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. 2017 Nov 17;8(65):109120–109134. doi: 10.18632/oncotarget.22621

Figure 3. PKM2 phosphorylates H2AX at Ser139 in vitro.

Figure 3

(A) Purified recombinant PKM2-WT or PKM2-R399E mutant was incubated with recombinant GST-H2AX in the presence of PEP or ATP. H2AX, γ-H2AX and PKM2 were detected by immunoblotting. (B) The in vitro kinase reactions were performed by incubating recombinant PKM2 or ATM with WT GST-H2AX or GST-H2AX-S139A mutant in the presence of PEP (for PKM2) or ATP (for ATM). (C) PKM2 (0.25μM) was incubated with varied purified H2AX in the presence of excess PEP at room temperature for 2min. The reaction mixtures were then analyzed by Western blot using indicated antibodies (upper panel). The phosphorylated H2AX protein bands were quantified by densitometry and the kinetic constants were determined by fitting the data with Michaelis-Menten equation using GraphPad Prism. (D) Phosphorylation of GST-H2AX by recombinant PKM2 in the presence of FBP, ADP or PEP was detected by immunoblotting. (E, F) The in vitro kinase assay was carried out by mixing WT PKM2 or its mutants as indicated with GST-H2AX. The protein bands on the gels were quantified by densitometry and shown on the bottom panel (D, F). (G, H) Phosphorylation of recombinant H2AX by ATM or PKM2 in the presence of escalating concentration of PKM2 (G) or ATM (H) was analyzed by in vitro kinase assay. Immunoblots were performed for indicated proteins. All these experiments were repeated at least for three times with the same results.