Proviral Insertion in Murine Lymphomas 2 (PIM2) Oncogene Phosphorylates Pyruvate Kinase M2 (PKM2) and Promotes Glycolysis in Cancer Cells

Pyruvate kinase M2 (PKM2) is a key player in the Warburg effect of cancer cells. However, the mechanisms of regulating PKM2 are not fully elucidated. Here, we identified the protein-serine/threonine kinase PIM2, a known oncogene, as a novel binding partner of PKM2. The interaction between PIM2 and PKM2 was confirmed by multiple biochemical approaches in vitro and in cultured cells. Importantly, we found that PIM2 could directly phosphorylate PKM2 on the Thr-454 residue, resulting in an increase of PKM2 protein levels. Compared with wild type, PKM2 with the phosphorylation-defective mutation displayed a reduced effect on glycolysis, co-activating HIF-1α and β-catenin, and cell proliferation, while enhancing mitochondrial respiration of cancer cells. These findings demonstrate that PIM2-dependent phosphorylation of PKM2 is critical for regulating the Warburg effect in cancer, highlighting PIM2 as a potential therapeutic target.     

Reciprocal Regulation of Protein Kinase and Pyruvate Kinase Activities of Pyruvate Kinase M2 by Growth Signals

Pyruvate kinase isoform M2 (PKM2) is an enzyme-catalyzing conversion of phosphoenolpyruvate to pyruvate in the glycolysis pathway. It was demonstrated that PKM2 interacts with tyrosine phosphopeptide, and the interaction with the tyrosine phosphopeptide affects the pyruvate kinase activity of PKM2. Our experiments suggest that PKM2 is also an active protein kinase (Gao, X., Wang, H., Yang, J. J., Liu, X., and Liu, Z. R. (2012) Mol. Cell 45, 598–609). We report here that growth signals reciprocally regulate the pyruvate kinase and protein kinase activities of PKM2 by different mechanisms. On the one hand, growth signals induce protein tyrosine phosphorylations. The tyrosine-phosphorylated protein(s) regulates the conversion of pyruvate kinase and protein kinase of PKM2 by directly interacting with PKM2. Binding of the tyrosyl-phosphorylated proteins at the fructose 1,6-bisphosphate-binding site converts the tetrameric PKM2 to a dimer. On the other hand, growth stimulations also lead to PKM2 phosphorylation, which consequently regulates the conversion of protein kinase and pyruvate kinase activities. Growth factor stimulations significantly increase the dimer/tetramer PKM2 ratio in cells and consequently activate the protein kinase activity of PKM2. Our study suggests that the conversion between the pyruvate kinase and protein kinase activities of PKM2 may be an important mechanism mediating the effects of growth signals in promoting cell proliferation.     

Missense Mutations in Pyruvate Kinase M2 Promote Cancer Metabolism,Oxidative Endurance,Anchorage Independence,and Tumor Growth in a Dominant Negative Manner

The present study was designed to examine the functional relevance of two heterozygous mutations (H391Y and K422R), observed earlier by us in the Bloom syndrome condition. Cells stably expressing exogenous wild-type or mutant PKM2 (K422R or H391Y) or co-expressing both wild type and mutant (PKM2-K422R or PKM2-H391Y) were assessed for cancer metabolism and tumorigenic potential. Interestingly, cells co-expressing PKM2 and mutant (K422R or H391Y) showed significantly aggressive cancer metabolism as compared with cells expressing either wild-type or mutant PKM2 independently. A similar trend was observed for oxidative endurance, tumorigenic potential, cellular proliferation, and tumor growth. These observations signify the dominant negative nature of mutations. Remarkably, PKM2-H391Y co-expressed cells showed a maximal effect on all the studied parameters. Such a dominant negative impaired function of PKM2 in tumor development is not known; this study demonstrates for the first time the possible predisposition of Bloom syndrome patients with impaired PKM2 activity to cancer and the importance of studying genetic variations in PKM2 in the future to understand their relevance in cancer in general.     

Protein Tyrosine Phosphatase 1B Regulates Pyruvate Kinase M2 Tyrosine Phosphorylation

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.     

