Breakviews and Views

Regulation of Pyruvate Kinase M Gene Expression☆

Retinitis pigmentosa occurs due to mutations that cause rod photoreceptor degeneration. Once most rods are lost, gradual degeneration of cone photoreceptors occurs. Oxidative damage and abnormal glucose metabolism have been implicated as contributors to cone photoreceptor death. Herein, we show increased phosphorylation of key enzymes of glucose metabolism in the retinas of rd10 mice, a model of RP, and retinas of wild type mice with paraquat-induced oxidative stress, thereby inhibiting these key enzymes. Dietary supplementation with glucose and pyruvate failed to overcome the inhibition, but increased reducing equivalents in the retina and improved cone function and survival. Dichloroacetate reversed the increased phosphorylation of pyruvate dehydrogenase in rd10 retina and increased histone acetylation and levels of TP53-induced glycolysis and apoptosis regulator (TIGAR), which redirected glucose metabolism toward the pentose phosphate pathway. These data indicate that oxidative stress induced damage can be reversed by shifting glycolytic intermediates toward the pentose phosphate pathway which increases reducing equivalents and provides photoreceptor protection.

Pyruvate kinase muscle isoform 2 (PKM2) is a key glycolytic enzyme and transcriptional coactivator and is critical for tumor metabolism. In cancer cells, native tetrameric PKM2 is phosphorylated or acetylated, which initiates a switch to a dimeric/monomeric form that translocates into the nucleus, causing oncogene transcription. However, it is not known how these post-translational modifications (PTMs) disrupt the oligomeric state of PKM2. We explored this question via crystallographic and biophysical analyses of PKM2 mutants containing residues that mimic phosphorylation and acetylation. We find that the PTMs elicit major structural reorganization of the fructose 1,6-bisphosphate (FBP), an allosteric activator, binding site, impacting the interaction with FBP and causing a disruption in oligomerization. To gain insight into how these modifications might cause unique outcomes in cancer cells, we examined the impact of increasing the intracellular pH (pH_i) from ∼7.1 (in normal cells) to ∼7.5 (in cancer cells). Biochemical studies of WT PKM2 (wtPKM2) and the two mimetic variants demonstrated that the activity decreases as the pH is increased from 7.0 to 8.0, and wtPKM2 is optimally active and amenable to FBP-mediated allosteric regulation at pH_i 7.5. However, the PTM mimetics exist as a mixture of tetramer and dimer, indicating that physiologically dimeric fraction is important and might be necessary for the modified PKM2 to translocate into the nucleus. Thus, our findings provide insight into how PTMs and pH regulate PKM2 and offer a broader understanding of its intricate allosteric regulation mechanism by phosphorylation or acetylation.

Metabolic regulation influences cell proliferation. The influence of pyruvate kinase isoforms on tumor cells has been extensively studied, but whether PKM2 is required for normal cell proliferation is unknown. We examine how PKM2 deletion affects proliferation and metabolism in nontransformed, nonimmortalized PKM2-expressing primary cells. We find that deletion of PKM2 in primary cells results in PKM1 expression and proliferation arrest. PKM1 expression, rather than PKM2 loss, is responsible for this effect, and proliferation arrest cannot be explained by cell differentiation, senescence, death, changes in gene expression, or prevention of cell growth. Instead, PKM1 expression impairs nucleotide production and the ability to synthesize DNA and progress through the cell cycle. Nucleotide biosynthesis is limiting, as proliferation arrest is characterized by severe thymidine depletion, and supplying exogenous thymine rescues both nucleotide levels and cell proliferation. Thus, PKM1 expression promotes a metabolic state that is unable to support DNA synthesis.

Metabolic homeostasis requires integration of complex signaling networks which, when deregulated, contribute to metabolic syndrome and related disorders. Protein-tyrosine phosphatase 1B (PTP1B) has emerged as a key regulator of signaling networks that are implicated in metabolic diseases such as obesity and type 2 diabetes. In this review, we examine mechanisms that regulate PTP1B-substrate interaction, enzymatic activity and experimental approaches to identify PTP1B substrates. We then highlight findings that implicate PTP1B in metabolic regulation. In particular, insulin and leptin signaling are discussed as well as recently identified PTP1B substrates that are involved in endoplasmic reticulum stress response, cell–cell communication, energy balance and vesicle trafficking. In summary, PTP1B exhibits exquisite substrate specificity and is an outstanding pharmaceutical target for obesity and type 2 diabetes.

Protein-tyrosine phosphatase 1B (PTP1B) is a physiological regulator of glucose homeostasis and adiposity and is a drug target for the treatment of obesity and diabetes. Here we identify pyruvate kinase M2 (PKM2) as a novel PTP1B substrate in adipocytes. PTP1B deficiency leads to increased PKM2 total tyrosine and Tyr¹⁰⁵ phosphorylation in cultured adipocytes and in vivo. Substrate trapping and mutagenesis studies identify PKM2 Tyr-105 and Tyr-148 as key sites that mediate PTP1B-PKM2 interaction. In addition, in vitro analyses illustrate a direct effect of Tyr-105 phosphorylation on PKM2 activity in adipocytes. Importantly, PTP1B pharmacological inhibition increased PKM2 Tyr-105 phosphorylation and decreased PKM2 activity. Moreover, PKM2 Tyr-105 phosphorylation is regulated nutritionally, decreasing in adipose tissue depots after high-fat feeding. Further, decreased PKM2 Tyr-105 phosphorylation correlates with the development of glucose intolerance and insulin resistance in rodents, non-human primates, and humans. Together, these findings identify PKM2 as a novel substrate of PTP1B and provide new insights into the regulation of adipose PKM2 activity.

Background: Tyrosine phosphorylation of PKM2 inhibits its activity; however, the phosphatase(s) that regulates PKM2 phosphorylation remains unidentified.

Results: PKM2 is a novel PTP1B substrate and PKM2 Tyr-105 and Tyr-148 are key sites that mediate the interaction.

Conclusion: PTP1B regulates PKM2 tyrosine phosphorylation and activity in adipocytes.

Significance: These findings provide new insights into the regulation of adipose PKM2 activity.

The metastasis of cancer cells to distant sites is responsible for the vast majority of cancer mortalities yet the molecular mechanisms underlying this extraordinarily complicated process have yet to be sufficiently elucidated. Recently, it has become clear that cancer cells need to inhibit anoikis, a cell death program induced by loss of attachment to the extracellular matrix (ECM), in order to successfully metastasize. These studies have motivated additional research into the relationship between ECM-detachment and cell viability, much of which reveals integral connections between ECM-detachment and cell metabolism. This review serves to thoroughly discuss the signaling pathways and metabolic changes that are induced by ECM-detachment. In addition, the molecular mechanisms by which cancer cells can alter signaling and metabolism to survive in the absence of ECM-attachment will be highlighted. Furthermore, cell death mechanisms that have been observed or implicated in cells detached from the ECM will also be examined. In aggregate, the studies discussed in this review reveal that ECM-detachment can regulate cancer cell metabolism in a variety of distinct cell types and suggest that interfering with metabolism in ECM-detached cells may be a novel and effective chemotherapeutic approach to selectively inhibit tumor progression.