G-6-P). Although both G-1-P and G-6-P are important inter- Revised 9 June 2014 mediates for glucose and glycogen metabolism, the biological roles and regulatory mechanisms of Accepted 10 June 2014 PGM1 are largely unknown. In this study we found that T553 is obligatory for PGM1 stability and Available online 18 June 2014 the last C-terminal residue, T562, is critical for its activity. Interestingly, depletion of PGM1 was asso- Edited by Judit Ovádi ciated with declined cellular glycogen content and decreased rates of glycogenolysis and glycogen- esis. Furthermore, PGM1 depletion suppressed cell proliferation under long-term repetitive glucose depletion. Our results suggest that PGM1 is required for sustained cell growth during nutritional Keywords: Phosphoglucomutase changes, probably through regulating the balance of G-1-P and G-6-P in order to satisfy the cellular Glycogen demands during nutritional stress. Glycogenolysis Ó 2014 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved. Glycogenesis Nutritional stress

1. Introduction PGM1. Previous studies have shown that phosphorylated 108 of PGM1 acts as a phosphoryl group donor to produce Cancer cells often encounter metabolic stresses such as glucose G-1,6-BP [5], that of PGM in yeast appears to affect depletion or hypoxia and have to use alternative metabolic path- its distribution [12], and that p21-activated kinase1 (Pak1) phos- ways for survival and proliferation. Many involved in phorylates threonine 466 of PGM1 and enhances its enzymatic have been studied in the context of metabolic repro- activity [13]. gramming of cancer cells [1–3]. As a substrate and/or a product of PGM1, G-1-P is an important Phosphoglucomutase 1 (PGM1; EC 5.4.2.2) catalyzes the revers- intermediate in glycogen, galactose, , and glycolipid ible conversion between glucose-1-phosphate (G-1-P) and metabolism, therefore involved in various cellular functions glucose-6-phosphate (G-6-P) via the intermediate glucose-1,6-bis- [14–16]. Likewise, G-6-P acts as a fundamental metabolic interme- phosphate (G-1,6-BP) in the presence of Mg2+ [4,5]. PGM1, which diate for glycolysis as well as the pentose phosphate pathway to belongs to the phosphohexose mutase family, account for provide precursors for anabolic pathways and cofactors required 85–95% of the total PGM activity, and other isoforms, such as for cell proliferation [17]. Therefore, it is conceivable that PGM1 PGM2 and PGM3, are for remainder of PGM activity in most tissues plays a critical role in cellular functions including cell survival even though PGM2 mainly functions as phosphopentose mutase and proliferation by maintaining intracellular levels of G-1-P and [6–8]. PGM1 has two major isoforms from alternatively spliced G-6-P through their rapid interconversion, thus contributing to first with distinct tissue distributions [8]. There are several metabolic homeostasis. reports demonstrated that defects in PGM1 caused abnormal glyco- Recently, considerable attention has been paid to the role of gly- gen accumulation, impaired N-glycosylation and muscle symp- cogen metabolism in cancer cell survival and growth under hyp- toms such as exercise intolerance and rhabdomyolysis [9–11]. oxic and/or glucose depleted conditions [18–21]. Although PGM1 Although the classic enzymology of PGM1 has been extensively appears to be associated with hypoxia-induced accumulation of studied, there are limited studies on the enzymatic regulation of glycogen in cancer cells [18], the physiological role of PGM1 in can- cer cell survival and proliferation under conditions of nutrient stress is largely unknown. ⇑ Corresponding authors. Fax: +82 2 312 5041. In this study we present evidence supporting a new regulatory E-mail addresses: [email protected] (E. Koh), [email protected] (K.-S. Kim). mechanism of PGM1 stability and activity that is attributed to

http://dx.doi.org/10.1016/j.febslet.2014.06.034 0014-5793/Ó 2014 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved. E. Bae et al. / FEBS Letters 588 (2014) 3074–3080 3075

