Regulation of Phosphoglucomutase 1 Phosphorylation and Activity by a Signaling Kinase

Regulation of phosphoglucomutase 1 phosphorylation and activity by a signaling kinase

Anupama Gururaj1, Christopher J Barnes1, Ratna K Vadlamudi1 and Rakesh Kumar*,1

1Department of Molecular and Cellular Oncology, The University of Texas MD Anderson Cancer Center, Houston, TX 77030, USA

We have identified a novel mechanism of cross-talk ylation and fatty metabolism have also been reported, between cell signaling and metabolic pathways, whereby which results in increased uptake of glucose, altered the signaling kinase p21-activated kinase 1 (Pak1)binds redox state in the cells and induction of cell survival to, phosphorylates and enhances the enzymatic activity of pathways (Unwin et al., 2003). Inside the cell, following phosphoglucomutase 1 (PGM), an important regulatory the conversion of glucose to glucose-6-phosphate (G-6- enzyme in cellular glucose utilization and energy home- P)(via hexokinase or phosphoglucomutase (PGM)),the ostasis. Pak1 and PGM were colocalized in model cell major pathways of glucose metabolism include glyco- systems and showed functional interactions in a physiolo- lysis and the pentose phosphate pathway. Although it gical setting. Strong direct interaction of PGM with Pak1 has been recognized that metabolic fluxes between but not Pak2, Pak3, or Pak4 was observed. PGM binding glycolysis and the pentose phosphate pathway are was within 75–149 amino acids (aa)of Pak1, while Pak1 particularly relevant for balancing cellular requirements binding to PGM was in the N-terminal 96 aa. Pak1- for energy and reducing equivalent homeostasis, a mediated phosphorylation of PGM selectively on threo- lacuna exists in our understanding of the manner in nine 466 significantly increased PGM enzymatic activity which the cell regulates the rates of these two basic and could be blocked by transfection with a dominant- metabolic pathways in response to various extracellular negative Pak1 expression vector and by Pak1-specific stimuli. small inhibitory RNA. Stable transfection of PGM into The biochemical pathways required to utilize glucose PGM-deficient K-562 leukemia cells further demonstrated as a carbon and energy source are highly conserved from the role of Pak1 in regulating PGM activity. The results bacteria to humans. PGM (EC 2.7.5.1)is an evolutio- presented here provide new evidence that the cell signaling narily conserved enzyme that regulates one of the most kinase Pak1 is a novel regulator of glucose metabolism important metabolic carbohydrate trafficking points in through its phosphorylation and regulation of PGM prokaryotic and eukaryotic organisms, catalysing the bi- activity. These findings suggest a new mechanism whereby directional interconversion of glucose-1-phosphate growth factor signaling may coordinately integrate (G-1-P)and G-6-P. In one direction, G-1-P produced metabolic regulation with established signaling functions from sucrose catabolism is converted to G-6-P, the first of cell cycle regulation and cell growth. intermediate in glycolysis. In the other direction, Oncogene (2004) 23, 8118–8127. doi:10.1038/sj.onc.1207969 conversion of G-6-P to G-1-P generates a substrate for Published online 20 September 2004 synthesis of UDP-glucose, which is required for synthesis of a variety of cellular constituents, including cell Keywords: signaling; PGM; glycolysis; cancer metabo- wall polymers and glycoproteins. PGM has been used lism extensively as a genetic marker for isozyme polymorph- ism among humans (Putt et al., 1993). PGM is known to be post-translationally modified by cytoplasmic glyco- sylation that does not seem to regulate its enzymatic activity but rather is implicated in the localization of the Introduction protein (Dey et al., 1994). Glucose 1,6 bisphosphate (Glc-1, 6-P2), a powerful regulator of carbohydrate Tumor cells typically demonstrate altered metabolism metabolism, has been demonstrated to be a potent and a disruption of regulation of glycolytic metabolism, activator of PGM (Boros et al., 2002). PGM is also respiration and a specific high glucose-utilizing meta- modified by phosphorylation on Ser108 as part of its bolic phenotype as compared to nontransformed cells. catalytic mechanism. However, no functional signifi- In particular, glycolytic metabolism is increased, as is cance, other than its role in the enzymatic process itself, the activity of the pentose phosphate cycle (Warburg, has been assigned to Ser108 phosphorylation, and a 1956). Concurrently, a reduction in oxidative phosphor- specific regulatory phosphorylation site and kinase have not been identified for PGM so far. Despite the critical *Correspondence: R Kumar; E-mail: [email protected] role of PGM in cellular carbon flux and energy Received 11 March 2004; revised 1 June 2004; accepted 10 June 2004; homeostasis, little is known regarding its functional published online 20 September 2004 regulation. PGM regulation by Pak1 signaling A Gururaj et al 8119 p21-activated kinase 1 (Pak1)is activated by growth (Figure 1c, top panel). Co-transfection of the auto- factor receptor tyrosine kinases via Rac or Cdc42 inhibitory fragment of Pak1 (83–149 amino acids (aa)) (Adam et al., 1998, 2000; Vadlamudi et al., 2000), by that acts in a dominant negative manner (Vadlamudi nonreceptor tyrosine kinases and by lipids (Bagheri- et al., 2002)resulted in decreased PGM phosphorylation Yarmand et al., 2001). The