Pyruvate kinase isoform M2 (PKM2)

PKM2 is nuclear localized upon SAICAR accumulation. PKM2-SAICAR increases PKM2's susceptibility to SAICAR binding via phosphorylating and activating Erk1/2. Furthermore, to cause mitogen-induced cell proliferation and persistent Erk1/2 activation, PKM2-SAICAR was required. Inducing H3 T11 and Erk1/2 phosphorylation requires and is satisfied by SAICAR-PKM2 interaction[2]. When cells are starved of glucose, the amount of SAICAR within them rises oscillatorily, which in turn causes cancer cells to activate PKM2. Additionally, in glucose-limited conditions, the SAICAR-PKM2 connection increases the survival of cancer cells. Adsl-kd cells and cells overexpressing PAICS fare better under glucose limitation, however paics-kd cells perished sooner than control-kd cells. Under glucose-restricted environments, SAICAR increases cancer cell survival.

---

Pyruvate kinase isoform M2 (PKM2) plays an important role in the growth and metabolic reprogramming of cancer cells in stress conditions. Here, we report that SAICAR (succinylaminoimidazolecarboxamide ribose-5′-phosphate, an intermediate of the de novo purine nucleotide synthesis pathway) specifically stimulates PKM2. Upon glucose starvation, cellular SAICAR concentration increases in an oscillatory manner and stimulates PKM2 activity in cancer cells. Changes in SAICAR levels in cancer cells alter cellular energy level, glucose uptake, and lactate production. The SAICAR-PKM2 interaction also promotes cancer cell survival in glucose-limited conditions. SAICAR accumulation is not observed in normal adult epithelial cells or lung fibroblasts regardless of glucose conditions. This allosteric regulation may explain how cancer cells coordinate different metabolic pathways to optimize their growth in the nutrient-limited conditions commonly observed in the tumor microenvironment.[1]

---

Abnormal metabolism and sustained proliferation are hallmarks of cancer. Pyruvate kinase M2 (PKM2) is a metabolic enzyme that plays important roles in both processes. Recently, PKM2 was shown to have protein kinase activity phosphorylating histone H3 and promoting cancer cell proliferation. However, the mechanism and extent of this protein kinase in cancer cells remain unclear. Here, we report that binding of succinyl-5-aminoimidazole-4-carboxamide-1-ribose-5'-phosphate (SAICAR), a metabolite abundant in proliferating cells, induces PKM2's protein kinase activity in vitro and in cells. Protein microarray experiments revealed that more than 100 human proteins, mostly protein kinases, are phosphorylated by PKM2-SAICAR. In particular, PKM2-SAICAR phosphorylates and activates Erk1/2, which in turn sensitizes PKM2 for SAICAR binding through phosphorylation. Additionally, PKM2-SAICAR was necessary to induce sustained Erk1/2 activation and mitogen-induced cell proliferation. Thus, the ligand-induced protein kinase activity from PKM2 is a mechanism that directly couples cell proliferation with intracellular metabolic status[2].

Protein kinase assays [2]
The reaction solution [0.1–10 nM PKM2, 0–4 μM histone (NEB, typically 1 μM H3 monomer concentration), 150 μM PEP, 50 mM HEPES, pH 7.4, 100 mM potassium chloride, 6.2 mM magnesium acetate, and 5% glycerol] was incubated at 25°C for 1–120 minutes (typically 5 min). After the reaction, the solution was mixed with an equal volume of SDS-gel loading buffer (375 mM Tris-HCl, pH 8, 10% SDS, 50% glycerol, 1 mM DTT, 0.1% bromophenol blue) and incubated at 95°C for 10 minutes. All other protein kinase assays were performed as described, with a fixed concentration of PKM2 (10 nM), for 30 minutes, unless specifically noted otherwise. Solutions were subjected to SDS-PAGE, staining with ProQ Diamond, or Western blot analysis. For the determination of catalytic efficiency (kcat), a reaction was carried out with higher concentration of PKM2 (10 nM) for 1 hour, and the resulting signal was assumed to represent the completed reaction.

---

Protein microarray experiments [2]
Protein microarray experiments were carried out using Invitrogen Human protein microarrays, recombinant PKM2, and PEP in the presence and in the absence of SAICAR. Phosphorylated proteins were detected using ProQ Diamond stain. An extensive description of this experiment is provided in the Supplementary Information.