C-terminal threonine residues. Furthermore, we show that PGM1 2.7. Quantification of G-1-P and G-6-P knockdown cells exhibit significantly diminished cell proliferation under repeated exposure to a limited glucose supply. We propose G-1-P and G-6-P was quantified by slight modification of fluori- that PGM1 plays a role in metabolic adaptation against detrimental metric assay [25]. nutritional status by rapid interconversion of G-1-P and G-6-P, leading to persistent cell proliferation. 2.8. Cell proliferation assay

Cells were plated in a 12-well plate (1 Â 104 cells/well) and 2. Materials and methods exposed to intermittent 4-h pulses of 10 mM glucose between 20-h periods of glucose depletion. Cell numbers were counted 2.1. Cell culture and reagents every 24 h for 4 days using ADAM-MC (NANOENTEK, South Korea). HEK 293T, MCF-7 and Hela cells were obtained from the ATCC 2.9. Statistical analysis (Manassas, VA, USA) and cultured in DMEM containing 10% FBS at 37 °C under 5% CO . 2 All results are expressed as the mean ± S.D. of at least three independent experiments. 2.2. Plasmids and transfections Detailed materials and methods are included in the Supplemen- tary materials. Human PGM1 cDNA (isoform1, NM_002633) was cloned into pSG5-KHA2M1 for HA-tag at N-terminus, pET-21a and pQE for 6ÂHis tag at C-terminus and N-terminus, respectively. pLKO 3. Results shRNA plasmids targeting all three PGM1 isoforms were purchased from Sigma–Aldrich. For transient transfection, Lipofectamine™ 3.1. Intact C-terminus of PGM1 is critical for its enzymatic activity Reagent (Invitrogen) was used. To assess the enzymatic activity of PGM we used bacterial puri- fied recombinant PGM1 containing six histidine tags 2.3. Western blot analysis (6ÂHis) at the N- or C-terminus (Fig. 1A). As shown in Fig. 1B, N-terminus 6ÂHis tagged PGM1 (6ÂHis-PGM1) had PGM enzy- Cultured cells were harvested in lysis buffer (0.5% NP-40, matic activity, whereas PGM1 with a C-terminus 6ÂHis tag 150 mM NaCl, 50 mM Tris–HCl, pH 7.4, 20 mM b-glycerophos- (PGM1-6ÂHis) completely lost activity. Addition of a potent activa- phate, 1 mM Na VO , 10 mM NaF) containing a protease inhibitor 3 4 tor of PGM1, G-1,6-BP, to the reaction mixture markedly cocktail (Roche). concentrations were determined by Brad- increased activity of 6ÂHis-PGM1 as expected, but no enhance- ford assay [22] or bicinconinic acid (BCA) assay [23]. ment was observed with PGM1-6ÂHis (Fig. 1C). These results imply that an intact C-terminal region of PGM1 is critical for enzy- 2.4. Recombinant protein expression matic function.

pQE30-PGM1 and pET21a-PGM1 was transformed into Rosset- 3.2. Characterization of the functional significances of PGM1 ta2 pLacI strain and BL21 (DE3) pLysS strain, respectively. Soluble C-terminal threonine residues recombinant proteins were purified using Ni–NTA agarose (Qiagen). The C-terminal domain of the functionally similar enzyme phosphoglycerate mutase (PGAM), which catalyzes the conversion 2.5. Phosphoglucomutase (PGM) activity assay of 3-phosphoglycerate (3PG) to 2-phosphoglycerate (2PG) through 2,3-bisphosphoglycerate (BPG) as an intermediate, is known to Measurement of G-6-P coupled with the G-6-P dehydrogenase affect catalytic efficiency [26–28]. Based on our observation that was used for the activity assay [24]. addition of C-terminal 6ÂHis tags to PGM1 totally compromised its enzymatic activity (Fig. 1), we assumed that the C-terminus of 2.6. Glycogen quantification PGM1 similarly contains critical residues that play a regulatory role in enzymatic activity. Glycogen was quantified using a starch assay kit (Sigma– The C-terminus of PGM1 contains repeated threonine residues, Aldrich) according to the manufacturer’s instructions. TXXTXXTXXT, that are highly conserved among different species