Pak1 enzyme is conserved in response to sphingomyelinase, indicating that Pak1 functionally through evolution from yeast to mammals. was responsible, at least in part, for the phosphorylation Wide ranges of biological activities have been shown to of PGM in vivo. Immunoprecipitation of Pak1 from the result from Pak1 phosphorylation of downstream same lysates and subsequent kinase assay using myelin substrates, including cell cytoskeletal reorganization basic protein as substrate (Figure 1c, lower panel) leading to increased motility, enhanced cell survival confirmed that Pak1 was activated upon stimulation and cross-talk with signaling pathways that influence with lipids and sphingomyelinase. In this panel, the gene expression (Kumar and Vadlamudi, 2002; Bokoch, activity of Pak1 was reduced as expected when the 2003). Pak1 participates in the activation of NADPH autoinhibitory fragment of Pak1 was transfected. oxidase by phosphorylation of serine residues on p47phox To identify the site(s)of Pak1 phosphorylation in that are required for assembly and activity of the PGM, GST-tagged PGM deletion constructs were enzyme (Knaus et al., 1995; Bokoch, 2003). The active subjected to in vitro kinase assay using recombinant enzyme complex uses NADPH to produce reactive Pak1. The C-terminal protein fragments containing oxygen intermediates that function in cell signaling, host 418–574 aa (i.e., deletion constructs numbers 3 and 4) defense and cell redox balance. Recently, it has also been were efficiently phosphorylated, while deletion of this reported that Pak1 inhibits phosphoglycerate mutase region (i.e., deletion constructs numbers 5–7)resulted in (PGAM), an enzyme of the glycolytic pathway causing a abrogation of phosphorylation (Figure 1d). Surpris- transient switch from glycolysis to pentose phosphate ingly, the fragment containing 90–574 aa (deletion pathway utilization and enhanced NADPH production construct number 2)was not phosphorylated. This in neutrophils (Shalom-Barak and Knaus, 2002; Bo- could be because partial deletion of very few amino koch, 2003). acids could create an overhang that could mask the Since the flux of glucose in the cells through the phosphorylation site and make it inaccessible for glycolytic pathway is responsive to mitogens, we phosphorylation by Pak1. However, the other deletion hypothesized that Pak1, a major regulatory kinase that constructs are smaller and could have a more open coordinates divergent pathways in response to growth structure that leaves the phosphorylation site exposed. It factor signaling, may influence the metabolic shift in could thus be concluded that the Pak1 phosphorylation cells by modulating the activities of essential enzymes of site on PGM was located between 418 and 574 aa. In this glucose metabolism. Here we investigate the regulation C-terminal domain, three potential Ser or Thr Pak1 of PGM phosphorylation and enzymatic activity by phosphorylation consensus sites are present. Site-direc- Pak1. ted mutagenesis of these potential Ser or Thr consensus phosphorylation sites to Ala in the full-length GST- tagged PGM protein followed by in vitro phosphorylation of the purified mutant GST fusion proteins by Pak1 Results resulted in abrogation of phosphorylation only in the Phosphorylation of PGM by Pak1 T466A mutant, while S459A and S504A mutants were phosphorylated to the same extent as the wild-type To test our hypothesis that PGM is a phosphorylation protein (Figure 1e). This suggested that Pak1 phosphor- substrate for Pak1, we first confirmed that there were ylates PGM on only one site and that this phosphoryla- several potential Pak1 phosphorylation consensus mo- tion site was threonine (Thr)466. The above results tifs (R/K XX S/T)within the PGM amino-acid sequence indicate that PGM is indeed a bonafide substrate for (Figure 1a). An in vitro kinase assay was then carried out Pak1 in vivo, with a single phosphorylation site at Thr using purified Pak1 enzyme and GST–PGM protein as 466. The stretch of amino acids surrounding the Pak1 substrate to test Pak1 kinase activity against PGM. As phosphorylation site in PGM shows a high degree of shown in Figure 1b, wild-type Pak1 readily phosphory- conservation both in terms of amino-acid sequence and lated GST–PGM, while recombinant kinase dead-Pak1 structure across a wide range of evolutionarily divergent enzyme (K299R)was unable to phosphorylate GST– organisms (Figure 1f): thus, Pak1 phosphorylation of PGM. The calculated phosphorylation stoichiometry PGM may be an evolutionarily conserved regulatory was B0.65 mol of phosphate/mol of PGM (see Materi- mechanism. als and methods for calculations). In order to validate the phosphorylation of PGM in vivo, we transfected a Identification of PGM as a Pak1 interacting protein T7-tagged PGM expression vector into MCF-7 cells and metabolically labeled the cells with 32P-labeled ATP. Initial in vitro PGM and Pak1 protein–protein interac- Immunoprecipitation of T7-PGM followed by SDS– tion was established by a GST-pulldown assay, where in PAGE and autoradiography indicated that PGM exists vitro translated PGM co-precipitated with GST–Pak1 in a phosphorylated form in vivo. Lipids and sphingo- (Figure 2a, upper panels). Conversely, in vitro translated myelinase, signals that are known to activate Pak1 in Pak1 also interacted with GST–PGM in the GST- MCF-7 cells, stimulated PGM phosphorylation pulldown assay (Figure 2a, lower panels). To determine

