Nuclear PKM2 regulates β-catenin transactivation upon EGFR activation

The embryonic pyruvate kinase M2 (PKM2) isoform is highly expressed in human cancer. In contrast to the established role of PKM2 in aerobic glycolysis or the Warburg effect, its non-metabolic functions remain elusive. Here we demonstrate, in human cancer cells, that epidermal growth factor receptor (EGFR) activation induces translocation of PKM2, but not PKM1, into the nucleus, where K433 of PKM2 binds to c-Src-phosphorylated Y333 of β-catenin. This interaction is required for both proteins to be recruited to the CCND1 promoter, leading to HDAC3 removal from the promoter, histone H3 acetylation and cyclin D1 expression. PKM2-dependent β-catenin transactivation is instrumental in EGFR-promoted tumour cell proliferation and brain tumour development. In addition, positive correlations have been identified between c-Src activity, β-catenin Y333 phosphorylation and PKM2 nuclear accumulation in human glioblastoma specimens. Furthermore, levels of β-catenin phosphorylation and nuclear PKM2 have been correlated with grades of glioma malignancy and prognosis. These findings reveal that EGF induces β-catenin transactivation via a mechanism distinct from that induced by Wnt/Wingless and highlight the essential non-metabolic functions of PKM2 in EGFR-promoted β-catenin transactivation, cell proliferation and tumorigenesis.     

Association of the Shc and Grb2/Sem5 SH2-containing proteins is implicated in activation of the Ras pathway by tyrosine kinases.

The mammalian shc gene encodes two overlapping, widely expressed proteins of 46 and 52K, with a carboxy-terminal SH2 domain that binds activated growth factor receptors, and a more amino-terminal glycine/proline-rich region. These shc gene products (Shc) are transforming when overexpressed in fibroblasts. Shc proteins become phosphorylated on tyrosine in cells stimulated with a variety of growth factors, and in cells transformed by v-src (ref. 2), suggesting that they are tyrosine kinase targets that control a mitogenic signalling pathway. Here we report that tyrosine-phosphorylated Shc proteins form a specific complex with a non-phosphorylated 23K polypeptide encoded by the grb2/sem-5 gene. The grb2/sem-5 gene product itself contains an SH2 domain, which mediates binding to Shc, and is implicated in activation of the Ras guanine nucleotide-binding protein by tyrosine kinases in both Caenorhabditis elegans and mammalian cells. Consistent with a role in signalling through Ras, shc overexpression induced Ras-dependent neurite outgrowth in PC12 cells. These results suggest that Shc tyrosine phosphorylation can couple tyrosine kinases to Grb2/Sem-5, through formation of a Shc-Grb2/Sem-5 complex, and thereby regulate the mammalian Ras signalling pathway.     

A quantitative protein interaction network for the ErbB receptors using protein microarrays

Although epidermal growth factor receptor (EGFR; also called ErbB1) and its relatives initiate one of the most well-studied signalling networks, there is not yet a genome-wide view of even the earliest step in this pathway: recruitment of proteins to the activated receptors. Here we use protein microarrays comprising virtually every Src homology 2 (SH2) and phosphotyrosine binding (PTB) domain encoded in the human genome to measure the equilibrium dissociation constant of each domain for 61 peptides representing physiological sites of tyrosine phosphorylation on the four ErbB receptors. This involved 77,592 independent biochemical measurements and provided a quantitative protein interaction network that reveals many new interactions, including ones that fall outside of our current view of domain selectivity. By slicing through the network at different affinity thresholds, we found surprising differences between the receptors. Most notably, EGFR and ErbB2 become markedly more promiscuous as the threshold is lowered, whereas ErbB3 does not. Because EGFR and ErbB2 are overexpressed in many human cancers, our results suggest that the extent to which promiscuity changes with protein concentration may contribute to the oncogenic potential of receptor tyrosine kinases, and perhaps other signalling proteins as well.     