Fig. 1. Loss of PGM activity in PGM1 with C-terminal 6ÂHis tag. (A) Bacterial recombinant PGM1 constructs containing 6ÂHis tags localized at the C- or N-terminus. Measurement of PGM activity without 20 lM G-1,6-BP (B) or with G-1,6-BP in the enzyme assay reaction mixture (C). The results are representative of at least three independent experiments conducted in triplicate. 3076 E. Bae et al. / FEBS Letters 588 (2014) 3074–3080

Fig. 2. Functional significances of PGM1 C-terminal threonine residues. (A) Alignment of the C-terminal sequences of PGM1 showing conserved threonine residues. (B) Wild type and threonine mutants of PGM1 constructs, pSG5-KHA2M1-hPGM1, which have N-terminus HA tag, were transiently expressed and whole cell lysates of HEK293T cells were used for Western blot analysis. # indicates endogenous PGM1 protein expression. Green fluorescence protein (GFP) expression vector was co-transfected to evaluate transfection efficiency. Each plasmid was transfected in triplicate. Dividing lines on blot data indicate non-adjacent lanes of the same blot. (C) Recombinant PGM1 proteins of wild type, T553E and T553A mutants. Lysates of E. coli expressing recombinant proteins were fractionated into soluble supernatant (S) and pellet (P) by centrifugation and subjected to SDS–PAGE. Two separate colonies were tested for T553E and T553A mutants (#1 and #2). M: protein marker (D) PGM activity assay using HEK293T cell lysate. PGM1 activity of mock was measured and the activity of each mutant was obtained by subtraction of mock activity from measured value of each sample (E) PGM activity by measuring the conversion of G-1-P to G-6-P with affinity column purified recombinant PGM1 proteins in the presence or absence of 20 lM G-1,6-BP. (F) Reverse PGM1 activity by measuring the conversion of G-6-P to G-1-P with purified recombinant PGM1 proteins in the presence of G-1,6-BP. (G) PGM activity was assayed with purified recombinant PGM1 proteins (100 ng of WT and 200 ng of T562A) in the presence of indicated concentrations of G-1,6-BP. The results are representative of at least three separate experiments conducted in triplicate. A unit of activity was defined as the amount of enzyme required for the formation of 1 lmol of G-6-P per minute.

(Fig. 2A). Because threonine residues can be post-translationally to 2ÂHA tag at N-terminus. The expression level of exogenous wild modified by , we individually replaced each threo- type PGM1 was much higher than that of endogenous enzyme. nine residue with glutamic acid (E) or alanine (A) to analyze its However, expression of T553 mutants, particularly T553E, were functional consequences. significantly reduced relative to wild type protein (Fig. 2B). No dif- To assay the enzymatic activity of the mutated PGM1, each ferences in GFP expression indicate that the altered expression of mutant was expressed along with GFP in HEK293T cells (Fig. 2B). the mutants were not due to transfection efficiency. To further Exogenous PGM1 migrated slower than endogenous PGM1 due assess the role of T553 in protein expression, we attempted to E. Bae et al. / FEBS Letters 588 (2014) 3074–3080 3077 express T553 mutants in Escherichia coli. As shown in Fig. 2C, a sig- G-1,6-BP, resulting in dephospho-PGM1 inactive form. Taken nificant proportion of wild type recombinant PGM1 was soluble together, our data suggest that the T562 residue is critically form. However, T553E mutant protein was mostly expressed as required for efficient catalytic reaction of PGM1. insoluble aggregates forming inclusion bodies. Likewise, the T553A mutation suppressed expression of the soluble form com- 3.3. PGM1 affects glycogen metabolism in cancer cells under pared with wild type. These results suggest that T553 is a critical conditions of glucose depletion residue for folding of PGM protein into the correct conformation. Notably, mutation (T562E, and T562A) or deletion of T562 Recent studies have suggested a critical role of glycogen in can- (DT562) markedly attenuated PGM activity whereas mutation of cer cell survival and proliferation [18–21]. Although PGM1 acts as a T556 and T559 did not affect activity (Fig. 2D). Wild