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Figure 1 PGM is phosphorylated by Pak1 in vitro and in vivo.(a)Amino-acid sequence of PGM showing potential Pak1 phosphorylation sites (in bold and underlined). (b)Wild-type Pak1 and kinase dead-Pak1 mutant (K299R)were used in a Pak1 in vitro kinase assay with GST–PGM as a substrate. The top panel indicates Pak autophosphoryaltion. GST alone was used as a negative control. The lower two panels are Ponceau stained blots showing the GST-tagged PGM proteins and GST protein. (c)MCF-7 cells were transiently transfected with T7-PGM, co-transfected with the Pak1 inhibitory fragment (83–149 aa), and, after 24 h, the cells were serum starved for another 24 h, labeled with 32P-orthophosphate overnight and then treated with the lipid, sphingosine (100 mM)or sphingomylinase (SPM, 1 U/ml medium)for 30 min to stimulate Pak1 activity. In vivo phosphorylation of T7-PGM was assessed by immunoprecipitating the cell lysates with T7 antibody. The immunoprecipitates were resolved on a SDS–PAGE gel, and transferred to membranes. After autoradiography, the membranes were blotted with T7 antibody. Pak1 was immunoprecipitated from the same lysates using Pak1-speciﬁc antibody and an in vitro kinase reaction was carried out with MBP as substrate (lower panel). (d)GST- tagged PGM deletion constructs were used as substrates for Pak1 in an in vitro kinase assay. (e)Three PGM mutant proteins with mutation of putative phosphorylation sites, PGS459A, PGMT466A and PGMS504A, were used as substrates in the Pak1 in vitro kinase assay. (f)Sequence alignment of the region of PGM near the Pak phosphorylation site

the speciﬁcity of Pak1 and PGM interaction, GST– (1–270 aa)or pGBD-Pak1 (271–570 aa)and pGAD- PGM was used in GST-pulldown assay along with in PGM (full length)showed that the N-terminal half of vitro translated Pak1, Pak2, Pak3 and Pak4. The Pak1 interacted in vivo with PGM, enabling the yeast strongest PGM binding was observed with Pak1, cells to grow in stringent (–A–H–L–T)conditions. A although Pak3, which shares high sequence homology known Pak1 interacting protein, Rac1, was used as a with Pak1, also weakly interacted with PGM (Figure 2b). positive control (Figure 3a). This in vivo interaction was Pak2 and Pak4, on the other hand, did not signiﬁcantly further substantiated using co-immunoprecipitation interact with PGM (Figure 2b). studies. Immunoprecipitation of Pak1 and Western Next we examined in vivo protein–protein interaction blotting for PGM in MCF-7 cells showed that the of Pak1 and PGM using two approaches. A one-on-one endogenous proteins interact with one another in a yeast co-transformation assay using either pGBD-Pak1 physiological setting (Figure 3b). This interaction was

