Figure 3: Serine202 and serine203 are phosphorylated by PKM2.
From: Pyruvate Kinase M2 Activates mTORC1 by Phosphorylating AKT1S1
(A) MS/MS spectra of tryptic peptides from the PKM2-treated HEK293T proteome that led to the identification of phosphorylation of serine202 (left) or serine 203 (right) of AKT1S1. (B) Flag-tagged AKT1S1 and Myc-tagged PKM2 were co-expressed in HEK293T cells. The PKM2 co-purified with the AKT1S1 was detected by Myc antibody. (C) Flag-tagged AKT1S1 and HA-tagged PKM2 were co-expressed in HEK293T cells. The AKT1S1 co-purified with the PKM2 was detected by Flag antibody. (D)Purified AKT1S1 was treated with recombinant PKM2 in the presence and absence of PEP, and the levels of P-Ser of AKT1S1 in the reaction mixture after treatment were determined. Numerical values below the gels indicate quantification of the bands relative to untreated AKT1S1 (hereinafter). (E) Flag-tagged AKT1S1 was co-expressed with PKM2. P-ser levels of AKT1S1 from cells cultured with and without TEPP-46 (100 nM) supplementation were determined. (F) Flag-tagged AKT1S1 was co-expressed with PKM2 or PKM2Y105F (Y105F), P-ser levels of AKT1S1 purified from different cells were determined. (G) Flag-tagged AKT1S1 was co-expressed with either HA-tagged PKM2 or HA-tagged PKM2K270 mutant (K270M) in HeLa cells. The P-Ser levels of Flag bead-purified AKT1S1 from each culture were determined and quantified. (I) Purified AKT1S1 was treated with either purified PKM2 or purified K270M. The P-S202, P-S203 and P-S202/203 levels of each treated AKT1S1 were determined by site-specific antibodies and quantified. The relative intensities of phosphorylation signals were normalized to those of untreated AKT1S1. (J) Flag-tagged PKM2 or Flag-tagged K270M was overexpressed in HEK293T cells. The endogenous P-S202/203 levels of AKT1S1 of each culture were determined and the relative intensities of P-S202/203 signals were normalized to that of HEK293T cells. (K) The endogenous P-S202/203 levels of HEK293T cells before and after PKM2 knockdown by independent shRNAs were compared. The PKM2 knockdown efficiency was confirmed by western blot.