The M2 splice isoform of pyruvate kinase is important for cancer metabolism and tumour growth

Many tumour cells have elevated rates of glucose uptake but reduced rates of oxidative phosphorylation. This persistence of high lactate production by tumours in the presence of oxygen, known as aerobic glycolysis, was first noted by Otto Warburg more than 75 yr ago. How tumour cells establish this altered metabolic phenotype and whether it is essential for tumorigenesis is as yet unknown. Here we show that a single switch in a splice isoform of the glycolytic enzyme pyruvate kinase is necessary for the shift in cellular metabolism to aerobic glycolysis and that this promotes tumorigenesis. Tumour cells have been shown to express exclusively the embryonic M2 isoform of pyruvate kinase. Here we use short hairpin RNA to knockdown pyruvate kinase M2 expression in human cancer cell lines and replace it with pyruvate kinase M1. Switching pyruvate kinase expression to the M1 (adult) isoform leads to reversal of the Warburg effect, as judged by reduced lactate production and increased oxygen consumption, and this correlates with a reduced ability to form tumours in nude mouse xenografts. These results demonstrate that M2 expression is necessary for aerobic glycolysis and that this metabolic phenotype provides a selective growth advantage for tumour cells in vivo.     

High tyrosine kinase activity in normal nonproliferating cells

Protein phosphorylation at serine and threonine residues has been implicated in the regulation of many cellular processes. More recently, tyrosine residue phosphorylation has been shown to be associated with stimulation of cell proliferation, including viral transformation and stimulation by epidermal growth factors (EGF), platelet-derived growth factor (PDGF) and other compounds related to cellular growth such as insulin and dimethyl sulphoxide. To compare protein kinases and phosphoproteins of normal and leukaemic human haematopoietic cells in vivo and in vitro, we first have investigated the percentages of phosphoserine, phosphothreonine and phosphotyrosine obtained after hydrolysis of proteins from different blood cell fractions phosphorylated in vitro. We report here that phosphotyrosine formed less than 1% of the soluble fractions from polymorphonuclear cells, mononuclear cells (80% circulating lymphocytes, 20% monocytes), blood platelets and red blood cells (not shown). Surprisingly, high percentages of phosphorylated tyrosine were found only in the particulate fractions from non-proliferating anuclear cells, platelets and red blood cells.     

A functional RNAi screen for regulators of receptor tyrosine kinase and ERK signalling

Receptor tyrosine kinase (RTK) signalling through extracellular-signal-regulated kinases (ERKs) has pivotal roles during metazoan development, underlying processes as diverse as fate determination, differentiation, proliferation, survival, migration and growth. Abnormal RTK/ERK signalling has been extensively documented to contribute to developmental disorders and disease, most notably in oncogenic transformation by mutant RTKs or downstream pathway components such as Ras and Raf. Although the core RTK/ERK signalling cassette has been characterized by decades of research using mammalian cell culture and forward genetic screens in model organisms, signal propagation through this pathway is probably regulated by a larger network of moderate, context-specific proteins. The genes encoding these proteins may not have been discovered through traditional screens owing, in particular, to the requirement for visible phenotypes. To obtain a global view of RTK/ERK signalling, we performed an unbiased, RNA interference (RNAi), genome-wide, high-throughput screen in Drosophila cells using a novel, quantitative, cellular assay monitoring ERK activation. Here we show that ERK pathway output integrates a wide array of conserved cellular processes. Further analysis of selected components-in multiple cell types with different RTK ligands and oncogenic stimuli-validates and classifies 331 pathway regulators. The relevance of these genes is highlighted by our isolation of a Ste20-like kinase and a PPM-family phosphatase that seem to regulate RTK/ERK signalling in vivo and in mammalian cells. Novel regulators that modulate specific pathway outputs may be selective targets for drug discovery.     