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Figure 2 Identification of PGM as a Pak1-interacting protein. (a) Pak1 cDNA encoding 1–545 aa was translated in vitro and 35S- labeled Pak1 was incubated with GST–PGM in the GST-pulldown assays. The interaction was analysed by SDS–PAGE followed by autoradiography (upper panels). Full-length PGM was translated in vitro and 35S-PGM was subjected to GST-pulldown assay using GST-tagged Pak1. The precipitated proteins were analysed by SDS–PAGE and autoradiography (lower panels). In each figure, the upper panel indicates the 35S-labeled proteins, while the lower Figure 3 In vivo interaction of PGM–Pak1. (a)Yeast cells were two panels show Ponceau staining of the blots to visualize the GST co-transfected with control Gal4 activation domain (GAD)vector proteins. (b)PGM binding to Pak isoforms in a GST-pulldown or GAD-PGM along with Gal4 DNA-binding domain (GBD) assay using GST–PGM and 35S-labeled Pak1 or Pak2 (upper vector, GBD-Pak1 (1–270 aa)or GBD-Pak1 (270–545 aa).Co- panels)or Pak3 or Pak4 (lower panels) transformants were plated on selection plates lacking leucine and tryptophan (LT, first panel)or lacking AHLT (last panel).Yeast colony growth was recorded after 72 h. GAD-Rac1 was used as a positive control. (b)Cell lysates from MCF-7 cells that were either dependent on serum factors, since immunoprecipitation serum starved or treated with 10% serum were used for immunoprecipitation with antibodies against endogenous Pak1 in the absence of 10% serum did not pull down any and immunoprecipitates were analysed by SDS–PAGE, followed PGM protein. Furthermore, co-transfected T7-PGM by Western blotting with a PGM-specific antibody (upper panel). (red)and myc-Pak1 (green)were distinctly colocalized in The lower panel shows Western blot for Pak1 as a control for the cell upon stimulation by epidermal growth factor immunoprecipitation. (c)Localization of T7-PGM (red),myc-Pak1 (green)and actin (blue)in serum-starved and EGF-stimulated (EGF, a mitogenic activator of Pak1), as evidenced by MCF-7 cells confocal scanning microscopy (Figure 3c). Curiously, the two proteins seemed to localize together more in the edges of the cells towards the cell membrane upon EGF treatment. Thus, PGM and Pak1 interact physically to GST were used for the GST-pulldown assay with in with each other in the cells, enabling Pak1 to phosphor- vitro translated Pak1 to define the Pak1-binding domain ylate PGM. in PGM. We mapped the Pak1-binding domain in PGM to the N-terminal region (1–96 aa), as this region was Mapping of PGM and Pak1-binding sites sufficient for strong binding with Pak1 and removal of this region of PGM abolished binding (Figure 4b). To elucidate the binding regions of Pak1 for PGM, a series of GST-tagged deletion constructs of Pak1 were Functional consequence of Pak-mediated phosphorylation used in a GST-pulldown assay with in vitro translated of PGM PGM. Deletion of the N-terminus of Pak1 completely abolished the binding of PGM with Pak1, indicating In order to verify if Pak1-mediated PGM phosphoryla- that PGM-binding site in Pak1 is localized in this region tion altered the biological activity of PGM, PGM (Figure 4a, Lane 2). The Pak1 autoinhibitory domain activity was assayed using purified recombinant PGM (75–149 aa)was sufficient for binding to PGM protein with and without preincubation in a Pak1 in (Figure 4a, lane 7), raising the possibility that PGM vitro kinase assay. Results indicate that the specific binding may facilitate Pak1 activation. Since the binding activity of PGM increased twofold above basal levels region was localized to the autoinhibitory domain of after it was phosphorylated in vitro by the Pak1 enzyme Pak1, it is reasonable to speculate that PGM may only (Figure 5a). The phosphorylation of PGM by Pak1 was bind to Pak1 in its open, active conformation. We next verified by SDS–PAGE and autoradiography using 20% mapped the Pak1-binding site in PGM. The full-length of the kinase reaction product. The phosphorylation by PGM–GST fusion protein but not GST efficiently Pak1 did not change the kinetics of the PGM reaction interacted with the 35S-labeled full-length Pak1 protein. but increased the rate of the reaction, since the Deletion mutants of PGM conjugated at the N-terminus saturation time for the reaction did not change. The

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Figure 5 Functional consequence of Pak-mediated phosphorylation of PGM. (a)Recombinant GST-tagged PGM was phosphorylated in vitro using recombinant Pak1 enzyme and PGM activity was assayed before and after in vitro Pak1 phosphorylation. GST was used as a negative control. (b)Recombinant GST-tagged PGM was phosphorylated in vitro using either recombinant Pak1 or kinase dead-Pak1 (K299R)enzymes and PGM activity was assayed before and after in vitro Pak1 phosphorylation. GST was used as a negative control. (c)Recombinant GST-tagged PGMT466A was phosphorylated in vitro using either recombinant Pak1 or kinase dead-Pak1 (K299R)enzymes and PGM activity was assayed before and after in vitro Pak1 phosphorylation. GST was used as a negative control. (d)Average velocity calculations for reactions performed in (b, c). A plot of average velocity versus time was used for linear regression ﬁt and the velocity of the reaction was calculated. m is the slope of the reaction and R2 gives the regression line ﬁt. (e)MCF-7 cells were transiently transfected with the pCDNA vector or with the T7-PGM T466A phosphorylation Figure 4 Mapping of PGM- and Pak1-binding sites. (a)Deletion mutant vector. The cells were serum starved for 48 h and then constructs of GST–Pak1 fusion proteins were incubated with in stimulated with EGF for 10 min. Cell lysates were assayed for vitro translated 35S-PGM, and binding was analysed by GST- PGM activity and normalized to fold activity of the enzyme. Equal pulldown assay, followed by SDS–PAGE and autoradiography. amounts of protein were used for the enzyme assay. Values are Lower panel depicts Ponceau staining of the blot. (b)GST–PGM averages of at least three independent estimations and are fusion proteins were incubated with in vitro translated 35S-Pak1. expressed as fold activity7s.e.m. Binding was analysed by GST-pulldown assay, followed by SDS– PAGE and autoradiography. Lower panel depicts Ponceau staining of the blot

linear fit was obtained when average velocity ([NADH]/ time) versus time was plotted, which was used to specific activity of PGM did not change from basal calculate the average velocity of each reaction levels when PGM activity was assayed in the presence of (Figure 5d). The average velocity of PGM activity was recombinant kinase dead-Pak1 enzyme (K299R) approximately twofold higher with Pak1 phosphoryla- (Figure 5b). T466A-PGM mutant (which cannot be tion. Likewise, treatment of MCF-7 cells with a phosphorylated by Pak1)showed no changes in its physiological signal that activates Pak1 (EGF)and specific activity as compared to the basal activity of increases phosphorylation of PGM (data not shown) PGM after in vitro phosphorylation by either recombi- caused an increase in the activity of PGM in these cells nant Pak1 or kinase dead-Pak1 enzymes (Figure 5c). A (Figure 5e). To demonstrate that the changes in