type and all gatekeeper in the metabolic pathway between glycogen and glu- of these mutants were efficiently expressed almost same levels cose by catalyzing the interconversion of G-1-P and G-6-P, there in HEK293T cells (Fig. S1A). In particular, T562E and DT562 muta- are few studies focusing on PGM1 function with respect to glyco- tions almost completely abolished enzyme activity even in the gen content. On the basis of a recent report showing that induction presence of G-1,6-BP. It is also notable that the severely repressed of glycogen storage and PGM1 expression are simultaneously activity of the T562A mutant was rescued by the addition of G-1, occurred under hypoxic conditions [18], we investigated the direct 6-BP. To exclude the effects of contaminants in HEK293T whole cell effects of PGM1 on the cellular levels of glycogen. lysates, bacterial soluble recombinant PGM1 proteins were homo- To define a possible role of PGM1 in cellular glycogen metabo- geneously purified and their specific activities in both directions, lism, we performed knockdown of PGM1 in MCF7 cells and quanti- conversion of G-1-P to G-6-P and vice versa, were measured fied the glycogen content. Compared with cells expressing negative (Figs. 2E,F and S1B). Consistent with the results from HEK293 cells, control shRNA (shNC), PGM1 knockdown cells (shPGM1) had lower mutation of T556 and T559 did not significantly affect PGM enzy- glycogen in high-glucose medium with no significant difference in matic activity. In marked contrast, the T562E mutant completely glucose uptake (Figs. 3A,B and S2). However, the overexpression of lost PGM activity even in the presence of G-1,6-BP. T562A mutant wild type PGM1 or catalytically defective T562E mutant failed to protein also showed severely decreased activity but the addition of increase the glycogen content indicating that the endogenous G-1,6-BP induced its activity in much higher folds than wild type PGM1 seems to be enough to reach the equilibrium state of glyco- enzyme (Fig. 2E). To precisely examine the response to G-1,6-BP gen level (Fig. S3). Moreover, the rate of glycogenolysis induced by in T562A mutant, we investigated the effect of concentrations of glucose deprivation was markedly decreased in shPGM1 cells com- G-1,6-BP on PGM activity of T562A mutant. Activity of the T562A pared with shNC cells, even though cellular glycogen was almost mutant was linearly elevated according to the increased concentra- completely depleted within 24 h after glucose starvation in both tions of G-1,6-BP up to 20 nM, and further increased up to 100 nM, cell lines with no changes in PGM1 expression level in control cells whereas the activity of wild type PGM1 was saturated below 5 nM (Figs. 3C and S4). In control shNC cells, the glycogen repletion was G-1,6-BP (Fig. 2G). These results indicate that the T562A mutation almost completed within 2 h after replenishment of glucose due to might destabilize the interaction with intermediary product, high rate of glycogen synthesis, whereas PGM1 knockdown

Fig. 3. PGM1 affects cellular glycogen content and its turnover rates. (A) MCF7 stable cells infected with lentivirus expressing shRNA PGM1 (shPGM1) or control shRNA (shNC) were assessed for PGM1 protein levels by Western blotting. GAPDH was used as an internal control. (B) Glycogen content in cells cultured at 25 mM glucose were measured and expressed as ng/lg protein. (C) Rate of glycogenolysis was measured upon glucose deprivation. Cells were cultured in 25 mM glucose containing medium for 24 h, washed with PBS, and incubated in medium containing no glucose. Intracellular glycogen levels were measured at the indicated times. (D) Rate of glycogen synthesis was measured upon 10 mM glucose replenishment after 24 h of glucose depletion. The average and S.D. of triplicates were represented. 