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.     

Prolactin inhibits activity of pyruvate kinase M2 to stimulate cell proliferation

Mitogenic and prosurvival effects underlie the tumorigenic roles of prolactin (PRL) in the pathogenesis of breast cancer. PRL signaling is mediated through its receptor (PRLr). A proteomics screen identified the pyruvate kinase M2 (PKM2), a glycolytic enzyme known to play an important role in tumorigenesis, as a protein that constitutively interacts with PRLr. Treatment of cells with PRL inhibited pyruvate kinase activity and increased the lactate content in human cells in a manner that was dependent on the abundance of PRLr, activation of Janus kinase 2, and tyrosine phosphorylation of the intracellular domain of PRLr. Knockdown of PKM2 attenuated PRL-stimulated cell proliferation. The extent of this proliferation was rescued by the knock-in of the wild-type PKM2 but not of its mutant insensitive to PRL-mediated inhibition. We discuss a hypothesis that the inhibition of PKM2 by PRL contributes to the PRL-stimulated cell proliferation.     

A Novel Pyruvate Kinase M2 Activator Compound that Suppresses Lung Cancer Cell Viability under Hypoxia

Pyruvate kinase M2 isoform (PKM2), a rate-limiting enzyme in the final step of glycolysis, is known to be associated with the metabolic rewiring of cancer cells, and considered an important cancer therapeutic target. Herein, we report a novel PKM2 activator, PA-12, which was identified via the molecular docking-based virtual screening. We demonstrate that PA-12 stimulates the pyruvate kinase activity of recombinant PKM2 in vitro, with a half-maximal activity concentration of 4.92 μM, and effectively suppresses both anchorage-dependent and -independent growth of lung cancer cells in non-essential amino acid-depleted medium. In addition, PA-12 blocked the nuclear translocalization of PKM2 in lung cancer cells, resulting in the inhibition of hypoxia response element (HRE)-mediated reporter activity as well as hypoxia-inducible factor 1 (HIF-1) target gene expression, eventually leading to the suppression of cell viability under hypoxia. We also verified that the effects of PA-12 were dependent on PKM2 expression in cancer cells, demonstrating the specificity of PA-12 for PKM2 protein. Taken together, our data suggest that PA-12 is a novel and potent PKM2 activator that has therapeutic implications for lung cancer.     

Interferon-inducible mouse Mx1 protein that confers resistance to influenza virus is GTPase.

The murine Mx1 protein is an interferon-inducible nuclear protein and confers resistance to influenza virus infection even though the resistance mechanism is yet unclear. The Mx1 protein contains a tripartite GTP-binding domain consisting of GXXXXGKS, DXXG, and T/NKXD motifs. In the GTPase gene superfamily such as p21ras protein, signal-transducing G protein, and translation elongation factor, the GTPase activity plays a key role in each protein function. Here we show that GTPase activity is indeed associated with the intact Mx1 protein purified from Escherichia coli expressing Mx1 cDNA. Amino acid substitution within the GTP-binding motif led to significant reduction in the GTPase activity. Yeast vacuolar protein sorting (VPS1) protein and the rat microtubule-associated mechanochemical enzyme dynamin were found to be homologous to Mx1 not only in the tripartite GTP-binding motif, but also in the amino-terminal region of approximately 300 amino acids in length. The function of Mx1 is discussed in comparison with these proteins.     

Antiviral determinants of rat Mx GTPases map to the carboxy-terminal half.

Rat Mx2 and rat Mx3 are two alpha/beta interferon-inducible cytoplasmic GTPases that differ in three residues in the amino-terminal third, which also contains the tripartite GTP-binding domain, and that differ in five residues in the carboxy-terminal quarter, which also contains a dimerization domain. While Mx2 is active against vesicular stomatitis virus (VSV), Mx3 lacks antiviral activity. We mapped the functional difference between Mx2 and Mx3 protein to two critical residues in the carboxy-terminal parts of the molecules. An exchange of either residue 588 or 630 of Mx2 with the corresponding residues of Mx3 abolished anti-VSV activity, and the introduction of the two Mx2 residues on an Mx3 background partially restored anti-VSV activity. These results are consistent with the facts that Mx2 and Mx3 have similar intrinsic GTPase activities and that the GTPase domain of Mx3 can fully substitute for the GTPase domain of Mx2. Nevertheless, the amino-terminal third containing the GTP-binding domain is necessary for antiviral activity, since an amino-terminally truncated Mx2 protein is devoid of anti-VSV activity. Furthermore, Fab fragments of a monoclonal antibody known to neutralize antiviral activity block GTPase activity by binding an epitope in the carboxy-terminal half of Mx2 or Mx3 protein. The results are consistent with a two-domain model in which both the conserved amino-terminal half and the less-well-conserved carboxy-terminal half of Mx proteins carry functionally important domains.     