Oncogene PGM regulation by Pak1 signaling A Gururaj et al 8123 EGF-stimulated Pak1 kinase activity were necessary for increased PGM activity, either pcDNA (vector control) or T7-tagged phosphorylation point mutant, PGMT466A was transfected into MCF-7 cells and the cells were stimulated using EGF for 10 min. The cell lysates were collected and assayed for PGM activity. Cells expressing the pcDNA plasmid alone showed an increase in PGM activity upon EGF stimulation, as seen in nontransfected MCF-7 cells. Overexpression of the mutant PGM that could not be phosphorylated by Pak1 led to the inhibition of signal-stimulated activation of PGM in vivo (Figure 5e), indicating that phosphorylation of PGM by Pak1 enhances the activity of PGM in vivo. In order to elucidate the modulation of PGM by Pak1 in vivo, we utilized two cell lines expressing differing amounts of Pak1 as model systems. MCF-7 cells express modest amounts of Pak1 while MDA-MB-435 express high amounts of Pak1 (Adam et al., 1998). Endogenous PGM activity in these cell lines was correlated with Pak1 and PGM protein expression (Figure 6a). An increase in PGM activity was correlated with higher Pak1 and PGM expression in the MDA-MB-435 cells. When Pak1-specific siRNA (Barnes et al., 2003)was transfected to downregulate Pak1 expression, PGM activity was reduced by 40% in MCF-7 cells and 30% in MDA- MB-435 cells (Figure 6b). Since the experiment was carried out in the presence of 10% serum, the results indicate that, under physiological conditions, Pak1 is required for PGM activity. The basal activity of PGM in MCF-7 cells grown in 0% serum did not change in the Figure 6 Modulation of PGM activity by Pak1 signaling. (a) presence of Pak1-siRNA (Figure 6c). However, the Lysates of MCF-7 cells and MDA-MB-435 cells were assayed for PGM activity. PGM and Pak1 protein levels in these cells were EGF-stimulated increase in activity of PGM was determined by Western blot analysis using specific antibodies for abrogated with Pak1 knockdown. Thus, it can be the proteins. (b)Control siRNA and Pak1-specific siRNA (5)were concluded that Pak1 activity is important for activation transfected into MCF-7 and MDA-MB-435 cells and the cells were of PGM through Pak1-dependent signaling pathways harvested after 48 h. Cell lysates were prepared and PGM activity in the cells was determined as described in Materials and methods. but not necessary for basal activity of the enzyme. Cell lysates were also analysed for expression of Pak1 by SDS– We next used previously characterized MCF-7 cells PAGE, followed by Western blotting using Pak1-specific antibody. stably transfected with a tetracycline-inducible domi- (c)MCF-7 cells were transfected with siRNA for Pak1 and control nant-active Pak1 plasmid (Vadlamudi et al., 2000)to siRNA. After 24 h, the cells were serum starved and stimulated test the potential role of Pak1 regulation of PGM in with EGF for 10 min. Cell lysates were then assayed for PGM activity. Equal amounts of protein were used for the enzyme assay. vivo. Doxycycline treatment of these cells for 24 h Values are averages of at least three independent estimations and increased protein expression of the inducible domi- are expressed as fold activity7s.e.m. (d)MCF-7 cells stably nant-active Pak1, with no change in PGM protein transfected with a tetracycline-inducible dominant-active Pak1 expression (Figure 6d). However, Pak1 induction was plasmid were used for induction of Pak1 expression by addition of doxycycline (1 mg/ml medium)for 24 h. Lysates were prepared with correlated with a significant increase in PGM activity and without doxycycline and PGM activity in the cell lysates was (Figure 6d). In control experiments using the parental analysed. Equal amounts of protein were used for the enzyme cell line, no induction of Pak1 protein, PGM protein, or assay. Values are averages of at least three independent estimations PGM enzymatic activity was observed with doxycycline and are expressed as fold activity7s.e.m. Cell lysates were also treatment (data not shown). These results indicate that used for Western blot analysis of PGM and Pak1 protein expression using specific Pak1 and PGM antibodies (lower panel) Pak1 specifically regulates PGM activity in vivo. The K562 leukemia cell line is spontaneously deficient in PGM (Tomkins et al., 1997). These cells do not express PGM protein, as detected by Western blot, and show very little or no PGM activity (Figure 7a). We transfected K562 cells (Figure 7c). The time course of stably transfected T7-tagged PGM into the K562 cells stimulation indicated that Pak1 was activated prior to and the stable clones demonstrated PGM activity peak activation of PGM in these cells. To examine the (Figure 7a). Although both the parental K-562 cells influence of PGM expression on the growth character- and the PGM-wild-type transfected K562 cells showed istics of K-562 cells, we measured the proliferation activation of Pak1 upon stimulation with sphingosine rate of both vector-transfected and PGM-transfected (Figure 7b), PGM activity was stimulated only in PGM- cells. The K-562 cells with PGM1 overexpression

