Michel Veron

NEMO specifically recognizes K63-linked poly-ubiquitin chains through a new bipartite ubiquitin-binding domain

An important property of NEMO, the core element of the IKK complex involved in NF‐κB activation, resides in its ability to specifically recognize poly‐ubiquitin chains. A small domain called NOA/UBAN has been suggested to be responsible for this property. We recently demonstrated that the C‐terminal Zinc Finger (ZF) of NEMO is also able to bind ubiquitin. We show here by ZF swapping and mutagenesis that this represents its only function. While neither NOA nor ZF shows any preference for K63‐linked chains, we demonstrate that together they form a bipartite high‐affinity K63‐specific ubiquitin‐binding domain. A similar domain can be found in two other proteins, Optineurin and ABIN2, and can be freely exchanged with that of NEMO without interfering with its activity. This suggests that the main function of the C‐terminal half of NEMO is to specifically bind K63‐linked poly‐ubiquitin chains. We also demonstrate that the recently described binding of NEMO to linear poly‐ubiquitin chains is dependent on the NOA alone and does not require the presence of the ZF.

X-ray structure of nucleoside diphosphate kinase.

The X‐ray structure of a point mutant of nucleoside diphosphate kinase (NDP kinase) from Dictyostelium discoideum has been determined to 2.2 A resolution. The enzyme is a hexamer made of identical subunits with a novel mononucleotide binding fold. Each subunit contains an alpha/beta domain with a four stranded, antiparallel beta‐sheet. The topology is different from adenylate kinase, but identical to the allosteric domain of Escherichia coli ATCase regulatory subunits, which bind mononucleotides at an equivalent position. Dimer contacts between NDP kinase subunits within the hexamer are similar to those in ATCase. Trimer contacts involve a large loop of polypeptide chain that bears the site of the Pro‐‐‐‐Ser substitution in Killer of prune (K‐pn) mutants of the highly homologous Drosophila enzyme. Properties of Drosophila NDP kinase, the product of the awd developmental gene, and of the human enzyme, the product of the nm23 genes in tumorigenesis, are discussed in view of the three‐dimensional structure and of possible interactions of NDP kinase with other nucleotide binding proteins.

Culmination in dictyostelium is regulated by the cAMP-dependent protein kinase

We placed a specific inhibitor of cyclic AMP-dependent protein kinase (PKA) under the control of a prestalk-specific promoter. Cells containing this construct form normally patterned slugs, but under environmental conditions that normally trigger immediate culmination, the slugs undergo prolonged migration. Slugs that eventually enter culmination do so normally but arrest as elongated, hairlike structures that contain neither stalk nor spore cells. Mutant cells do not migrate to the stalk entrance when codeveloped with wild-type cells and show greatly reduced inducibility by DIF, the stalk cell morphogen. These results suggest that the activity of PKA is necessary for the altered pattern of movement of prestalk cells at culmination and their differentiation into stalk cells. We propose a model whereby a protein repressor, under the control of PKA, inhibits precocious induction of stalk cell differentiation by DIF and so regulates the choice between slug migration and culmination.

Activation of the prespore and spore cell pathway of Dictyostelium differentiation by cAMP-dependent protein kinase and evidence for its upstream regulation by ammonia.

Expression of a dominant inhibitor of the Dictyostelium cAMP‐dependent protein kinase in prespore cells blocks their differentiation into spore cells. The resultant structures comprise a normal stalk supporting a bolus of cells that fail to express a sporulation‐specific gene and that show greatly reduced levels of expression of several prespore‐specific genes. The latter result suggests that in addition to activating spore formation, the cAMP‐dependent protein kinase may play a role in initial prespore cell differentiation. Development of the strain expressing the dominant inhibitor is hypersensitive to the inhibitory effects of ammonia, the molecule that is believed to repress entry into culmination during normal development. This result supports a model whereby a decrease in ambient ammonia concentration at culmination acts to elevate intracellular cAMP and hence induce terminal differentiation.

Multiple roles for cAMP-dependent protein kinase during Dictyostelium development☆

The cAMP-dependent protein kinase (PKA) holoenzyme of Dictyostelium comprises a single regulatory (R) and catalytic (C) subunit, and both proteins increase in concentration during cellular aggregation. In order to determine the role of the kinase, we have constructed mutants of the R subunit that are defective in cAMP binding, in inhibition of the C subunit, or in both functions. Analysis of these mutants suggests that overexpression of the unmutated R subunit, which is known to block development, occurs by direct inactivation of the C subunit rather than by an effect on intracellular cAMP levels. Cells with an inactive C subunit (PKA- cells) are defective in cAMP relay, the production of cAMP in response to extracellular cAMP stimulation. This presumably accounts for their inability to undertake aggregation. When mixed with wild-type cells, PKA- cells migrate toward the signalling centre but remain confined to the periphery of the tight aggregate and are lost from the back of the migratory slug. This suggests that PKA may be required during the late, multicellular stages of development. Consistent with this, we find that a number of postaggregative genes are not expressed in PKA- cells, even when they are allowed to synergise with normal cells.