3078 E. Bae et al. / FEBS Letters 588 (2014) 3074–3080 retarded the glycogenesis rate and prolonged the recovery of gly- energy demand for cell survival and proliferation [14–21]. Given cogen storage after glucose replenishment up to 6 h (Fig. 3D). that PGM1 affects the rate of G-1-P and glycogen turnover, we These results demonstrate that the glycogen levels were rapidly hypothesized that PGM1 maintains cell growth under the extracel- modulated in response to changes in extracellular glucose level lular nutritional fluctuations by enabling the interconversion of and that PGM1 is closely involved in cellular glycogen metabolism. G-1-P and G-6-P during metabolic adaptation (Fig. 4A). To examine the consequences of PGM1 depletion we generated 3.4. PGM1 is required for maintenance of cell proliferation during stable PGM1 knockdown MCF7 and Hela cells (Fig. S4). As shown in repetitive glucose depletion Fig. 4B and C, PGM1 knockdown did not affect cell proliferation in standard culture condition. Since prolonged glucose depletion According to previous reports, glycogen appears to be used induced cell death in both shNC and shPGM1, we developed a pro- when extracellular glucose levels are insufficient to meet the tocol that reflects repetitive nutritional stress by exposing cells to

Fig. 4. PGM1 is required for cell proliferation under repetitive glucose depletion. (A) Simple diagram showing utilization of G-1-P and G-6-P in the cellular environment. (B–E) MCF7 and Hela cells stably infected with lentivirus expressing shRNA PGM1 (shPGM1) or control shRNA (shNC) were cultured in 24 mM glucose containing medium (B and C) or nutrient stress conditions (periodic glucose depletion as described in Section 2) (D and E). Cell number was counted at 24-h intervals. The results are representative of at least two separate experiments conducted in triplicate. (F–H) Glycogen, G-1-P and G-6-P contents in MCF7 cells (j: NC and h: shPGM1). Cells were harvested at 2 h after 10 mM glucose refeeding (R) and 2 h after glucose deprivation (D) as denoted in D. E. Bae et al. / FEBS Letters 588 (2014) 3074–3080 3079 intermittent pulse of 10 mM glucose for 4 h between 20-h period glucose in cardiac muscle through the last 52 C-terminal residues of glucose depletion in order to evaluate the role of PGM1 in met- of PGM1 including T562, although effects of this interaction on abolic adaptation against long-term nutritional stress without PGM activity was not determined [30]. In skeletal muscle, PGM1 occurring cell death (Fig. S5). Cell numbers were counted every was reported to be inhibited by interaction with sarcoplasmic 24 h for 4 days. reticulum fraction, of which the blocking by treatment with deter- Notably, shNC cells continued to grow under conditions of gent recovered PGM1 activity, although the precise domain target- repetitive glucose depletion whereas cells lacking PGM1 showed ing to membrane was not determined [31]. These reports provide severely suppressed growth (Fig. 4D and E). This result could be the possibility that the interactions of C-terminus of PGM1 with expected by the fact that PGM1 mediates glycogen storage and gly- cytoskeletal proteins or membrane can be the regulatory mecha- cogen turnover rate as shown in Fig. 3C and D. To evaluate this nism as well as the covalent modification. hypothesis, glycogen, G-6-P, and G-1-P were measured in shNC In the present study we demonstrated that PGM1 knockdown and shPGM1 cells at the indicated time point in Fig. 4D (denoted is associated with decreased basal cellular glycogen levels by R and D). Addition of 10 mM glucose for 2 h after glucose deple- (Fig. 3B) but not totally depleted probably through the comple- tion for 20 h rapidly increased the glycogen, and then following 2 h mentation of PGM isoforms present in the cells. In addition, of glucose deprivation abruptly decreased glycogen in NC cells. shPGM1 showed suppression of glucose depletion-induced glyco- This dynamic change in glycogen level according to glucose avail- genolysis and glucose supplementation-induced glycogenesis ability was severely blunted by PGM1 knockdown (Fig. 4F). The lev- (Fig. 3C and