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Figure 8 Putative effects of Pak1-mediated phosphorylation of PGM on conformational changes near the enzymatic active site. (a) The three-dimensional rendering of the rabbit PGM crystal structure (PDB 3PMG, created using the SwissProt PDBViewer) was examined for localization of the Thr 466 Pak1 phosphorylation site (shown)in relation to the active site. Phosphorylation of Thr 466 may disrupt interstrand hydrogen bonding (b)and instead create intrastrand hydrogen bonding (c), resulting in a putative contraction of a beta strand and beta turn overlying the active site entrance and increasing access to the active site (arrow)

Figure 7 Validation studies using K562/PGM stable clones. (a) Stable clones of PGM in K-562 leukemia cells were generated and expression of PGM and Pak1 in these cells was checked by Western blot analysis. PGM activity in the stable clones was assayed as covalently modiﬁed by phosphorylation. Substitution of described in the Materials and methods section. (b)K-562 cells and Glu for Thr 466 using SwissProt analysis software, in PGM-expressing-K-562 clone 3 were treated with sphingosine order to mimic the addition of a charged phosphate (100 mM)for 10 min, then assayed for Pak1 kinase activity after group, resulted in the dissolution of hydrogen bonding immunoprecipitation of Pak1 using MBP as substrate. (c)PGM activity in parental K-562 cells and PGM-expressing-K-562 clone 3 between beta sheets 12 and 13 (Figure 8b), and a gain of was assayed after stimulation with sphingosine at different time intrachain hydrogen bonds in beta strand 13 (Figure 8c). intervals. (d)K-562 and PGM-overexpressing clone 3 were plated This potential structural alteration with the addition of at a density of 2 Â 104 cells and cells were counted after 4 days a phosphate group could cause a shift in a beta strand interval. Values are averages of three independent experiments and and loop that lie over one end of the PGM active site are expressed as average cell number7s.e.m. and thus alter access to this region, potentially increasing the enzymatic activity. In addition, alteration of the intracellular localization of PGM by physiological signals that activate Pak1 may also inﬂuence enzymatic activity (data not shown). Thus, Pak1 may induce demonstrated a twofold increased growth rate as com- relocalization of PGM to areas of glycogen storage or pared with vector-transfected control cells (Figure 7d). energy utilization in addition to increasing PGM activity These results from K562 cells strongly support our in the intact cell. hypothesis that Pak1 regulates the activity of PGM in vivo.

Putative phosphorylation-induced changes in PGM conformation Discussion Examination of the three-dimensional crystal structure It has been shown that, in transformed cells, there is of rabbit PGM (Figure 8a)showed that Thr 466 is in a increased breakdown of glycogen to G-1-P, which can position fully accessible to the solvent and could thus be then enter the glycolytic pathway through the activity of