---

Phosphorylation of PKM2 [2]
Recombinant PKM2 (1 μM) and recombinant GST-Erk1 (10 nM) were incubated together in a buffer containing 1 mM ATP, 50 mM Tris-HCl, pH 7.0, 150 mM NaCl, 1 mM DTT, 1 mM MgCl2 for 30 minutes at 25°C. The reaction solution was passed through glutathione-agarose resin to deplete GST-Erk1. Unbound proteins were eluted with 50 mM Tris, pH 8.0, 150 mM NaCl, 1 mM DTT, 1 mM MgCl2 buffer. Fractions were then subjected to SDS-PAGE and either Coomassie or Pro-Q diamond staining. Fractions containing phosphorylated PKM2 were identified using Pro-Q diamond staining. Concentration of phosphorylated PKM2 was determined by measuring A280 in denaturing conditions (Edelhoch, 1969) using a calculated extinction coefficient (28,910 M−1 cm−1).

---

Erk1/2 activity assay [2]
A 10 μL reaction (25 nM rPKM2, 1 μM rErk1, 150 μM PEP, 50 mM HEPES pH 7.4, 100 mM potassium chloride, 10% glycerol, 6.2 mM magnesium chloride, and 1 mM DTT) was incubated for 30 minutes at room temperature. The Erk1 reaction was serially diluted using water, and 8 μL of diluted solution was added to 32 μL Omnia Kinase Assay solution (10 μM Omnia Peptide 17, 1 mM ATP, 0.2 mM DTT, and proprietary buffer components from the vendor) in a black transparent bottom 96-well plate. The emission at 485 nm (excitation at 360 nm) was recorded every minute for 60 minutes at 30°C using a Tecan Infinity M2 fluorescence microplate reader.

Western Blot [2]
Western blots were performed as described by AbCam (http://www.abcam.com/ps/pdf/protocols/WB-beginner.pdf). In brief, cells were lysed in ice-cold RIPA buffer (20 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1% NP-40, 1% sodium deoxycholate) supplemented with 1 mM phenylmethylsulfonyl fluoride and 1 mM sodium fluoride, and total protein amount was measured using a Pierce 660 reagent with bovine serum albumin as a standard. Proteins were separated by SDS-PAGE, and transferred to nitrocellulose membranes. The membrane was blocked with phosphate-buffered saline (PBS) supplemented with 5% bovine serum albumin, and probed with the appropriate primary antibody overnight at 4°C. After primary antibody incubation, membranes were probed with horseradish peroxidase (HRP) conjugated goat anti-rabbit IgG or goat anti-mouse IgG secondary antibodies (Bio-Rad). HRP-conjugated secondary antibodies were then detected using commercial chemiluminescence substrates. Chemiluminescence images were acquired using a FluorChem M FM0455 imager.

[1]. SAICAR stimulates pyruvate kinase isoform M2 and promotes cancer cell survival in glucose-limited conditions. Science. 2012 Nov 23;338(6110):1069-72.

[2]. SAICAR induces protein kinase activity of PKM2 that is necessary for sustained proliferative signaling of cancer cells. Mol Cell. 2014 Mar 6;53(5):700-9.

SAICAR is a 1-(phosphoribosyl)imidazolecarboxamide resulting from the formal condesation of the darboxy group of 5-amino-1-(5-O-phosphono-beta-D-ribofuranosyl)-1H-imidazole-4-carboxylic acid with the amino group of L-aspartic acid. It has a role as an Escherichia coli metabolite and a mouse metabolite. It is functionally related to a succinic acid. It is a conjugate acid of a SAICAR(4-).
SAICAR is a metabolite found in or produced by Escherichia coli (strain K12, MG1655).
Saicar has been reported in Homo sapiens, Schizosaccharomyces pombe, and Apis cerana with data available.
SAICAR (or (S)-2-[5-Amino-1-(5-phospho-D-ribosyl)imidazole-4-carboxamido]succinate) is a substrate for the multifunctional protein ADE2. SAICAR is an intermediate in purine metabolism. (S)-2-[5-Amino-1-(5-phospho-D-ribosyl)imidazole-4-carboxamido]succinate is converted from 5-Amino-1-(5-phospho-D-ribosyl) imidazole-4-carboxylate via phosphoribosylaminoimidazole-succinocarboxamide synthase [EC: 6.3.2.6] or SAICAR synthase. This enzyme catalyses the seventh step out of ten in the biosynthesis of purine nucleotides. The appearance of succinylaminoimidazolecarboxamide riboside (SAICAriboside) and succinyladenosine (S-Ado) in cerebrospinal fluid, urine, and to a lesser extent in plasma is characteristic of a heritable deficiency Adenylosuccinate lyase deficiency. .
SAICAR is a metabolite found in or produced by Saccharomyces cerevisiae.