Spred is a Sprouty-related suppressor of Ras signalling

Cellular proliferation, and differentiation of cells in response to extracellular signals, are controlled by the signal transduction pathway of Ras, Raf and MAP (mitogen-activated protein) kinase. The mechanisms that regulate this pathway are not well known. Here we describe two structurally similar tyrosine kinase substrates, Spred-1 and Spred-2. These two proteins contain a cysteine-rich domain related to Sprouty (the SPR domain) at the carboxy terminus. In Drosophila, Sprouty inhibits the signalling by receptors of fibroblast growth factor (FGF) and epidermal growth factor (EGF) by suppressing the MAP kinase pathway. Like Sprouty, Spred inhibited growth-factor-mediated activation of MAP kinase. The Ras-MAP kinase pathway is essential in the differentiation of neuronal cells and myocytes. Expression of a dominant negative form of Spred and Spred-antibody microinjection revealed that endogenous Spred regulates differentiation in these types of cells. Spred constitutively associated with Ras but did not prevent activation of Ras or membrane translocation of Raf. Instead, Spred inhibited the activation of MAP kinase by suppressing phosphorylation and activation of Raf. Spred may represent a class of proteins that modulate Ras-Raf interaction and MAP kinase signalling.     

Silencing of TGF-beta signalling by the pseudoreceptor BAMBI.

Members of the transforming growth factor-beta (TGF-beta) superfamily, including TGF-beta, bone morphogenetic proteins (BMPs), activins and nodals, are vital for regulating growth and differentiation. These growth factors transduce their signals through pairs of transmembrane type I and type II receptor kinases. Here, we have cloned a transmembrane protein, BAMBI, which is related to TGF-beta-family type I receptors but lacks an intracellular kinase domain. We show that BAMBI is co-expressed with the ventralizing morphogen BMP4 (refs 5, 6) during Xenopus embryogenesis and that it requires BMP signalling for its expression. The protein stably associates with TGF-beta-family receptors and inhibits BMP and activin as well as TGF-beta signalling. Finally, we provide evidence that BAMBI's inhibitory effects are mediated by its intracellular domain, which resembles the homodimerization interface of a type I receptor and prevents the formation of receptor complexes. The results indicate that BAMBI negatively regulates TGF-beta-family signalling by a regulatory mechanism involving the interaction of signalling receptors with a pseudoreceptor.     

Protection of telomeres through independent control of ATM and ATR by TRF2 and POT1

When telomeres are rendered dysfunctional through replicative attrition of the telomeric DNA or by inhibition of shelterin, cells show the hallmarks of ataxia telangiectasia mutated (ATM) kinase signalling. In addition, dysfunctional telomeres might induce an ATM-independent pathway, such as ataxia telangiectasia and Rad3-related (ATR) kinase signalling, as indicated by the phosphorylation of the ATR target CHK1 in senescent cells and the response of ATM-deficient cells to telomere dysfunction. However, because telomere attrition is accompanied by secondary DNA damage, it has remained unclear whether there is an ATM-independent pathway for the detection of damaged telomeres. Here we show that damaged mammalian telomeres can activate both ATM and ATR and address the mechanism by which the shelterin complex represses these two important DNA damage signalling pathways. We analysed the telomere damage response on depletion of either or both of the shelterin proteins telomeric repeat binding factor 2 (TRF2) and protection of telomeres 1 (POT1) from cells lacking ATM and/or ATR kinase signalling. The data indicate that TRF2 and POT1 act independently to repress these two DNA damage response pathways. TRF2 represses ATM, whereas POT1 prevents activation of ATR. Unexpectedly, we found that either ATM or ATR signalling is required for efficient non-homologous end-joining of dysfunctional telomeres. The results reveal how mammalian telomeres use multiple mechanisms to avoid DNA damage surveillance and provide an explanation for the induction of replicative senescence and genome instability by shortened telomeres.     

Platelet-derived growth factor stimulates synthesis of PtdIns(3,4,5)P3 by activating a PtdIns(4,5)P2 3-OH kinase.

Although the hormone-stimulated synthesis of 3-phosphorylated inositol lipids is known to form an intracellular signalling system, there is no consensus on the crucial receptor-regulated event in this pathway and it is still not clear which of the intermediates represent potential output signals. We show here that the key step in the synthesis of 3-phosphorylated inositol lipids in 3T3 cells stimulated by platelet-derived growth factor is the activation of a phosphatidylinositol(4,5)-bisphosphate (3)-hydroxy (PtdIns(4,5)P2 3-OH) kinase. A similar conclusion has been applied to explain the actions of formyl-Met-Leu-Phe on neutrophils, and it may be that receptors that couple through intrinsic tyrosine kinases or through G proteins stimulate the same step in 3-phosphorylated inositol lipid metabolism. The close parallel between these two mechanisms for the activation of PtdIns(4,5)P2 3-OH kinase and those described for the activation of another key signalling enzyme, phospholipase C (ref. 7), focuses attention on the product of the PtdIns(4,5)P2 3-OH kinase, PtdIns(3,4,5)P3, as a possible new second messenger.     

