Lack of Evidence for PKM2 Protein Kinase Activity

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 [32P]-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 [32P]-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. [Display omitted] •PEP-dependent phosphorylation was assayed in cell lysates using radioactive PEP•Trace adenine nucleotides can confound PEP-dependent phosphorylation assay results•PEP-dependent phosphorylation is rare and is not dependent on PKM2•We are unable to detect PKM2 protein kinase activity in a variety of contexts Hosios et al. use radioactive assays to investigate PEP-dependent phosphorylation. While some proteins can be labeled by phosphate from PEP, this activity is not dependent upon PKM2. They did not observe protein kinase activity involving this metabolic enzyme, and reports of PKM2 protein kinase activity may be partially explained by ATP regeneration by pyruvate kinase.