Pyruvate kinase M2 facilitates colon cancer cell migration via the modulation of STAT3 signalling

Understanding the mechanisms of colorectal cancer (CRC) metastatic progression is essential to reducing its morbidity and mortality. Pyruvate kinase (PK) catalyses the final step of glycolysis and has been identified as a critical regulator of glucose consumption. However, the mechanisms and roles of PKM1 and PKM2 in the regulation of CRC cell migration and cell adhesion remain elusive. Here, we report that PKM2 rather than PKM1 drives CRC cell migration and cell adhesion, whereas PKM attenuation reverses these phenomena. Furthermore, the overexpression of PKM2 significantly increases the expression of N-cadherin, MMP-2, MMP-9, STAT3, Snail-2, pFAK and active β1-integrin, while E-cadherin expression is suppressed. More importantly, the results indicated that PKM2 overexpression facilitates STAT3 nuclear translocation, and it is required for PKM2 function in the regulation of migration and adhesion associated signalling. In addition, the dimeric form of PKM2, which lacks the pyruvate kinase activities but possesses protein kinase activity, is critical for CRC cell migration and cell adhesion. Overall, this study suggests that PKM2 overexpression promotes CRC cell migration and cell adhesion by regulating STAT3-associated signalling and that PKM2 may serve as a therapeutic target for CRC metastasis.     

PKMζ is necessary and sufficient for synaptic clustering of PSD-95

The persistent activity of protein kinase Mzeta (PKMζ), a brain-specific, constitutively active protein kinase C isoform, maintains synaptic long-term potentiation (LTP). Structural remodeling of the postsynaptic density is believed to contribute to the expression of LTP. We therefore examined the role of PKMζ in reconfiguring PSD-95, the major postsynaptic scaffolding protein at excitatory synapses. In primary cultures of hippocampal neurons, PKMζ activity was critical for increasing the size of PSD-95 clusters during chemical LTP (cLTP). Increasing PKMζ activity by overexpressing the kinase in hippocampal neurons was sufficient to increase PSD-95 cluster size, spine size, and postsynaptic AMPAR subunit GluA2. Overexpression of an inactive mutant of PKMζ did not increase PSD-95 clustering, and applications of the ζ-pseudosubstrate inhibitor ZIP reversed the PKMζ-mediated increases in PSD-95 clustering, indicating that the activity of PKMζ is necessary to induce and maintain the increased size of PSD-95 clusters. Thus the persistent activity of PKMζ is both necessary and sufficient for maintaining increases of PSD-95 clusters, providing a unified mechanism for long-term functional and structural modifications of synapses.     

GTPase Activity of MxB Contributes to Its Nuclear Location,Interaction with Nucleoporins and Anti-HIV-1 Activity

The human myxovirus resistance 2(Mx2/Mx B) protein, a member of interferon(IFN)-inducible dynamin-like large GTPases, restricts a number of virus infections. Inhibition of these viruses occurs at poorly-defined steps after viral entry and has a common requirement for Mx B oligomerization. However, the GTPase activity is essential for the anti-viral effects of Mx B against herpesviruses and HBV but not HIV-1. To understand the role of Mx B GTPase activity, including GTP binding and GTP hydrolysis, in restriction of HIV-1 infection, we genetically separated these two functions and evaluated their contributions to restriction. We found that both the GTP binding and hydrolysis function of Mx B involved in the restriction of HIV-1 replication. The GTPase activity of Mx B contributed to its nuclear location, interaction with nucleoporins(NUPs) and HIV-1 capsids. Furthermore, Mx B disrupted the association between NUPs and HIV-1 cores dependently upon its GTPase activity. The function of GTPase activity was therefore multi-faceted, led to fundamentally distinct mechanisms employed by wild-type Mx B and GTPase activity defective Mx B mutations to restrict HIV-1 replication.     

