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. 2013 Apr 9;110(15):5881-6.
doi: 10.1073/pnas.1217157110. Epub 2013 Mar 25.

M2 pyruvate kinase provides a mechanism for nutrient sensing and regulation of cell proliferation

Hugh P Morgan  1 , Francis J O'Reilly, Martin A Wear, J Robert O'Neill, Linda A Fothergill-Gilmore, Ted Hupp, Malcolm D Walkinshaw
Affiliations

M2 pyruvate kinase provides a mechanism for nutrient sensing and regulation of cell proliferation

Hugh P Morgan et al. Proc Natl Acad Sci U S A. .

Abstract

We show that the M2 isoform of pyruvate kinase (M2PYK) exists in equilibrium between monomers and tetramers regulated by allosteric binding of naturally occurring small-molecule metabolites. Phenylalanine stabilizes an inactive T-state tetrameric conformer and inhibits M2PYK with an IC50 value of 0.24 mM, whereas thyroid hormone (triiodo-L-thyronine, T3) stabilizes an inactive monomeric form of M2PYK with an IC50 of 78 nM. The allosteric activator fructose-1,6-bisphosphate [F16BP, AC50 (concentration that gives 50% activation) of 7 μM] shifts the equilibrium to the tetrameric active R-state, which has a similar activity to that of the constitutively fully active isoform M1PYK. Proliferation assays using HCT-116 cells showed that addition of inhibitors phenylalanine and T3 both increased cell proliferation, whereas addition of the activator F16BP reduced proliferation. F16BP abrogates the inhibitory effect of both phenylalanine and T3, highlighting a dominant role of M2PYK allosteric activation in the regulation of cancer proliferation. X-ray structures show constitutively fully active M1PYK and F16BP-bound M2PYK in an R-state conformation with a lysine at the dimer-interface acting as a peg in a hole, locking the active tetramer conformation. Binding of phenylalanine in an allosteric pocket induces a 13° rotation of the protomers, destroying the peg-in-hole R-state interface. This distinct T-state tetramer is stabilized by flipped out Trp/Arg side chains that stack across the dimer interface. X-ray structures and biophysical binding data of M2PYK complexes explain how, at a molecular level, fluctuations in concentrations of amino acids, thyroid hormone, and glucose metabolites switch M2PYK on and off to provide the cell with a nutrient sensing and growth signaling mechanism.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
Allosteric nutrient sensing mechanism also regulates cellular proliferation. X-ray structures of the tetrameric Phe-bound T-state (B) and F16BP-activated R-state (D) are shown as cartoons, with each 50-kDa protomer represented by a rectangular shape showing the effector and active sites. M2PYK exists in equilibrium between tetrameric (C) and enzymatically inactive monomer (A) forms (gray arrows). Phenylalanine (Phe, cyan square) and the thyroid hormone T3 (orange) act as allosteric inhibitors and prevent the tetramer adopting an active R-state conformation. The activator F16BP (green square) clamps the tetramer in an enzymatically active conformation. (E) Addition of F16BP to HCT-116 cells inhibits proliferation, whereas both inhibitors of M2PYK (T3 and Phe) stimulate proliferation.
Fig. 2.
Fig. 2.
Oligomeric states of M1PYK and M2PYK. (A) Analytical gel-filtration elution profile observed for 10-µL sample injection of 0.1 mg mL−1 M1PYK in the absence (black) and presence (red) of 500 µM F16BP. (B) Same experiment as in A but for M2PYK. (C) Determination of the molar mass of M2PYK using SEC-MALS. Solid black line indicates the trace from the refractive index detector, and red dots are the weight-averaged molecular masses for each 0.5-s slice analyzed. A total of 200 µg of M2PYK was injected onto a Superdex 200 10/300 column. Flow rate was 0.5 mL min−1. (D) Concentration response curves observed for the titration of PEP against M2PYK in the presence (blue line) or absence (black line) of saturated F16BP. Error bars are derived from three independent repeat experiments. (E) Analytical gel-filtration elution profile observed for 10-µL sample injection of 0.1 mg mL−1 M2PYK in the absence (black) and presence (red) of 10 µM T3. (F) Analytical gel-filtration elution profiles observed for 10-µL sample injection of 0.5 mg mL−1 M2PYK in the absence (black) and presence (red) of 5 mM Phe. Experiments in which Phe was added to the running buffer could not be monitored at 214 nm because Phe saturated the absorbance. Monitoring M2PYK (that has a low extinction coefficient) at 280 nm required a higher M2PYK concentration, which increased lower limits from 0.1 mg/mL to 0.5 mg/mL. Thermal shift assays performed at 0.5 mg/mL M2PYK (Fig. S2) provide complementary results to those observed by gel filtration.
Fig. 3.
Fig. 3.
M1PYK forms a tighter C-C interface than M2PYK. (A) The M2PYK tetramer is shown with the active and effector sites indicated. The bottom right chain has been colored to aid the identification of domains: domain-A (blue = residues 25–116 and 220–402), domain-B (gray = residues 117–219), and domain-C (red = residues 403–531). The large (A-A) and small (C-C) interfaces between monomers are shown as dashed lines. (B) Enlarged view of the dimer interface showing a comparison of the M1PYK and M2PYK side chains. M1PYK hydrogen bonds are shown in red and those of M2PYK in black (1). Gln439 is repelled by Met408 in M1PYK allowing Glu409 to form a salt bridge with Lys421 (2). (3) Cα2 of M1PYK is 2.5 Å closer to Lys421 than Cα2 of M2PYK. (C) Splice variant amino acids between M1PYK and M2PYK.
Fig. 4.
Fig. 4.
Allosteric inhibitor phenylalanine locks the M2PYK tetramer in the T-state. All ligands and interacting amino acids are shown as sticks; hydrogen bonds are shown as dashed red lines. The resolution of the electron density (Fo-Fc map, shown in green) map is 2.9 Å and is contoured at 3σ. (A) A cartoon representation of the T-state tetramer (M2PYK-Phe). (B) Enlargement of the Phe binding site. (C) Side view (rotated 90° to that of A) of the superposed R-state (M2PYK-ATP/OX/F16BP) and T-state structures (M2PYK-Phe). (D) Enlargement of the C-C interface highlighting conformational changes and side chain movements (arrows) as the protomers rotate from R- (effector bound) to T- (Phe bound) state.
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