Proteomic Profiling of Endothelial Invasion Revealed Receptor for Activated C Kinase 1 (RACK1) Complexed with Vimentin to Regulate Focal Adhesion Kinase (FAK)

Angiogenesis is critical for many physiological and pathological processes. To identify molecules relevant to angiogenesis, we performed a proteomic screen comparing invading versus non-invading endothelial cells in three-dimensional collagen matrices. We found up-regulated levels of receptor for activated C kinase 1 (RACK1) and the intermediate filament protein vimentin that correlated with increased endothelial cell invasion. Because both RACK1 and vimentin have been linked to focal adhesion kinase (FAK), we investigated whether this pathway regulated invasion. RACK1 depletion reduced invasion responses, and this was associated with attenuated activation of FAK. Knockdown of vimentin significantly decreased levels of phosphorylated and total FAK. Treatment with a pharmacological inhibitor of FAK dose-dependently reduced invasion, indicating a crucial role for FAK activity during invasion. Because RACK1 and vimentin were both up-regulated with sphingosine 1-phosphate treatment, required for invasion, and regulated FAK, we tested whether they complexed together. RACK1 complexed with vimentin, and growth factors enhanced this interaction. In addition, RACK1, vimentin, and FAK formed an intermolecular complex in invading endothelial cultures in three dimensions in response to stimulation by sphingosine 1-phosphate and growth factors. Moreover, depletion of RACK1 decreased the association of vimentin and FAK, suggesting that RACK1 was required for stabilizing vimentin-FAK interactions during sprouting. Silencing of vimentin and RACK1 decreased cell adhesion and focal contact formation. Taken together, these results demonstrate that proangiogenic signals converge to enhance expression and association of RACK1 and vimentin, which regulated FAK, resulting in successful endothelial sprout formation in three-dimensional collagen matrices.     

Dominant Negative Mutations Affect Oligomerization of Human Pyruvate Kinase M2 Isozyme and Promote Cellular Growth and Polyploidy

This study was designed to understand the mechanism and functional implication of the two heterozygous mutations (H391Y and K422R) of human pyruvate kinase M2 isozyme (PKM₂) observed earlier in a Bloom syndrome background. The co-expression of homotetrameric wild type and mutant PKM₂ in the cellular milieu resulting in the interaction between the two at the monomer level was substantiated further by in vitro experiments. The cross-monomer interaction significantly altered the oligomeric state of PKM₂ by favoring dimerization and heterotetramerization. In silico study provided an added support in showing that hetero-oligomerization was energetically favorable. The hetero-oligomeric populations of PKM₂ showed altered activity and affinity, and their expression resulted in an increased growth rate of Escherichia coli as well as mammalian cells, along with an increased rate of polyploidy. These features are known to be essential to tumor progression. This study provides insight in understanding the modulated role of large oligomeric multifunctional proteins such as PKM₂ by affecting cellular behavior, which is an essential observation to understand tumor sustenance and progression and to design therapeutic intervention in future.     

Lactate Engages Receptor Tyrosine Kinases Axl,Tie2, and Vascular Endothelial Growth Factor Receptor 2 to Activate Phosphoinositide 3-Kinase/Akt and Promote Angiogenesis

Although a high level of lactate is quintessential to both tumors and wound healing, the manner by which lactate impacts endothelial cells to promote angiogenesis and thereby create or restore vascular perfusion to growing tissues has not been fully elucidated. Here we report that lactate activated the PI3K/Akt pathway in primary human endothelial cells. Furthermore, activating this signaling pathway was required for lactate-stimulated organization of endothelial cells into tubes and for sprouting of vessels from mouse aortic explants. Lactate engaged the PI3K/Akt pathway via ligand-mediated activation of the three receptor tyrosine kinases Axl, Tie2, and VEGF receptor 2. Neutralizing the ligands for these receptor tyrosine kinases, pharmacologically inhibiting their kinase activity or suppressing their expression largely eliminated the ability of cells and explants to respond to lactate. Elucidating the mechanism by which lactate communicates with endothelial cells presents a previously unappreciated opportunity to improve our understanding of the angiogenic program and to govern it.     