D). These results strongly imply that PGM1 is closely els of G-1-P and G-6-P were also dynamically regulated by glucose involved in the modulation of glycogen content in response to availability and PGM1 knockdown impeded this regulation (Fig. 4G extracellular glucose levels. Most studies on cellular glycogen con- and H). These data imply that PGM1 is required for sustained pro- tent have focused on the activities of glycogen synthase [32] and liferation of cells exposed to nutrient stress by regulating glycogen glycogen phosphorylase [18–20]. Recently, protein phosphatase 1, metabolism. regulatory subunit 3C (PPP1R3C) was reported to promote glyco- gen accumulation under hypoxia [21], and another study showed 4. Discussion that PGM1 expression was increased as a target of HIF-1 to promote the first step of glycogenesis in hypoxia [18]. To the best PGM1 catalyzes the reversible conversion between G-1-P and of our knowledge, our study provides the first demonstration that G-6-P. Although G-1-P and G-6-P are important metabolic inter- PGM1 knockdown directly affects glycogen metabolism in cancer mediates of glucose and glycogen metabolism, and glycogen cells. metabolism in cancer cells was recently focused on recently To delineate the role of PGM1 in cell proliferation under condi- [14,18–20], the biological roles and regulatory mechanisms of tions of fluctuating energy stress, we exposed cells to long-term PGM1 are largely unknown. repetitive nutritional stress by providing intermittent 4-h pulses In this study, we present evidence for important roles of C-ter- of 10 mM glucose between 20-h periods of glucose depletion. minal threonine residues in PGM1 function. Furthermore, we pro- Under these conditions, control cells persistently continued to pose that PGM1 functions as a rheostat of permissive and grow, whereas PGM1 knockdown cells exhibited marked growth detrimental nutritional status by fine-tuning G-1-P turnover with repression (Fig. 4D and E). This result could be expected by our respect to cell proliferation (Fig. 4A). observations that PGM1 is required for rapid recovery of glycogen We provide several lines of evidence that the C-terminal threo- after glucose depletion (Figs. 3D and 4F) which in turn enable cells nine residues of PGM1 are critical for its stability and activity. We to survive followed by glucose depleted conditions. Taken together, showed that addition of C-terminal 6ÂHis tags to PGM1 totally PGM1 is required for the proliferation of cells that are exposed to compromised enzymatic activity (Fig. 1). C-terminal T533 muta- long-term metabolic stress, probably through regulating the flux tions decreased the expression level in animal cells and led to of G-1-P and G-6-P in response to cellular demands incurred by misfolded aggregates in E. coli (Fig. 2B and C). Since it is well doc- changes in the extracellular energy status. umented that mutations that markedly perturb protein folding Several recent studies have shown that glycogen accumulation trigger rapid hydrolysis [29], we concluded that the T533 residue is induced for cell survival and proliferation under hypoxia and/or is critical for PGM protein into the correct conformation. Deletion glucose-depleted conditions [18–21]. Therefore, it appears that of T562 or T562E mutation completely abolished PGM activity cancer cells store energy in glycogen reservoirs for glycolysis, and the T562A mutant protein required a high concentration of nucleic acid and de novo fatty acid synthesis when extracellular G-1,6-BP for enzymatic activity (Fig. 2D–G). The reaction catalyzed glucose levels are insufficient to meet the energy demands. We by PGM involves sequential formation of a complex of G-1-P and showed a critical role of PGM1 in cell proliferation using an exper- phosphorylated PGM1, conversion to G-1,6-BP and dephosphoryl- imental model that reflects the dynamic nutritional status of ated PGM1, and the final production of G-6-P and phosphorylated repetitive glucose