Interferon-induced Mx proteins form oligomers and contain a putative leucine zipper.

Interferons induce a number of different proteins that mediate the antiproliferative, antiviral, and immunomodulatory functions of interferons. At least three different proteins mediate the antiviral response, and one of them, Mx protein, specifically inhibits the replication of influenza virus and (vesicular stomatitis virus). Mouse and rat Mx1 proteins are nuclear, whereas other presently known Mx proteins are cytoplasmic. The cellular functions of Mx proteins are unknown, but all of them contain a consensus GTP binding site. Very little information is available on the structure and characteristics of the mouse Mx1 protein itself. For biochemical characterization, we expressed mouse Mx1 protein in a baculovirus system and purified it to homogeneity. The purified protein as well as the authentic murine cellular Mx1 protein exists in dimers and trimers in the presence of dissociating solvents, whereas in physiological buffers they form aggregates. Cross-linking experiments done on Mx-expressing cells from various species revealed that mouse, rat, and human Mx proteins exist predominantly in trimers. Amino acid sequence analysis shows that all known Mx proteins have conserved leucine repeats typical for a leucine zipper at their COOH-terminal end. In vitro translation of chimeric catechol O-methyltransferase-Mx1 gene constructs revealed that the leucine zipper domain of Mx1 protein is responsible for the oligomerization. The COOH terminus also functions as a nuclear localization signal. Microinjection of purified oligomers into the cell cytoplasm resulted in a fast accumulation of the protein in the resulted in a fast accumulation of the protein in the nucleus. Immunoelectron microscopy revealed that nuclear murine Mx1 protein exists in distinct, electron-dense structures separate from nuclear membrane, and chromatin, or nucleolus. These observations reveal that a COOH-terminal leucine zipper domain is an important structural element of all Mx proteins. Its relevance to the biology and functions of Mx proteins is presently not known.     

Isozymes of protein kinase C in human neutrophils and their modification by two endogenous proteinases

Two major protein kinase C (PKC) isozymes, accounting for approximately 95% of the total activity in human neutrophils, were separated by hydroxyapatite chromatography and were identified as beta-PKC (60% of the total) and alpha-PKC (35% of the total). No gamma-PKC was detected. A minor Ca2+/phospholipid requiring kinase that eluted from hydroxyapatite after alpha-PKC did not react significantly with any of the specific antisera employed for identification. Modification of beta-PKC or the minor PKC isozyme by calpain yielded Ca2+/phospholipid-independent forms (PKM) that retained only 50% of the original activities. In contrast, PKM formed from alpha-PKC retained full catalytic activity. For each native isozyme the rate of conversion by calpain was accelerated in the presence of Ca2+ and the lipid effectors, and the PKM form generated in each case was resistant to further digestion by calpain. All three PKC isozymes were also modified by a neutral serine proteinase isolated from human neutrophils, with this proteinase the major effect being loss of kinase activity, via a transient production of a Ca2+/phospholipid-independent form. This neutral serine proteinase appears to be localized at sites of interaction of cytoskeletal proteins with the cell membrane. Following stimulation of intact neutrophils with phorbol 12-myristate 13-acetate complete loss of native cytosolic kinase activity was observed, with recovery of approximately 30% of the original activity as a cytosolic Ca+/phospholipid independent form, presumably PKM. Loss of native PKC activity was greatest for the beta-isozyme. In cells stimulated by fMet-Leu-Phe approximately 60% of the original PKC activity was recovered as native cytosolic PKC and 30% as cytosolic PKM. Inhibitors of calpain reduced the extent of down-regulation of PKC, increased the proportion of PKC that remained associated with the plasma membrane and significantly reduced the proteolytically generated fully active PKM. Taken together, the in vitro and in vivo results suggest that calpain is involved primarily in the conversion of the PKC isozymes to the irreversibly activated PKM forms, and that the neutral serine proteinase may be the enzyme responsible for down-regulation, possibly via PKM as an intermediate.     

