PKM2 uses control of HuR localization to regulate p27 and cell cycle progression in human glioblastoma cells

Abstract

The M2 isoform of pyruvate kinase (PK) is upregulated in most cancers including glioblastoma. Although PKM2 has been reported to use dual kinase activities to regulate cell growth, it also interacts with phosphotyrosine (pY)-containing peptides independently of its kinase activity. The potential for PKM2 to use the binding of pY-containing proteins to control tumor growth has not been fully examined. We here describe a novel mechanism by which PKM2 interacts in the nucleus with the RNA binding protein HuR to regulate HuR sub-cellular localization, p27 levels, cell cycle progression and glioma cell growth. Suppression of PKM2 in U87, T98G and LN319 glioma cells resulted in increased p27 levels, defects in entry into mitosis, increased centrosome number, and decreased cell growth. These effects could be reversed by shRNA targeting p27. The increased levels of p27 in PKM2 knock-down cells were caused by a loss of the nuclear interaction between PKM2 and HuR, and a subsequent cytoplasmic re-distribution of HuR, which in turn led to increased cap-independent p27 mRNA translation. Consistent with these results, the alterations in p27 mRNA translation, cell cycle progression and cell growth caused by PKM2 suppression could be reversed in vitro and in vivo by suppression of HuR or p27 levels, or by introduction of forms of PKM2 that could bind pY, regardless of their kinase activity. These results define a novel mechanism by which PKM2 regulates glioma cell growth, and also define a novel set of potential therapeutic targets along the PKM2-HuR-p27 pathway.Pyruvate kinase (PK) is an enzyme that catalyzes the conversion of phosphoenolpyruvate (PEP) to pyruvate in the final and rate-limiting step of glycolysis. 1 PK is expressed in four isoforms, L, R, M1 and M2. 2 PKML and PKMR have a limited, tissue-specific pattern of expression, being expressed in liver and erythrocytes, respectively. PKM1 in contrast is expressed in tissues thought to have high energy requirements such as skeletal muscle and brain. 3 PKM1 forms homotetramers, which have a high affinity for PEP and generate high levels of pyruvate, 2 which in turn is funneled into the citric acid cycle for further production of energy. 2 PKM2 in contrast, is expressed in many differentiated tissues as well as in cells with a high rate of nucleic acid synthesis such as stem cells and many (but not all) tumors, including gliomas. 4,5 In these tissues PKM2 exists in both tetrameric and monomeric/dimeric forms. 6 The tetrameric form of PKM2 is the most active with regard to PK activity. 2 In tumors, however, growth-related signaling events lead to phosphorylation of PKM2 at Y105, 7 and increased binding of PKM2 to phospho-tyrosine (pY)-containing proteins. 8 These events, along with low levels of the PKM2 allosteric activator fructose 1,6 -bisphosphate, limit tetramer formation, limit PK activity, and in turn are associated with increased tumor growth. 2,4,7,8 Consistent with this idea, the complete deletion of PKM2 in models of breast cancer results in increased tumor gro...

EYA4 Promotes Cell Proliferation Through Downregulation of p27Kip1 in Glioma

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