depletion that might occur in the tumor micro- PGM1. Therefore, it is plausible that formation of a stable complex environment (Fig. 4A). Our observation that PGM1 affects glycogen between G-1,6-BP and dephosphorylated enzyme is important for synthesis and degradation indicates that PGM1 allows cancer cells the PGM1 catalytic reaction. If this complex is unstable, dissocia- to overcome nutrient-depleted conditions by facilitating adequate tion of G-1,6-BP will result in dephosphorylated PGM1 that has use of glycogen. However, we cannot exclude the possibility that no catalytic activity by itself. Thus, T562 residue might play a G-1-P is also required for the synthesis of UDP-glucose and UDP- key role in maintaining a stable complex between G-1,6-BP and galactose to produce and glycolipids which are dephosphorylated PGM1. important for cellular functions [9,14–16]. Therefore, beneficial PGM1 is known to be phosphorylated on serine 108 acting as a effects of galactose supplementation in shPGM1 cells need to be phosphoryl group donor in the production of G-1,6-BP [5], and on observed in future studies. threonine 466 by p21-activated kinase1 (Pak1) contributing to Taken together, our results suggest that PGM1 provides the enhanced activity [13]. Although present study has not elucidated ability to sustain cell growth throughout frequent changes in nutri- whether T562 is involved in the regulation of PGM activity in vivo, tional supply, probably through regulating the balance of G-1-P the previous reports provide the possibility. PGM1 is known to and G-6-P in order to satisfy the fluctuating cellular demands interacts with ZASP/Cyper in response to starvation of serum or incurred by nutritional stress. 3080 E. Bae et al. / FEBS Letters 588 (2014) 3074–3080

Acknowledgements [14] Hakomori, S. (1985) Aberrant glycosylation in cancer cell membranes as focused on glycolipids: overview and perspectives. Cancer Res. 45, 2405– 2414. We thank Dr. Jeon for performing FDG uptake assay. This work [15] Gahmberg, C.G. and Hakomori, S.I. (1973) Altered growth behavior of was supported by a National Research Foundation of Korea (NRF) malignant cells associated with changes in externally labeled glycoprotein Grant funded by the Korean Government (MSIP) (NRF-2011-003 and glycolipid. Proc. Natl. Acad. Sci. USA 70, 3329–3333. [16] Novelli, G. and Reichardt, J.K. (2000) Molecular basis of disorders of human 0086 and NRF-2011-0016206). galactose metabolism: past, present, and future. Mol. Genet. Metab. 71, 62–65. [17] Ward, P.S. and Thompson, C.B. (2012) Metabolic reprogramming: a cancer Appendix A. Supplementary data hallmark even warburg did not anticipate. Cancer Cell 21, 297–308. [18] Pelletier, J., Bellot, G., Gounon, P., Lacas-Gervais, S., Pouyssegur, J. and Supplementary data associated with this article can be found, in Mazure, N.M. (2012) Glycogen synthesis is induced in hypoxia by the hypoxia-inducible factor and promotes cancer cell survival. Front. Oncol. 2, the online version, at http://dx.doi.org/10.1016/j.febslet.2014.06. 18. 034. [19] Favaro, E. et al. (2012) Glucose utilization via glycogen phosphorylase sustains proliferation and prevents premature senescence in cancer cells. Cell Metab. References 16, 751–764. [20] Lee, W.N.P. et al. (2004) Metabolic sensitivity of pancreatic tumour cell apoptosis to glycogen phosphorylase inhibitor treatment. Br. J. Cancer 91, [1] Moon, J.S., Kim, H.E., Koh, E., Park, S.H., Jin, W.J., Park, B.W., Park, S.W. and Kim, 2094–2100. K.S. (2011) Kruppel-like factor 4 (KLF4) activates the transcription of the gene [21] Shen, G.M., Zhang, F.L., Liu, X.L. and Zhang, J.W. (2010) Hypoxia-inducible for the platelet isoform of phosphofructokinase (PFKP) in breast cancer. J. Biol. factor 1-mediated regulation of PPP1R3C promotes glycogen accumulation in Chem. 286, 23808–23816. human MCF-7 cells under hypoxia. FEBS Lett. 584, 4366–4372. [2] Hitosugi, T. et al. (2012) Phosphoglycerate mutase 1 coordinates glycolysis and [22] Bradford, M.M. (1976) A rapid and sensitive method for the quantitation of biosynthesis to promote tumor growth. Cancer Cell 22, 585–600. microgram quantities of protein utilizing the principle of protein-dye binding. [3] Christofk, H.R. et al. (2008) The M2 splice isoform of pyruvate is Anal. Biochem. 