Figure 2. PKM2 Directly Interacts with Histone H3 and Phosphorylates H3-T11.

Please also see supplemental Figures S5A and S5B.

Immunoprecipitation and immunoblotting analyses were performed with the indicated antibodies.

(A) Pull-down analyses were performed by mixing purified immobilized His-PKM2 on nickel agarose beads with purified non-tagged recombinant histone H3 or histone H2A.

(B) U87/EGFR cells were treated with or without EGF (100 ng/ml) for 6 h.

(C) In vitro phosphorylation was analyzed by mixing recombinant WT PKM2, PKM2 K367M, or PKM1 with purified recombinant WT H3 or H3-T11A in the presence of PEP or ATP.

(D) Purified recombinant His-histone H3 was phosphorylated by PKM2 in vitro and was analyzed by mass spectrometry. Mass spectrometric analysis of a tryptic fragment at m/z 533.258 (mass error was -0.98 ppm) matched to the doubly-charged peptide 9-KSTGGKAPR-17, suggesting that T11 was phosphorylated. The Sequest score for this match was Xcorr = 2.74; Mascot scores were 46, expectation value 5.1e-4. The pRS score was 116, site probability was 99.1%. The presence of the b₂⁺ ion at 258.2 indicates the S10 residue is unmodified; the presence of the y₇⁺ at 808.2 is in agreement with the assignment of the phosphorylation site to T11.

(E, F) U87/EGFR cells expressing PKM2 shRNA were reconstituted by the expression of WT rPKM2, rPKM2 K367M, or rPKM2 K433E (E) and treated with or without EGF (100 ng/ml) for 6 h (F).