---

Based on these results, we conclude that at least some aspects of PKM2’s role in tumor biology can be explained by SAICAR-mediated allosteric stimulation. This feature of PKM2 may allow cells to adjust their energy generation in response to nutritional and metabolic demands. When sufficient levels of glucose are provided, diverting glycolytic intermediates to biosynthetic processes like the pentose phosphate pathway can promote cell growth. However, in energy-depleting conditions such as nutrient-limitation, continued diversion of glycolytic intermediates to biosynthetic processes can hamper cells by depleting cellular energy below the minimal level required. The SAICAR-PKM2 interaction described here can be a molecular mechanism for this delicate balance. Ribose-5-phosphate, the starting material of nucleotide biosynthesis, is produced from the pentose phosphate pathway, which diverts glycolytic intermediates away from energy production. Connecting PKM2’s PK activity with an intermediate of the de novo purine nucleotide biosynthesis pathway could give cells fine-tuned control of their metabolism in demanding conditions. SAICAR is produced from L-aspartate, a byproduct of glutaminolysis, which is known to be important in cancer cell metabolism, and the cleavage of SAICAR yields fumarate, which contributes to the citric acid cycle in mitochondria. SAICAR is therefore positioned to convey cellular metabolic demands to PKM2.[1]

---

In addition to previously known histone H3 (Yang et al., 2012a), we also identified many human proteins as potential substrates for PKM2-SAICAR, with the vast majority of proteins involved in the regulation of cell proliferation. Further investigation on how PKM2-SAICAR recognizes its substrates may reveal a previously unrecognized feature common to these proteins involved in cell cycle regulation. Previous work on PKM2 has revealed an interaction between MAPK activity and PKM2, showing that Erk1/2 phosphorylates PKM2 (Yang et al., 2012b). In our work, the reciprocal reaction – phosphorylation of MAPK protein kinases by PKM2 – also occurs in vitro and in cells upon the binding of SAICAR to PKM2. Our result challenges the view that PKM2 is a passive recipient of MAPK protein kinase signaling and suggests that PKM2 is a more active component that is necessary for the MAPK protein kinase signaling. Intricately related to MAPK activity is cell cycle control and proliferation. As has been well documented, malignant tumor cells frequently display sustained proliferation. Many different mechanisms such as mutations in genes encoding MAPK signaling contribute to the sustained proliferation of cancer cells. However, many types of cancer cells show sustained proliferation without showing any mutations in these genes. The altered metabolism associated with most cancer cells, including upregulation of PKM2, may thus be a mechanism to sustain cell proliferation in those cells.[2]

C13H19N4O12P

454.28336

454.074

C, 34.37; H, 4.22; N, 12.33; O, 42.26; P, 6.82

3031-95-6

3031-95-6

160666

Purple to purplish red solid powder

-4.3

8

14

9

30

718

5

OC(C[C@H](NC(C1N=CN([C@@H]2O[C@H](COP(=O)(O)O)[C@@H](O)[C@H]2O)C=1N)=O)C(=O)O)=O

NAQGHJTUZRHGAC-ZZZDFHIKSA-N

InChI=1S/C13H19N4O12P/c14-10-7(11(22)16-4(13(23)24)1-6(18)19)15-3-17(10)12-9(21)8(20)5(29-12)2-28-30(25,26)27/h3-5,8-9,12,20-21H,1-2,14H2,(H,16,22)(H,18,19)(H,23,24)(H2,25,26,27)/t4-,5+,8+,9+,12+/m0/s1

N-((5-Amino-1-(5-O-phosphono-beta-D-ribofuranosyl)-1H-imidazol-4-yl)carbonyl)-L-aspartic acid

SAICAR; SAICAR; Succino-AICAR; SAICAriboside; UNII-K1PVR64RIF; K1PVR64RIF; SAICA riboside; 5-Amino-4-imidazole-N-succinocarboxamide ribonucleotide; ...; 3031-95-6; Succino-AICAR.

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: Please store this product in a sealed and protected environment (e.g. under nitrogen), avoid exposure to moisture and light.

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

DMSO : ~220 mg/mL (~484.28 mM)
H2O : ~100 mg/mL (~220.13 mM)

Solubility in Formulation 1: ≥ 5.5 mg/mL (12.11 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 55.0 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

---

Solubility in Formulation 2: ≥ 5.5 mg/mL (12.11 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 55.0 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.

---

View More

Solubility in Formulation 3: ≥ 5.5 mg/mL (12.11 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 55.0 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

---

 (Please use freshly prepared in vivo formulations for optimal results.)