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)imidazolium carboxamide, formed by the condensation of the darboxy group of 5-amino-1-(5-O-phosphono-β-D-furanoribosyl)-1H-imidazolium-4-carboxylic acid with the amino group of L-aspartic acid. It is a metabolite in both E. coli and mice. Its function is related to succinic acid. It is the conjugate acid of SAICAR(4-). SAICAR is a metabolite found or produced in E. coli strains (K12, MG1655). It has been reported in humans, fission yeast, and Apis cerana, with relevant data. SAICAR (or (S)-2-[5-amino-1-(5-phospho-D-ribosyl)imidazolium-4-carboxamido]succinic acid) is a substrate of the multifunctional protein ADE2. SAICAR is an intermediate in purine metabolism. (S)-2-[5-amino-1-(5-phosphate-D-ribosyl)imidazol-4-carboxylic acid]succinate is derived from 5-amino-1-(5-phosphate-D-ribosyl)imidazol-4-carboxylic acid via phosphoribosylaminoimidazolium-succinocarboxamide synthase [EC: 6.3.2.6] or SAICAR synthase. This enzyme catalyzes the seventh step of the ten-step reaction in purine nucleotide biosynthesis. The presence of (small amounts) succinylaminoimidazolium-carboxamide nucleoside (SAICAriboside) and succinoadenosine (S-Ado) in cerebrospinal fluid, urine, and plasma is characteristic of hereditary adenylate succinate lyase deficiency. SAICAR is a metabolite found or produced in Saccharomyces cerevisiae. Based on these results, we conclude that the role of PKM2 in tumor biology can be explained, at least in some respects, by SAICAR-mediated allosteric stimulation. This property of PKM2 may enable cells to regulate their energy production according to nutritional and metabolic needs. When glucose levels are sufficient, the transfer of glycolytic intermediates to biosynthetic processes such as the pentose phosphate pathway can promote cell growth. However, under energy-depleted conditions, such as nutrient restriction, the continuous transfer of glycolytic intermediates to biosynthetic processes can lead to cell energy levels below the minimum required level, thereby hindering cell growth. The SAICAR-PKM2 interaction described in this article may be the molecular mechanism for maintaining this delicate balance. Ribose-5-phosphate is the starting material for nucleotide biosynthesis, produced by the pentose phosphate pathway, which transfers glycolytic intermediates away from energy production pathways. Linking the PK activity of PKM2 to intermediates in the de novo synthesis of purine nucleotides allows for fine-tuning of cellular metabolism under harsh conditions. SAICAR is generated from L-aspartate, a byproduct of glutamine breakdown, which is known to play an important role in cancer cell metabolism. SAICAR cleavage produces fumarate, which participates in the citric acid cycle in mitochondria. Therefore, SAICAR can transfer cellular metabolic needs to PKM2. [1] In addition to histone H3 (Yang et al., 2012a), which was previously known, we have identified many human proteins as potential substrates for PKM2-SAICAR, most of which are involved in the regulation of cell proliferation. Further research into how PKM2-SAICAR recognizes its substrates may reveal a previously undiscovered characteristic shared by these proteins involved in cell cycle regulation. Previous studies on PKM2 have revealed the interaction between MAPK activity and PKM2, showing that Erk1/2 can phosphorylate PKM2 (Yang et al., 2012b). In our study, phosphorylation of MAPK protein kinase by PKM2 also occurred in vitro and in vivo when SAICAR binds to PKM2. Our results challenge the view that PKM2 is a passive receptor for MAPK protein kinase signaling and suggest that PKM2 is a more active component of MAPK protein kinase signaling. Cell cycle regulation and proliferation are closely related to MAPK activity. It is well known that malignant tumor cells often exhibit persistent proliferation. Many different mechanisms, such as mutations in genes encoding MAPK signaling, can lead to persistent proliferation of cancer cells. However, many types of cancer cells can continue to proliferate even without any mutations in these genes. Therefore, most cancer cell-related metabolic changes, including the upregulation of PKM2, may be a mechanism by which these cells maintain proliferation. [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.)