A model for intracellular translocation of protein kinase C involving synergism between Ca2+ and phorbol esters

The activation of protein kinase C by diacylglycerol and by tumour promoters has implicated this enzyme in transmembrane signalling and in the regulation of the cell cycle. In vitro studies revealed that catalytic activity requires the presence of calcium and phospholipids with a preference for phosphatidylserine. Diacylglycerol and tumour promoters such as phorbol esters bind to the enzyme, leading to its activation while sharply increasing its affinity for Ca2+ and phospholipid. Addition of diacylglycerol analogues or phorbol esters to intact cells results in the phosphorylation of specific polypeptides. Several cellular processes, including hormone and neurotransmitter release and receptor down-regulation, are modulated by the activation of protein kinase C, while phorbol ester-induced stimulation of the enzyme in whole cells has been associated with its translocation from the cytoplasm to the plasma membrane. Moreover, the use of Ca2+ ionophores has revealed an apparent synergism between Ca2+ mobilization and protein kinase C activation. This synergism has recently also been found to apply to receptor down-regulation (ref. 23 and accompanying paper). Here we describe a reconstitution system in which intracellular translocation of protein kinase C and the synergism between Ca2+ and enzyme activators can be studied. The results suggest a rationale for concomitant Ca2+ mobilization and diacylglycerol formation in response to some hormones, neurotransmitters and growth factors.     

No T-cell tyrosine protein kinase signalling or calcium mobilization after CD4 association with HIV-1 or HIV-1 gp120.

The T lymphocyte surface protein CD4 is an integral membrane glycoprotein noncovalently associated with the tyrosine protein kinase p56lck. In normal T cells, surface association of CD4 molecules with other CD4 molecules or other T-cell surface proteins, such as the T-cell antigen receptor, stimulates the activity of the p56lck tyrosine kinase, resulting in the phosphorylation of various cellular proteins at tyrosine residues. Thus, the signal transduction in T cells generated through the surface engagement of CD4 is similar to that observed for the class of growth factor receptors possessing endogenous tyrosine kinase activity. As CD4 is also the cellular receptor for the human immunodeficiency virus (HIV), binding of the virus or gp120 (the virus surface protein responsible for specific CD4+ T-cell association) could mimic the types of immunological interactions that have previously been found to stimulate p56lck and trigger T-cell activation pathways. We have evaluated this possibility and report here that binding of HIV-1 or the virus glycoprotein gp120 to CD4+ human T cells fails to elicit detectable p56lck-dependent tyrosine kinase activation and signalling, alterations in the composition of cellular phosphotyrosine-containing proteins, or changes in intracellular Ca2+ concentration.     

Crystal structure of enolase indicates that enolase and pyruvate kinase evolved from a common ancestor

Enolase or 2-phospho-D-glycerate hydrolase catalyses the dehydration of 2-phosphoglycerate to phosphoenolpyruvate, which in turn is converted by pyruvate kinase to pyruvate. We describe here the crystallographic determination of the structure of yeast enolase at high resolution (2.25 A) and an analysis of the structural homology between enolase, pyruvate kinase and triose phosphate isomerase. Each of the two subunits of enolase forms two distinctive domains. The larger domain (residues 143-420) is a regular 8-fold beta/alpha-barrel, as first found in triose phosphate isomerase, and later in pyruvate kinase and 11 other functionally different enzymes. An analysis of the molecular geometries of enolase and pyruvate kinase based on the roughly 8-fold symmetry of the barrel showed a structural homology better than expected for proteins related by convergent evolution. We argue that enolase and pyruvate kinase have evolved from a common ancestral multifunctional enzyme which could process phosphoenolpyruvate in both directions along the glycolytic pathway. There is structural and sequence evidence that muconate lactonizing enzyme later evolved from enolase.     