Overexpression of nucleoside diphosphate kinase (nm23) in solid tumours

The product of the nm23-H1 gene, reported to be related to the metastatic potential of tumour cells, was recently identified as the nucleoside diphosphate (NDP) kinase A (Gilles et al., 1991, J Biol Chem, 266, 8784-8789). An analysis of the enzyme by activity measurement and immunological techniques using polyclonal antibodies raised against the NDP kinase A purified from human erythrocytes, was performed on 39 human tissue specimens. Markedly increased activity and higher level of the protein were observed in extracts of solid tumours as compared to the corresponding normal tissues (P less than 0.01). An intense immunolabelling of tumoral cells was observed in sections of the malignant tumours and of some but not all benign neoplasia. The staining is observed in noninvasive and invasive ductal breast carcinomas with or without lymph node involvement as well as in colon and cervix carcinomas and in a case of metastatic melanoma. Therefore, NDP kinase A level is increased in neoplastic tissues but no correlation with metastatic potential could be demonstrated.

Crystal structure of the Awd nucleotide diphosphate kinase from Drosophila

BACKGROUND Nucleotide diphosphate kinase (NDP kinase) is a phosphate transfer enzyme involved in cell regulation and in animal development. Drosophila NDP kinase is the product of the abnormal wing disc (awd) developmental gene, a point mutation in which can produce the killer of prune (K-pn) conditional lethal phenotype. The highly homologous mammalian genes control metastasis and a human NDP kinase acts as a transcription factor. RESULTS The X-ray structure of the Awd protein prepared from Drosophila was solved at 2.4 A resolution by molecular replacement from the homologous Dictyostelium protein. Both are hexamers, and both have the same fold and the same active site. Subunit contacts differ as a result of sequence changes in the carboxy-terminal segment and in the loop that is the site of the K-pn mutation. CONCLUSIONS Regulatory properties of animal NDP kinases depend on interactions with other macromolecules, such as DNA and the product of the Drosophila prune gene. The Awd structure suggests an allosteric mechanism of action of NDP kinase where DNA is the effector and the protein undergoes a major conformational change, possibly dissociating to dimers.

Activation of G-proteins by receptor-stimulated nucleoside diphosphate kinase in Dictyostelium.

Recently, interest in the enzyme nucleoside diphosphate kinase (EC2.7.4.6) has increased as a result of its possible involvement in cell proliferation and development. Since NDP kinase is one of the major sources of GTP in cells, it has been suggested that the effects of an altered NDP kinase activity on cellular processes might be the result of altered transmembrane signal transduction via guanine nucleotide‐binding proteins (G‐proteins). In the cellular slime mould Dictyostelium discoideum, extracellular cAMP induces an increase of phospholipase C activity via a surface cAMP receptor and G‐proteins. In this paper it is demonstrated that part of the cellular NDP kinase is associated with the membrane and stimulated by cell surface cAMP receptors. The GTP produced by the action of NDP kinase is capable of activating G‐proteins as monitored by altered G‐protein‐receptor interaction and the activation of the effector enzyme phospholipase C. Furthermore, specific monoclonal antibodies inhibit the effect of NDP kinase on G‐protein activation. These results suggest that receptor‐stimulated NDP kinase contributes to the mediation of hormone action by producing GTP for the activation of GTP‐binding proteins.

The Zinc Finger of NEMO Is a Functional Ubiquitin-binding Domain

NEMO (NF-κB essential modulator) is a regulatory protein essential to the canonical NF-κB signaling pathway, notably involved in immune and inflammatory responses, apoptosis, and oncogenesis. Here, we report that the zinc finger (ZF) motif, located in the regulatory C-terminal half of NEMO, forms a specific complex with ubiquitin. We have investigated the NEMO ZF-ubiquitin interaction and proposed a structural model of the complex based on NMR, fluorescence, and mutagenesis data and on the sequence homology with the polymerase η ubiquitin-binding zinc finger involved in DNA repair. Functional complementation assays and in vivo pull-down experiments further show that ZF residues involved in ubiquitin binding are functionally important and required for NF-κB signaling in response to tumor necrosis factor-α. Thus, our findings indicate that NEMOZFisa bona fide ubiquitin-binding domain of the ubiquitin-binding zinc finger type.

Structural basis for activation of α-boranophosphate nucleotide analogues targeting drug-resistant reverse transcriptase

AIDS chemotherapy is limited by inadequate intracellular concentrations of the active triphosphate form of nucleoside analogues, leading to incomplete inhibition of viral replication and the appearance of drug‐resistant virus. Drug activation by nucleoside diphosphate kinase and inhibition of HIV‐1 reverse transcriptase were studied comparatively. We synthesized analogues with a borano (BH3−) group on the α‐phosphate, and found that they are substrates for both enzymes. X‐ray structures of complexes with nucleotide diphosphate kinase provided a structural basis for their activation. The complex with d4T triphosphate displayed an intramolecular CH…O bond contributing to catalysis, and the Rp diastereoisomer of thymidine α‐boranotriphosphate bound like a normal substrate. Using α‐(Rp)‐boranophosphate derivatives of the clinically relevant compounds AZT and d4T, the presence of the α‐borano group improved both phosphorylation by nucleotide diphosphate kinase and inhibition of reverse transcription. Moreover, repair of blocked DNA chains by pyrophosphorolysis was reduced significantly in variant reverse transcriptases bearing substitutions found in drug‐resistant viruses. Thus, the α‐borano modification of analogues targeting reverse transcriptase may be of generic value in fighting viral drug resistance.