Phenotypic selection with an intrabody library reveals an anti-apoptotic function of PKM2 requiring Mitofusin-1

Bcl-2 family proteins control a decisive apoptotic event: mitochondrial outer membrane permeabilization (MOMP). To discover MOMP-regulating proteins, we expressed a library of intracellular single-chain variable fragments (scFvs) (intrabodies) and selected for those rescuing cells from apoptosis induced by BimS (the short isoform of Bim). One anti-apoptotic intrabody, intrabody 5 (IB5), recognized pyruvate kinase M2 (PKM2), which is expressed in cancer cells. PKM2 deletion ablated this clonogenic rescue; thus, IB5 activated a latent cytoprotective function of PKM2. This resulted not from pyruvate kinase activity per se but rather from the formation of an active tetrameric conformation of PKM2. A stably tetrameric PKM2 mutant, K422R, promoted cell survival even in the absence of IB5, and IB5 further increased survival. Mitochondria isolated from IB5-expressing cells were relatively resistant to MOMP in vitro. In cells, IB5 expression up-regulated Mitofusin-1 (Mfn1) and increased mitochondrial length. Importantly, Mfn1 deficiency abrogated IB5's cytoprotective effect. PKM2's anti-apoptotic function could help explain its preferential expression in human cancer. Author summary Proteins belonging to the Bcl-2 family regulate a common form of cell death known as apoptosis. Typically, these proteins function in apoptosis by controlling the formation of large pores in the mitochondrial outer membrane (MOM). While many proteins that regulate apoptosis have been identified over the years, some may still be unknown. Here, we used an unbiased approach in which we first expressed in cultured tumor cells a library of intracellular single-chain antibodies termed intrabodies. We then selected for intrabodies that allowed cells to evade apoptosis. We identified pyruvate kinase isoform M2 (PKM2), a major glycolytic enzyme that has been linked to cancer development, as the specific target of one such anti-apoptotic intrabody. We showed that the PKM2-specific intrabody promoted cell survival not by neutralizing its target but rather by activating an anti-apoptotic function of PKM2. While this cell survival function of PKM2 was not related to changes in the levels of Bcl-2 family proteins or to effects on the enzymatic activity of PKM2, we found that cell survival requires the increased expression of a MOM protein, Mitofusin-1 (Mfn1), known to regulate mitochondrial fusion. We conclude that this cell survival function of PKM2 could contribute to a role in cancer progression for this protein.