IGF1 regulates PKM2 function through Akt phosphorylation

Pyruvate kinase M2 (PKM2) acts at the crossroad of growth and metabolism pathways in cells. PKM2 regulation by growth factors can redirect glycolytic intermediates into key biosynthetic pathway. Here we show that IGF1 can regulate glycolysis rate, stimulate PKM2 Ser/Thr phosphorylation and decrease cellular pyruvate kinase activity. Upon IGF1 treatment we found an increase of the dimeric form of PKM2 and the enrichment of PKM2 in the nucleus. This effect was associated to a reduction of pyruvate kinase enzymatic activity and was reversed using metformin, which decreases Akt phosphorylation. IGF1 induced an increased nuclear localization of PKM2 and STAT3, which correlated with an increased HIF1α, HK2, and GLUT1 expression and glucose entrapment. Metformin inhibited HK2, GLUT1, HIF-1α expression and glucose consumption. These findings suggest a role of IGFIR/Akt axis in regulating glycolysis by Ser/Thr PKM2 phosphorylation in cancer cells.     

Beta‐elemene inhibits breast cancer metastasis through blocking pyruvate kinase M2 dimerization and nuclear translocation

Pyruvate kinase M2 (PKM2), playing a central role in regulating aerobic glycolysis, was considered as a promising target for cancer therapy. However, its role in cancer metastasis is rarely known. Here, we found a tight relationship between PKM2 and breast cancer metastasis, demonstrated by the findings that beta‐elemene (β‐elemene), an approved drug for complementary cancer therapy, exerted distinct anti‐metastatic activity dependent on PKM2. The results indicated that β‐elemene inhibited breast cancer cell migration, invasion in vitro as well as metastases in vivo. β‐Elemene further inhibited the process of aerobic glycolysis and decreased the utilization of glucose and the production of pyruvate and lactate through suppressing pyruvate kinase activity by modulating the transformation of dimeric and tetrameric forms of PKM2. Further analysis revealed that β‐elemene suppressed aerobic glycolysis by blocking PKM2 nuclear translocation and the expression of EGFR, GLUT1 and LDHA by influencing the expression of importin α5. Furthermore, the effect of β‐elemene on migration, invasion, PKM2 transformation, and nuclear translocation could be reversed in part by fructose‐1,6‐bisphosphate (FBP) and L‐cysteine. Taken together, tetrameric transformation and nuclear translocation of PKM2 are essential for cancer metastasis, and β‐elemene inhibited breast cancer metastasis via blocking aerobic glycolysis mediated by dimeric PKM2 transformation and nuclear translocation, being a promising anti‐metastatic agent from natural compounds.     

A non-canonical role for pyruvate kinase M2 as a functional modulator of Ca2+ signalling through IP3 receptors

Pyruvate kinase isoform M2 (PKM2) is a rate-limiting glycolytic enzyme that is widely expressed in embryonic tissues. The expression of PKM2 declines in some tissues following embryogenesis, while other pyruvate kinase isozymes are upregulated. However, PKM2 is highly expressed in cancer cells and is believed to play a role in supporting anabolic processes during tumour formation. In this study, PKM2 was identified as an inositol 1,4,5-trisphosphate receptor (IP₃R)-interacting protein by mass spectrometry. The PKM2:IP₃R interaction was further characterized by pull-down and co-immunoprecipitation assays, which showed that PKM2 interacted with all three IP₃R isoforms. Moreover, fluorescence microscopy indicated that both IP₃R and PKM2 localized at the endoplasmic reticulum. PKM2 binds to IP₃R at a highly conserved 21-amino acid site (corresponding to amino acids 2078–2098 in mouse type 1 IP₃R isoform). Synthetic peptides (denoted ‘TAT-D5SD’ and ‘D5SD’), based on the amino acid sequence at this site, disrupted the PKM2:IP₃R interaction and potentiated IP₃R-mediated Ca²⁺ release both in intact cells (TAT-D5SD peptide) and in a unidirectional ⁴⁵Ca²⁺ flux assay on permeabilized cells (D5SD peptide). The TAT-D5SD peptide did not affect the enzymatic activity of PKM2. Reducing PKM2 protein expression using siRNA increased IP₃R-mediated Ca²⁺ signalling in intact cells without altering the ER Ca²⁺ content. These data identify PKM2 as an IP₃R-interacting protein that inhibits intracellular Ca²⁺ signalling. The elevated expression of PKM2 in cancer cells is therefore not solely connected to its canonical role in glycolytic metabolism, rather PKM2 also has a novel non-canonical role in regulating intracellular signalling.