Lack of Evidence for PKM2 Protein Kinase Activity

Abstract

The role of pyruvate kinase M2 (PKM2) in cell proliferation is controversial. A unique function of PKM2 proposed to be important for the proliferation of some cancer cells involves the direct activity of this enzyme as a protein kinase; however, a detailed biochemical characterization of this activity is lacking. Using [(32)P]-phosphoenolpyruvate (PEP) we examine the direct substrates of PKM2 using recombinant enzyme and in vitro systems where PKM2 is genetically deleted. Labeling of some protein species from [(32)P]-PEP can be observed; however, most were dependent on the presence of ADP, and none were dependent on the presence of PKM2. In addition, we also failed to observe PKM2-dependent transfer of phosphate from ATP directly to protein. These findings argue against a role for PKM2 as a protein kinase.

Figure 1

Assessement of phosphoenolpyruvate (PEP) as a phosphate donor for protein phosphorylation. Hypotonic lysates and nuclear extracts from (A) H1299, (B) PKM2-null MEF, (C) U87, and (D) U87 EGFR-VIII cells were incubated for 1 h with impure [³²P]-PEP, and phosphorylated proteins analyzed by SDS-PAGE and autoradiography. Reactions were carried out with the addition of no enzyme, recombinant wild type (WT) or mutant (R399E) his-tagged PKM2 (each at 10 μg/mL) as indicated. In addition, no competitor or excess (1 mM) non-radioactive competitor PEP or ATP was also included where indicated. See also Figure S1.

Figure 2

Contaminating [γ-³²P]-ATP is the predominant phosphoryl donor when using crude [³²P]-PEP. (A) [³²P]-PEP synthesized from [γ-³²P]-ATP is contaminated by small amounts of [γ-³²P]-ATP, which can be separated by preparative HPLC. The purity of each fraction was analyzed by thin-layer chromatography and autoradiography. (B) The contaminating [γ-³²P]-ATP component of impure [³²P]-PEP is the phosphoryl donor for the majority of proteins phosphorylated by impure [³²P]-PEP. An H1299 hypotonic lysate was incubated for 1 h with recombinant PKM2 and different ³²P-containing metabolites as well as no competitor or excess (1 mM) non-radioactive competitor PEP or ATP as indicated. Phosphorylated proteins were analyzed by SDS-PAGE and autoradiography. See also Figure S2.

Figure 3

(A) Calf thymus histones were incubated for 1 h with impure [³²P]-PEP and no enzyme, recombinant wild type (WT) or mutant (R399E) PKM2 (each at 10 μg/mL) as indicated. An H1299 nuclear extract was also incubated with impure [³²P]-PEP with and without calf thymus histones added. Phosphorylated proteins were analyzed by SDS-PAGE and autoradiography. Arrows indicate phosphorylated proteins with apparent molecular weights similar to histones (core histones, 11-17 kDa, many histone H1 isoforms 25-35 kDa) (Shechter et al., 2007). (B) H1299 cell hypotonic lysates and nuclear extracts and calf thymus histones were incubated for 1 h with [³²P]-PEP and 10 μg/mL rPKM2 with or without 0.5 mM SAICAR as well as no competitor or excess (1 mM) cold competitor PEP or ATP. (C) Hypotonic lysates and nuclear extracts from H1299 cells and PKM2-expressing (flox/flox) or PKM2-null (−/−) MEFs were desalted and incubated for 1 h with HPLC-purified [³²P]-PEP with no additional enzymes or with added recombinant wild type or mutant (R399E) rPKM2 (each at 10 μg/mL). See also Figure S3.

Figure 4

Lack of evidence for an ATP-dependent protein kinase activity for PKM2. (A) H1299 and (B) PKM2-null MEF hypotonic lysates and nuclear extracts were used to identify substrates for PKM2 kinase activity. Lysates were fractionated on a 1mL Q Sepharose HP column (Inp., 1:10 dilution of column input; FT, flow through; 1-10, fractions 1-10), and incubated for 1 h with [γ-³²P]-ATP with or without the addition of 10 μg/mL rPKM2. Phosphorylated proteins were analyzed by SDS-PAGE and autoradiography. See also Figure S4.