Figure 3: PKM2 Phosphorylates MLC2 at Y118.

(a,c–g), Immunoblotting analyses were performed with the indicated antibodies. C, cytokinesis; I, interphase (no thymidine release). (a) WT His-PKM2, His-PKM2 K367M or His-PKM1 on agarose beads was incubated with purified Aurora B protein for a kinase assay; it was then washed in PBS three times and incubated with recombinant GST-MLC2 to proceed the kinase assay in the presence of ³²P-labeled PEP or ATP for autoradiography. (b) In vitro kinase assays were performed by mixing purified recombinant GST-MLC2 with His-PKM2. MLC2 was analysed by mass spectrometry. The results of a mass spectrometric analysis of a tryptic fragment at m/z 550.779 (mass error, −0.16 p.p.m.) matched those of the doubly charged peptide 111-GVLKADYVR-120, suggesting that Y118 was phosphorylated. The Sequest score for this match was Xcorr=2.83. The Mascot score was 51, and the expectation value was 1.1e−4. (c) In vitro kinase assays were performed by mixing the indicated GST-MLC2 proteins with WT His-PKM2 in the presence of ³²P-PEP for autoradiography. (d) U87 cells that had been synchronized by double thymidine block (2 mM) for 43 h were released for the indicated time periods. (e) U87 cells, with and without MLC2 shRNA expression, were reconstituted with or without expression of WT rMLC2, rMLC2 Y118F or rMLC2 Y152F (left panel). The cells were synchronized by double thymidine block (2 mM) for 43 h and then unreleased or released for 10 h (right panel). (f) U87 cells synchronized by double thymidine block (2 mM) for 43 h were unreleased or released for 9 h, followed by MG132 (25 μM) treatment for 1.5 h to arrest cells at metaphase. MG132 was replaced with AZD1152 (100 nM), Y-27632 (10 μM) or ML-7 (10 μM) for 30 min before cell harvesting. (g) U87 cells with PKM2 depletion were stably transfected with a vector expressing WT rPKM2 or rPKM2 T45A. These cells were synchronized by double thymidine block (2 mM) for 43 h and then unreleased or released for 10 h.