Epidermal growth factor receptor is a cellular receptor for human cytomegalovirus

Human cytomegalovirus (HCMV) is a widespread opportunistic herpesvirus that causes severe and fatal diseases in immune-compromised individuals, including organ transplant recipients and individuals with AIDS. It is also a leading cause of virus-associated birth defects and is associated with atherosclerosis and coronary restenosis. HCMV initiates infection and intracellular signalling by binding to its cognate cellular receptors and by activating several signalling pathways including those mediated by mitogen-activated protein kinase, phosphatidylinositol-3-OH kinase, interferons, and G proteins. But a cellular receptor responsible for viral entry and HCMV-induced signalling has yet to be identified. Here we show that HCMV infects cells by interacting with epidermal growth factor receptor (EGFR) and inducing signalling. Transfecting EGFR-negative cells with an EGFR complementary DNA renders non-susceptible cells susceptible to HCMV. Ligand displacement and crosslinking analyses show that HCMV interacts with EGFR through gB, its principal envelope glycoprotein. gB preferentially binds EGFR and EGFR-ErbB3 oligomeric molecules in Chinese hamster ovary cells transfected with erbB family cDNAs. Taken together, these data indicate that EGFR is a necessary component for HCMV-triggered signalling and viral entry.     

Notch-dependent VEGFR3 upregulation allows angiogenesis without VEGF-VEGFR2 signalling

Developing tissues and growing tumours produce vascular endothelial growth factors (VEGFs), leading to the activation of the corresponding receptors in endothelial cells. The resultant angiogenic expansion of the local vasculature can promote physiological and pathological growth processes. Previous work has uncovered that the VEGF and Notch pathways are tightly linked. Signalling triggered by VEGF-A (also known as VEGF) has been shown to induce expression of the Notch ligand DLL4 in angiogenic vessels and, most prominently, in the tip of endothelial sprouts. DLL4 activates Notch in adjacent cells, which suppresses the expression of VEGF receptors and thereby restrains endothelial sprouting and proliferation. Here we show, by using inducible loss-of-function genetics in combination with inhibitors in vivo, that DLL4 protein expression in retinal tip cells is only weakly modulated by VEGFR2 signalling. Surprisingly, Notch inhibition also had no significant impact on VEGFR2 expression and induced deregulated endothelial sprouting and proliferation even in the absence of VEGFR2, which is the most important VEGF-A receptor and is considered to be indispensable for these processes. By contrast, VEGFR3, the main receptor for VEGF-C, was strongly modulated by Notch. VEGFR3 kinase-activity inhibitors but not ligand-blocking antibodies suppressed the sprouting of endothelial cells that had low Notch signalling activity. Our results establish that VEGFR2 and VEGFR3 are regulated in a highly differential manner by Notch. We propose that successful anti-angiogenic targeting of these receptors and their ligands will strongly depend on the status of endothelial Notch signalling.     

Altered cytosol/membrane enzyme redistribution on interleukin-3 activation of protein kinase C

Interleukin-3 (IL-3) is a member of a family of growth and differentiation peptides, collectively referred to as colony-stimulating factors, which regulate haematopoiesis. IL-3 has been highly purified from medium conditioned by WEHI-3B cells, and recently the molecular cloning of complementary DNA for murine IL-3 has been reported. IL-3 seems to stimulate a wide range of colony-forming cells derived from murine bone marrow and has consequently been studied under a variety of names, including burst-promoting activity, mast cell growth factor, P-cell stimulating factor and multi-colony-stimulating factor. Here we present evidence that IL-3-receptor interaction stimulates the rapid and transient redistribution of protein kinase C (PK-C) from cytosol to plasma membrane in FDC-P1 cells. Phorbol myristate acetate (PMA) is shown to have a similar effect in these IL-3-dependent FDC-P1 cells. Our data suggest that IL-3 and phorbol esters share a common feature of transmembrane signalling crucial for growth and differentiation.