72, 248–254. important for cancer metabolism and tumour growth. Nature 452. 230-U74. [23] Smith, P.K. et al. (1985) Measurement of protein using bicinchoninic acid. [4] Najjar, V.A. and Pullman, M.E. (1954) The occurrence of a group transfer Anal. Biochem. 150, 76–85. involving enzyme (phosphoglucomutase) and substrate. Science 119, 631– [24] Lowry, O.H. and Passonneau, J.V. (1969) Phosphoglucomutase kinetics with 634. the of fructose, glucose, mannose, ribose, and galactose. J. Biol. [5] Pouyssegur, J., Shiu, R.P. and Pastan, I. (1977) Induction of two transformation- Chem. 244, 910–916. sensitive membrane polypeptides in normal fibroblasts by a block in [25] Zhu, A., Romero, R. and Petty, H.R. (2009) An enzymatic fluorimetric assay for glycoprotein synthesis or glucose deprivation. Cell 11, 941–947. glucose-6-phosphate: application in an in vitro Warburg-like effect. Anal. [6] Maliekal, P., Sokolova, T., Vertommen, D., Veiga-da-Cunha, M. and Van Biochem. 388, 97–101. Schaftingen, E. (2007) Molecular identification of mammalian phosphopen- [26] Winn, S.I., Watson, H.C., Harkins, R.N. and Fothergill, L.A. (1981) Structure and tomutase and glucose-1,6-bisphosphate synthase, two members of the alpha- activity of phosphoglycerate mutase. Philos. Trans. R. Soc. Lond. B Biol. Sci. D-phosphohexomutase family. J. Biol. Chem. 282, 31844–31851. 293, 121–130. [7] McAlpine, P.J., Hopkinson, D.A. and Harris, H. (1970) The relative activities [27] Walter, R.A., Nairn, J., Duncan, D., Price, N.C., Kelly, S.M., Rigden, D.J. and attributable to the three phosphoglucomutase loci (PGM1, PGM2, PGM3) in Fothergill-Gilmore, L.A. (1999) The role of the C-terminal region in human tissues. Ann. Hum. Genet. 34, 169–175. phosphoglycerate mutase. Biochem. J. 337 (Pt. 1), 89–95. [8] Putt, W., Ives, J.H., Hollyoake, M., Hopkinson, D.A., Whitehouse, D.B. and [28] Hallows, W.C., Yu, W. and Denu, J.M. (2012) Regulation of glycolytic enzyme Edwards, Y.H. (1993) Phosphoglucomutase 1: a gene with two promoters and phosphoglycerate mutase-1 by Sirt1 protein-mediated deacetylation. J. Biol. a duplicated first . Biochem. J. 296 (Pt. 2), 417–422. Chem. 287, 3850–3858. [9] Tegtmeyer, L.C. et al. (2014) Multiple phenotypes in phosphoglucomutase 1 [29] Goldberg, A.L. (2003) Protein degradation and protection against misfolded or deficiency. N. Engl. J. Med. 370, 533–542. damaged proteins. Nature 426, 895–899. [10] Stojkovic, T. et al. (2009) Muscle glycogenosis due to phosphoglucomutase 1 [30] Arimura, T. et al. (2009) Impaired binding of ZASP/Cypher with deficiency. N. Engl. J. Med. 361, 425–427. phosphoglucomutase 1 is associated with dilated cardiomyopathy. [11] Preisler, N. et al. (2013) Fat and carbohydrate metabolism during exercise in Cardiovasc. Res. 83, 80–88. phosphoglucomutase type 1 deficiency. J. Clin. Endocrinol. Metab. 98, E1235– [31] Lee, Y.S., Marks, A.R., Gureckas, N., Lacro, R., Nadal-Ginard, B. and Kim, D.H. E1240. (1992) Purification, characterization, and molecular cloning of a 60-kDa [12] Dey, N.B., Bounelis, P., Fritz, T.A., Bedwell, D.M. and Marchase, R.B. (1994) The phosphoprotein in rabbit skeletal sarcoplasmic reticulum which is an isoform glycosylation of phosphoglucomutase is modulated by source and heat of phosphoglucomutase. J. Biol. Chem. 267, 21080–21088. shock in Saccharomyces cerevisiae. J. Biol. Chem. 269, 27143–27148. [32] Pescador, N. et al. (2010) Hypoxia promotes glycogen accumulation through [13] Gururaj, A., Barnes, C.J., Vadlamudi, R.K. and Kumar, R. (2004) Regulation of hypoxia inducible factor (HIF)-mediated induction of glycogen synthase 1. phosphoglucomutase 1 phosphorylation and activity by a signaling kinase. Plos One 5. Oncogene 23, 8118–8127.