LKB1 Deficiency in Tie2-Cre-expressing Cells Impairs Ischemia-induced Angiogenesis

LKB1 is a tumor suppressor protein whose loss leads to HIF1α-mediated activation of a proangiogenic program in intestinal polyps. LKB1 is also protein kinase regulator of AMP-activated protein kinase (AMPK) signaling, which is essential for endothelial cell responses to tissue ischemia. To discern whether LKB1 signaling is either pro- or antiangiogenic, we investigated ischemia-induced revascularization in mice that were deficient for LKB1 in Tie2-Cre-expressing cells. Whereas homozygous deletion of LKB1 led to embryonic lethality, heterozygous LKB1-knock-out (KO) (Lkb1^flox/+;Tie2^Tg/+) mice were viable. Unchallenged heterozygous LKB1-KO mice displayed normal capillary density, but the revascularization of hind limb following ischemic surgery was significantly impaired as evaluated by laser Doppler flow and capillary density measurements. Reduction of LKB1 in cultured endothelial cells, using either small interfering RNA or an adenovirus expressing nonfunctional kinase-dead LKB1 protein, attenuated endothelial proliferation, migration, and differentiation into network structures on Matrigel that was accompanied by diminished AMPK phosphorylation at Thr-172. Conversely, adenovirus-mediated LKB1 overexpression (Ad-LKB1) augmented network structure formation, and this was associated with elevated AMPK phosphorylation. The augmented differentiation of endothelial cells into network structures induced by Ad-LKB1 was abrogated by the co-transduction of a dominant negative mutant of AMPK. These observations suggest that the LKB1-AMPK signaling axis in endothelial cells is a positive regulator of the revascularization response to tissue ischemia.     

Knock-in Mutation Reveals an Essential Role for Focal Adhesion Kinase Activity in Blood Vessel Morphogenesis and Cell Motility-Polarity but Not Cell Proliferation

Focal adhesion kinase (FAK) associates with both integrins and growth factor receptors in the control of cell motility and survival. Loss of FAK during mouse development results in lethality at embryonic day 8.5 (E8.5) and a block in cell proliferation. Because FAK serves as both a scaffold and signaling protein, gene knock-outs do not provide mechanistic insights in distinguishing between these modes of FAK function. To determine the role of FAK activity during development, a knock-in point mutation (lysine 454 to arginine (R454)) within the catalytic domain was introduced by homologous recombination. Homozygous FAK^R454/R454 mutation was lethal at E9.5 with defects in blood vessel formation as determined by lack of yolk sac primary capillary plexus formation and disorganized endothelial cell patterning in FAK^R454/R454 embryos. In contrast to the inability of embryonic FAK^−/− cells to proliferate ex vivo, primary FAK^R454/R454 mouse embryo fibroblasts (MEFs) were established from E8.5 embryos. R454 MEFs exhibited no difference in cell growth compared with normal MEFs, and R454 FAK localized to focal adhesions but was not phosphorylated at Tyr-397. In E8.5 embryos and primary MEFs, FAK R454 mutation resulted in decreased c-Src Tyr-416 phosphorylation. R454 MEFs exhibited enhanced focal adhesion formation, decreased migration, and defects in cell polarity. Within immortalized MEFs, FAK activity was required for fibronectin-stimulated FAK-p190RhoGAP association and p190RhoGAP tyrosine phosphorylation linked to decreased RhoA GTPase activity, focal adhesion turnover, and directional motility. Our results establish that intrinsic FAK activity is essential for developmental processes controlling blood vessel formation and cell motility-polarity but not cell proliferation. This work supports the use of FAK inhibitors to disrupt neovascularization.     