Oncogene PGM regulation by Pak1 signaling A Gururaj et al 8125 PGM (Boros et al., 2002). Recently, it was reported that, Pak1 as a regulatory switch operative in metabolic in neutrophils, Pak1 inhibits PGAM, an enzyme of the pathways, coordinately regulating cellular redox balance glycolytic pathway, resulting in a transient switch from (Shalom-Barak and Knaus, 2002). glycolysis to metabolism through the pentose phosphate There is a developing theme in cell survival research pathway (Shalom-Barak and Knaus, 2002)and an that regulation of apoptosis and glucose metabolism are increased cellular supply of NADPH. Thus, it is closely linked (Danial et al., 2003). Pak1 initiates cell attractive to speculate that Pak1 phosphorylation of survival pathways in part through the phosphorylation PGM acts in concert with its action on other Pak1 and inactivation of the proapoptotic protein, Bad substrates to cause changes required for a specific (Schurmann et al., 2000). We have now demonstrated metabolic shift towards the utilization of the pentose that Pak1 also regulates the enzymatic activation of phosphate pathway in tumor cells. A recent report of PGM, a critical regulator of cellular glucose utilization. kinetic measurements of PGM activity indicates that Also, cells that have higher expression and activity of PGM preferentially converts G-1-P to G-6-P even when PGM seem to have a distinct growth advantage over the two substrates are present in equimolar concentra- cells that do not express PGM (Figure 7d). Given the tion in the reaction and the equilibrium constant of the widespread expression and activation of Pak1 and reaction where G-6-P is used as a substrate is unfavor- PGM, the new mechanistic link between cell survival able (Gao and Leary, 2004). This gives further support and energy utilization is likely to have broad relevance to our hypothesis that Pak1-induced PGM activity to normal and pathological cellular function. would drive the PGM reaction towards formation of G-6-P and thus glycolysis and pentose phosphate pathways. However, since direct flux measurements Materials and methods using tracer techniques were not employed in our study, we are unable to conclude definitively on the direction of Cell culture and antibodies PGM activity in the current context. NADPH derived MCF-7 breast cancer cells and K562 leukemia cells were from the pentose phosphate pathway is used as a obtained from ATCC. The antibodies used were against Pak1 substrate or cofactor in diverse regulatory pathways in (Zymed), myc tag (MBL Laboratories), T7 tag (Novagen) and the cell. Also, reactive oxygen species produced as a PGM (a gift from Dr Richard B Marchase). DNA dye ToPro3 byproduct of NADPH oxidase activity (which is also and fluorescent second antibodies were from Molecular upregulated by Pak1; Kumar and Vadlamudi, Probes. Stock chemicals were from Sigma. MCF-7 cells and 2002; Bokoch, 2003)are known to act as signaling K562 cells were maintained in Dulbecco’s modified Eagle’s molecules and to downregulate protein phosphatase medium (DMEM)-F12 (1 : 1) supplemented with 10% fetal activity (Meng et al., 2002), effectively potentiating some calf serum. cell signaling pathways. Thus, regulation of NADPH levels in the cell by Pak1 via control of PGM, in concert In vitro transcription and translation, and GST-pulldown assay with other Pak1 regulatory targets, may result in a In vitro transcription and translation of the PGM or Pak1 global impact on cellular metabolic and regulatory protein was performed using the TNT-transcription–transla- pathways. tion system (Promega). The GST-pulldown assays were In this context, a recent finding that PGM is a performed by incubating GST protein alone or GST-test fusion proteins immobilized on GST beads with in vitro thioredoxin-interacting protein in cyanobacteria as- 35 sumes importance (Lindahl and Florencio, 2003). In translated S-labeled protein. Bound proteins were isolated by incubating the mixture for 2 h at 41C, washing 5 with NP40 these bacteria, PGM activity is shown to be inhibited Â lysis buffer, eluting the proteins with 2 Â SDS buffer, under oxidizing conditions and activated by DTT and separating them on sodium dodecyl sulfate (SDS)–PAGE reduced thioredoxin A. It has been shown earlier that and developing by autoradiography as described previously cells respond to oxidative stress by directed oxidation (Mazumdar et al., 2001). The amount of proteins present in the and inactivation of glycolytic enzymes, leading to a blot was visualized using Ponceau S stain. metabolic reshuffling of glucose equivalents through the pentose phosphate pathway. It has been proposed that Yeast one-on-one protein–protein interaction studies this metabolic shift could provide the necessary reducing Pak1 1–270 or 270–545 aa were amplified by polymerase chain power (NADPH2)for antioxidant defense mechanism reaction (PCR)from the full-length cDNA and subcloned into for the cells (Shanmuganathan et al., 2004). Considering a Gal4-binding domain fusion pGBD vector (Clontech). that cancer cells are subjected to chronic oxidative stress Likewise, full-length PGM cDNA was amplified by PCR and and Pak1 activates PGM activity to the same extent as subcloned into Gal4-activation domain fusion pGAD vector do thioredoxin and DTT, Pak1 could be heavily (Clontech). Protein–protein interaction was verified by one-on- involved in helping cancer cells cope with oxidative one transformations and selection on agar plates lacking insults in addition to mimicking a stress response in adenine, histidine, tryptophan and leucine (AHTL), and also these cells. These new data also give us a fundamental by b-galactosidase assay as described previously (Vadlamudi understanding of the intricate cross-talk between signal- et al., 2002). ing and metabolic pathways to bring about the physiological effect in the cells in response to an Immunofluorescence confocal assays extracellular signal. Pak1 regulation of PGM activity The cellular localization of T7-tagged PGM and myc-tagged also lends additional support to the proposed role of Pak1 was determined using indirect immunofluorescence as

Oncogene PGM regulation by Pak1 signaling A Gururaj et al 8126 described previously (Barnes et al., 2002; Kumar et al., 2002). Table 1 Nucleotide sequences of PCR primers

Briefly, cells were grown on glass coverslips and treated as 0 described. Cells were then fixed in phosphate-buffered 4% Primer 1 5 -GACCCCCGGCAGGAATTCGCCACCA TGGTG-30 paraformaldehyde and incubated with specific primary anti- Primer 2 50-GGGCAGTACCACTCGAGGCCTGTCTTC bodies followed by Alexa-546-labeled goat anti-mouse anti- TTAGG-30 body (red color)and Alexa-488-labelled goat anti-rabbit Primer 3 50-GGACAGAATGGAATTCTCTCCACC-30 antibody (green color, Molecular Probes). Cellular F-actin Primer 4 50-CCAACTGGCTGGAATTCTGGG-30 was localized using Alexa-633-labeled phalloidin (blue color, Primer 5 50-GTATGGCCGGAATTCCTTCACCAGG-30 Molecular Probes). Primer 6 50-GGATACAGCAGGGCTCGAGAGGATTCC-30 Primer 7 50-CCTGACTTCATGGTCTCGAGCAGGT CAGC-30 0 0 Pak kinase assay Primer 8 5 -GCTTCCACCTCCTCGAGATCATACCTGG-3 PGMS459A_F 50-GGGGAAGCAGTTCGCAGCAAATGA In vitro kinase assays using myelin basic protein (MBP)or CAAAG-30 GST–PGM fusion protein were performed in HEPES buffer PGMS459A_R 50-CTTTGTCATTTGCTGCGAACTGCTT 0 (50 mM HEPES, 10 mM MgCl ,2mM MnCl ,1mM DTT) CCCC-3 2 2 0 containing 1 mg of purified bacterially expressed GST–Pak1 PGMT466A_F 5 -GCAAATGACAAAGTTTACGCTGTGGA GAAGGCC-30 enzyme, 10 mCi of g-32P-ATP and 25 mM cold ATP. The PGMT466A_R 50-GGCCTTCTCCACAGCGTAAACTTTGTCA reaction was carried out in a volume of 30 ml for 30 min at TTTGC-30 301C, and then stopped by addition of 10 mlof4Â SDS buffer. PGMS504A_F 50-CGTCTTCCGACTGGCCGGCACTGGGA Reaction products were then analysed by SDS–PAGE gel and GTGCC-30 autoradiography. PGMS504A_R 50-GGCACTCCCAGTGCCGGCCAGTCGG AAGACG-30

