We investigated PKM2 as being a possible downstream effector of FGFR1 as a consequence of its vital purpose Survivin in cancer cell metabolism. Figure 1A shows a schematic illustration of PKM2 as well as tyrosine residues identified as phosphorylated in response to oncogenic FGFR1 signaling, these consist of Y83, Y105, Y148, Y175, Y370, and Y390. The MS spectrum of peptide fragments of PKM2 that contained the specified phospho Tyr residues is shown in fig. S1B. Previous phosphoproteomic studies have shown that PKM2 tyrosine residues Y83, Y105, and Y370 will also be phosphorylated in human leukemia KG 1a cells expressing FGFR1OP 2 FGFR1, a constitutively active fusion tyrosine kinase related to ins stem cell MPD.

Glutathione S transferase ?tagged PKM2 was tyrosine phosphorylated in 293T cells co transfected with plasmids encoding a constitutively active mutant type of ZNF198 FGFR1, PR/TK, by which an N terminal proline wealthy domain of ZNF198 is fused towards the C terminal FGFR1 GSK-3 beta pathway tyrosine kinase domain, and in ligand taken care of cells expressing FGFR1, but not in cells expressing GST PKM2 without the need of FGFR1. Also, the presence of FGFR1 wild type, but not a kinase dead mutant, significantly decreased the enzymatic activity of endogenous PKM2 in 293T cells. Overexpression of FGFR1 or its mutational activation has become implicated in many human solid tumors, such as breast cancer, pancreatic adenocarcinoma, and malignant astrocytoma. We found that remedy with the FGFR1 inhibitor TKI258 considerably enhanced PKM2 enzymatic activity in human myeloid leukemia KG 1a cells harboring the FOP2 FGFR1 fusion protein, as well as breast cancer MDA MB 134 cells and lung cancer NCI H1299 cells overexpressing FGFR1.

Collectively, these data recommend that FGFR1 may possibly right or indirectly phosphorylate and inhibit PKM2. Mutational Infectious causes of cancer evaluation exposed that expression of GST PKM2 wild kind or of numerous PKM2 mutants during which a Tyr residue was replaced having a Phe to abolish phosphorylation, such as Y83F, Y148F, Y175F, Y370F, and Y390F, resulted in comparable, improved PKM2 enzyme action compared with that in handle 293T cells, whereas substitution of Y105 led to substantially better PKM2 activation. To elucidate the role of FGFR1 in phosphorylation and inhibition of PKM2 in cancer cells, we used FGFR1 expressing human lung cancer H1299 cells to make mouse PKM2 wild sort, Y105F, and Y390F rescue cell lines as described by RNA interference?mediated steady knockdown of endogenous human PKM2 and rescue expression of Flag tagged mPKM2 variants.

Steady with all the information in Fig. 2A, mPKM2 Y105F showed enhanced enzymatic action inside the rescue cells compared with that of wild variety and Y390F mPKM2. We also produced an antibody that specifically recognizes PKM2 phospho Y105. This antibody detected PKM2 in 293T cells coexpressing FGFR1 wild form but not in cells coexpressing the KD mutant. Moreover, in an in vitro kinase assay, recombinant FGFR1 phosphorylated purified GST PKM2 at Y105, whereas phosphorylation of this site by rFGFR1 was not obvious within the GST PKM2 Y105F mutant.