Visualizing and Manipulating Focal Adhesion Kinase Regulation in Live Cells

Focal Adhesion Kinase (FAK) is essential for cell migration and plays an important role in tumor metastasis. However, the complex intermolecular and intramolecular interactions that regulate FAK activity at the focal adhesion remain unresolved. We have engineered a toolbox of FRET sensors that retain all of the individual FAK domains but modulate a key intramolecular regulatory interaction between the band 4.1/ezrin/radixin/moesin (FERM) and kinase domains of FAK. We demonstrate systematic control and quantitative measurement of the FERM-kinase interaction at focal adhesions, which in turn allows us to control cell migration. Using these sensors, we find that Tyr-397 phosphorylation, rather than kinase activity of FAK, is the key determinant of cell migration. Our sensors directly demonstrate, for the first time, a pH-dependent change in a protein-protein interaction at a macromolecular structure in live cells. The FERM-kinase interaction at focal adhesions is enhanced at acidic pH, with a concomitant decrease in Tyr-397 phosphorylation, providing a potential mechanism for enhanced migration of cancer cells.     

LKB1 Represses Focal Adhesion Kinase (FAK) Signaling via a FAK-LKB1 Complex to Regulate FAK Site Maturation and Directional Persistence

Liver kinase β1 (LKB1, also known as STK11) is a serine/threonine kinase that has multiple cellular functions including the regulation of cell polarity and motility. Murine proteomic studies show that LKB1 loss causes aberrant adhesion signaling; however, the mechanistic underpinnings of this relationship are unknown. We show that cells stably depleted of LKB1 or its co-activator STRADα have increased phosphorylation of focal adhesion kinase (FAK) at Tyr³⁹⁷/Tyr⁸⁶¹ and enhanced adhesion to fibronectin. LKB1 associates in a complex with FAK and LKB1 accumulation at the cellular leading edge is mutually excluded from regions of activated Tyr³⁹⁷-FAK. LKB1-compromised cells lack directional persistence compared with wild-type cells, but this is restored through subsequent pharmacological FAK inhibition or depletion, showing that cell directionality is mediated through LKB1-FAK signaling. Live cell confocal imaging reveals that LKB1-compromised cells lack normal FAK site maturation and turnover, suggesting that defects in adhesion and directional persistence are caused by aberrant adhesion dynamics. Furthermore, re-expression of full-length wild-type or the LKB1 N-terminal domain repressed FAK activity, whereas the kinase domain or C-terminal domain alone did not, indicating that FAK suppression is potentially regulated through the LKB1 N-terminal domain. Based upon these results, we conclude that LKB1 serves as a FAK repressor to stabilize focal adhesion sites, and when LKB1 function is compromised, aberrant FAK signaling ensues, resulting in rapid FAK site maturation and poor directional persistence.     

Src Kinase Determines the Dynamic Exchange of the Docking Protein NEDD9 (Neural Precursor Cell Expressed Developmentally Down-regulated Gene 9) at Focal Adhesions

Dynamic exchange of molecules between the cytoplasm and integrin-based focal adhesions provides a rapid response system for modulating cell adhesion. Increased residency time of molecules that regulate adhesion turnover contributes to adhesion stability, ultimately determining migration speed across two-dimensional surfaces. In the present study we test the role of Src kinase in regulating dynamic exchange of the focal adhesion protein NEDD9/HEF1/Cas-L. Using either chemical inhibition or fibroblasts genetically null for Src together with fluorescence recovery after photobleaching (FRAP), we find that Src significantly reduces NEDD9 exchange at focal adhesions. Analysis of NEDD9 mutant constructs with the two major Src-interacting domains disabled revealed the greatest effects were due to the NEDD9 SH2 binding domain. This correlated with a significant change in two-dimensional migratory speed. Given the emerging role of NEDD9 as a regulator of focal adhesion stability, the time of NEDD9 association at the focal adhesions is key in modulating rates of migration and invasion. Our study suggests that Src kinase activity determines NEDD9 exchange at focal adhesions and may similarly modulate other focal adhesion-targeted Src substrates to regulate cell migration.     