Determination of the stoichiometry of the in vitro phosphorylation of PGM The stoichiometry of phosphorylation of PGM was essentially performed as described previously, with minor modifications PGMS504A_F and PGMS504A_R for conversion of serine (Zhai and Comai, 1999). In brief, varying concentrations of 504 to alanine. PGM (1, 4, 7 and 10 mg)were subjected to in vitro phosphorylation in the presence of GST–Pak1 enzyme Transient transfection and electroporation as described above. After 30 min of incubation, the reaction mixture was resolved by SDS–polyacrylamide Transient transfection with the appropriate plasmids was gel electrophoresis (PAGE), and the protein was performed using Fugene-6 (Roche Biochemical, NJ, USA)as transferred onto a nitrocellulose membrane. The proteins were per the manufacturer’s instructions. In order to create K562- visualized by Ponceau S staining. The number of PGM stable clones, electroporation of the T7-tagged PGM moles of PGM used was obtained from the estimated expression vector into K562 cells was performed as previously amount of PGM and its known molecular weight. described (Adam et al., 2000). The band corresponding to the PGM protein was cut and the amount t of incorporated phosphate was quantitated with PGM activity assay a scintillation counter. According to the quantitation data, the Bacterially expressed GST–PGM, GST–PGMT466A and GST number of moles of phosphate being transferred to PGM was proteins were bead purified, eluted in 20 mM glutathione, obtained. The stoichiometry of PGM phosphorylation was dialysed in 0.2 M Tris–HCl, pH 7.3, then concentrated to 3 mg/ calculated as mole(s)of phosphate transferred per mole of ml. In all, 30 mg of protein with and without preincubation in a PGM. Pak1 or kinase-dead Pak1-K299R kinase reaction was used to measure the ability of PGM to convert G-1-P to G-6-P. Plasmid construction and mutagenesis Absorbance at 340 nm as a measure of NADH produced by G-6-P dehydrogenase was used as a final quantitative end GST and T7 tagged PGM constructs were generated by point (Takahashi et al., 2001). Reaction conditions were as subcloning the PGM cDNA (MGC-1682, ATCC)into the follows: 24 mM G-1-P, 5 mM NAD þ , 1 U G-6-P dehydrogen- EcoR1 and XhoI sites of pGEX 5X1 (Amersham)and ase, 10 mM EDTA, 5 mM MgCl2 and 50 mM Tris–HCl, pH 7.4. pcDNA3.1 (Invitrogen)vectors using primers 1 and 2 (please Control reactions of GST–PGM lacking the substrate G-1-P see Table 1 for the primer sequences). Pak1 deletion constructs and purified GST protein alone were run in parallel with have been previously described (Vadlamudi et al., 2002). PGM experimental reactions. PGM activity from cell lysates was deletion constructs were generated using the PCR followed by measured in a similar fashion, with 100 mg of protein used in a into the EcoR1 and Xho1 sites of pGEX 5X1 by using the 300 ml reaction. To obtain kinetic parameters, experimental following primer pairs (Table 1): (a) deletion no. 2 with data were fitted to theoretical equations using Excel software. primers 2 and 3; (b)deletion no. 3 with primers 2 and 4; (c) Average velocity versus time was plotted and a linear deletion no. 4 with primers 2 and 5; (d)deletion no. 5 with regression line fit was carried out. Average velocity of the primers 1 and 6; (e)deletion no. 6 with primers 1 and 7; and (f) reaction was calculated based on the equation obtained from deletion no. 7 with primers 1 and 8. Mutations on PGM full- the line fit. All values are reported as the average of three length expression vectors were generated by site-directed replicate experiments. mutagenesis using a Quick Change Mutagenesis Kit from Stratagene and GST–PGM as a backbone with the following Cell proliferation assay primers (Table 1): (a) PGMS459A_F and PGMS459A_R for conversion of serine 459 to alanine; (b)PGMT466A_F and K-562 cells and PGM clone-3 were plated in a six-well plate PGMT466A_R for conversion of Thr 466 to alanine; and (c) (2 Â 105 cells/well)and counted using a Coulter Counter after 4

Oncogene PGM regulation by Pak1 signaling A Gururaj et al 8127 days. The values are averages of three independent experi- quantitation of Western blotting was performed by using ments. software from SigmaGel.

Acknowledgements Reproducibility and quantitation We thank Dr Richard B Marchase for providing a PGM Results are representative of at least three replicate experi- antibody. This work was supported by the NIH grant CA ments. Bar charts are presented as the mean7s.e.m. Semi- 90970 and CA80066 (RK).

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