GPR126 Protein Regulates Developmental and Pathological Angiogenesis through Modulation of VEGFR2 Receptor Signaling

Angiogenesis, the formation of new blood vessels from pre-existing ones, is essential for development, wound healing, and tumor progression. The VEGF pathway plays irreplaceable roles during angiogenesis, but how other signals cross-talk with and modulate VEGF cascades is not clearly elucidated. Here, we identified that Gpr126, an endothelial cell-enriched gene, plays an important role in angiogenesis by regulating endothelial cell proliferation, migration, and tube formation. Knockdown of Gpr126 in the mouse retina resulted in the inhibition of hypoxia-induced angiogenesis. Interference of Gpr126 expression in zebrafish embryos led to defects in intersegmental vessel formation. Finally, we identified that GPR126 regulated the expression of VEGFR2 by targeting STAT5 and GATA2 through the cAMP-PKA-cAMP-response element-binding protein signaling pathway during angiogenesis. Our findings illustrate that GPR126 modulates both physiological and pathological angiogenesis through VEGF signaling, providing a potential target for the treatment of angiogenesis-related diseases.     

Transactivation of the Receptor-tyrosine Kinase Ephrin Receptor A2 Is Required for the Low Molecular Weight Hyaluronan-mediated Angiogenesis That Is implicated in Tumor Progression

Angiogenesis or the formation of new blood vessels is important in the growth and metastatic potential of various cancers. Therefore, understanding the mechanism(s) by which angiogenesis occurs can have important therapeutic implications in numerous malignancies. We and others have demonstrated that low molecular weight hyaluronan (LMW-HA, ∼2500 Da) promotes endothelial cell (EC) barrier disruption and angiogenesis. However, the mechanism(s) by which this occurs is poorly defined. Our data indicate that treatment of human EC with LMW-HA induced CD44v10 association with the receptor-tyrosine kinase, EphA2, transactivation (tyrosine phosphorylation) of EphA2, and recruitment of the PDZ domain scaffolding protein, PATJ, to the cell periphery. Silencing (siRNA) CD44, EphA2, PATJ, or Dbs (RhoGEF) expression blocked LMW-HA-mediated angiogenesis (EC proliferation, migration, and tubule formation). In addition, silencing EphA2, PATJ, Src, or Dbs expression blocked LMW-HA-mediated RhoA activation. To translate our in vitro findings, we utilized a novel anginex/liposomal targeting of murine angiogenic endothelium with either CD44 or EphA2 siRNA and observed inhibition of LMW-HA-induced angiogenesis in implanted Matrigel plugs. Taken together, these results indicate LMW-HA-mediated transactivation of EphA2 is required for PATJ and Dbs membrane recruitment and subsequent RhoA activation required for angiogenesis. These results suggest that targeting downstream effectors of LMW-HA could be a useful therapeutic intervention for angiogenesis-associated diseases including tumor progression.     

Src-mediated Post-translational Regulation of Endoglin Stability and Function Is Critical for Angiogenesis

Endoglin is a transforming growth factor β (TGF-β) co-receptor essential for angiogenesis and tumor vascularization. Endoglin modulates the crucial balance between pro- and anti-angiogenic signaling by activin receptor-like kinase (ALK) 1, 5, and TGF-β type II (TβRII) receptors. Despite its established role in physiology and disease, the mechanism of endoglin down-regulation remains unknown. Here we report that the conserved juxtamembrane cytoplasmic tyrosine motif (⁶¹²YIY⁶¹⁴) is a critical determinant of angiogenesis. Src directly phosphorylates this motif to induce endoglin internalization and degradation via the lysosome. We identified epidermal growth factor (EGF) and vascular endothelial growth factor (VEGF) as Src-activators that induce endoglin turnover following ⁶¹²YIY⁶¹⁴ phosphorylation. Interestingly, Src phosphorylation of endoglin-⁶¹²YIY⁶¹⁴ was also an important process for receptor down-regulation by TRACON105 (TRC105), an endoglin-targeting antibody currently in clinical trials. The regulation of ⁶¹²YIY⁶¹⁴ phosphorylation was critical for angiogenesis, as both the phosphomimetic and unphosphorylatable mutants impaired endothelial functions including proliferation, migration, and capillary tube formation. Collectively, these findings establish Src and pro-angiogenic mitogens as critical mediators of endoglin stability and function.     

Regulation of Vascular Endothelial Growth Factor-induced Endothelial Cell Migration by LIM Kinase 1-mediated Phosphorylation of Annexin 1

In this study, we obtained evidence indicating that annexin 1 is a new target of the p38/MAPKAP kinase-2 pathway and that it regulates endothelial cell migration in response to vascular endothelial growth factor (VEGF). These conclusions are supported by a series of substantiating experiments. First, by two-dimensional gel electrophoresis and mass spectrometry, we identified annexin 1 as a protein whose phosphorylation is induced by VEGF and is impaired by inhibiting p38. Second, using in vitro kinase assays and in vivo phosphorylation assays, we found that VEGF-mediated activation of LIM kinase 1 downstream of the p38 pathway triggers the phosphorylation of annexin 1. Third, VEGF-induced cell migration and tube formation in Matrigel are inhibited following small interfering RNA-mediated knockdown of annexin 1. Fourth, both processes are rescued in cells expressing an annexin 1 construct insensitive to the small interfering RNA knockdown. Finally, the VEGF/annexin 1-mediated cell migration is impaired by inhibiting p38. We therefore conclude that phosphorylation of annexin 1 regulates the angiogenic effect that is associated with the activation of the p38/LIM kinase 1 axis by VEGF.     

Lipopolysaccharide (LPS)-mediated Angiopoietin-2-dependent Autocrine Angiogenesis Is Regulated by NADPH Oxidase 2 (Nox2) in Human Pulmonary Microvascular Endothelial Cells

Sepsis-mediated endothelial Angiopoeitin-2 (Ang2) signaling may contribute to microvascular remodeling in the developing lung. The mechanisms by which bacterial cell wall components such as LPS mediate Ang2 signaling in human pulmonary microvascular endothelial cells (HPMECs) remain understudied. In HPMEC, LPS-induced Ang2, Tie2, and VEGF-A protein expression was preceded by increased superoxide formation. NADPH oxidase 2 (Nox2) inhibition, but not Nox4 or Nox1 inhibition, attenuated LPS-induced superoxide formation and Ang2, Tie2, and VEGF-A expression. Nox2 silencing, but not Nox4 or Nox1 silencing, inhibited LPS-mediated inhibitor of κ-B kinase β (IKKβ) and p38 phosphorylation and nuclear translocation of NF-κB and AP-1. In HPMECs, LPS increased the number of angiogenic tube and network formations in Matrigel by >3-fold. Conditioned media from LPS-treated cells also induced angiogenic tube and network formation in the presence of Toll-like receptor 4 blockade but not in the presence of Ang2 and VEGF blockade. Nox2 inhibition or conditioned media from Nox2-silenced cells attenuated LPS-induced tube and network formation. Ang2 and VEGF-A treatment rescued angiogenesis in Nox2-silenced cells. We propose that Nox2 regulates LPS-mediated Ang2-dependent autocrine angiogenesis in HPMECs through the IKKβ/NF-κB and MAPK/AP-1 pathways.     

Inactivation of Pyruvate Dehydrogenase Kinase 2 by Mitochondrial Reactive Oxygen Species

Reactive oxygen species are byproducts of mitochondrial respiration and thus potential regulators of mitochondrial function. Pyruvate dehydrogenase kinase 2 (PDHK2) inhibits the pyruvate dehydrogenase complex, thereby regulating entry of carbohydrates into the tricarboxylic acid (TCA) cycle. Here we show that PDHK2 activity is inhibited by low levels of hydrogen peroxide (H₂O₂) generated by the respiratory chain. This occurs via reversible oxidation of cysteine residues 45 and 392 on PDHK2 and results in increased pyruvate dehydrogenase complex activity. H₂O₂ derives from superoxide (O₂^˙̄), and we show that conditions that inhibit PDHK2 also inactivate the TCA cycle enzyme, aconitase. These findings suggest that under conditions of high mitochondrial O₂^˙̄ production, such as may occur under nutrient excess and low ATP demand, the increase in O₂^˙̄ and H₂O₂ may provide feedback signals to modulate mitochondrial metabolism.