Ataxia telangiectasia (AT) is a rare human autosomal recessive disorder with pleiotropic phenotypes, including neuronal degeneration, immune dysfunction, premature ageing and increased cancer risk. The gene mutated in AT, ATM, encodes a putative lipid or protein kinase. Most of the human AT patient phenotypes are recapitulated in Atm-deficient mice. Cells derived from Atm-/- mice, like those from AT patients, exhibit abnormal response to ionizing radiation. One of the known responses to ionizing radiation is the activation of a nuclear tyrosine kinase encoded by the c-abl proto-oncogene. Ionizing radiation does not activate c-Abl in cells from AT patients or in thymocytes or fibroblasts from the Atm-deficient mice. Ectopic expression of a functional ATM kinase domain corrects this defect, as it phosphorylates the c-Abl tyrosine kinase in vitro at Ser 465, leading to the activation of c-Abl. A mutant c-Abl with Ser 465 changed to Ala 465 is not activated by ionizing radiation or ATM kinase in vivo. These findings identify the c-Abl tyrosine kinase as a downstream target of phosphorylation and activation by the ATM kinase in the cellular response to ionizing radiation.

Genotoxic stress triggers signalling pathways that mediate either the protection or killing of affected cells. Whereas induction of p53 involves events in the cell nucleus, the activation of transcription factors AP-1 and NF-kappaB by ultraviolet radiation is mediated through membrane-associated signalling proteins, ruling out a nuclear signal. An early event in AP-1 induction by ultraviolet radiation is activation of Jun kinases (JNKs), which mediate the induction of the immediate-early genes c-jun and c-fos. The JNKs have also been proposed to mediate the apoptopic response to genotoxins. The non-receptor tyrosine kinase c-Abl is also activated by genotoxic stress. To understand the relationship between these events, we compared the activation of p53, JNK and c-Abl by several DNA-damaging agents in murine fibroblasts. We found that whereas p53 was induced by every genotoxic stimulus tested, c-Abl was activated by most stimuli except ultraviolet irradiation and JNK was strongly stimulated only by ultraviolet light and the alkylating agent methyl methanesulphonate. Activation of JNK by this alkylating agent was normal in c-Abl-null cells but was reduced in c-Src-null cells. Unlike p53 induction, c-Abl activation occurs in the S phase of the cell cycle and does not affect cell proliferation. These findings show that signals generated by genotoxins are transduced by multiple, independent pathways. Only p53 appears to be a universal sensor of genotoxic stress.

Cell-cycle checkpoints help to protect the genomes of proliferating cells under genotoxic stress. In multicellular organisms, cell proliferation is often directed toward differentiation during development and throughout adult homeostasis. To prevent the formation of differentiated cells with genetic instability, we hypothesized that genotoxic stress may trigger a differentiation checkpoint. Here we show that exposure to genotoxic agents causes a reversible inhibition of myogenic differentiation. Muscle-specific gene expression is suppressed by DNA-damaging agents if applied prior to differentiation induction but not after the differentiation program is established. The myogenic determination factor, MyoD (encoded by Myod1), is a target of the differentiation checkpoint in myoblasts. The inhibition of MyoD by DNA damage requires a functional c-Abl tyrosine kinase (encoded by Abl1), but occurs in cells deficient for p53 (transformation-related protein 53, encoded by Trp53) or c-Jun (encoded by the oncogene Jun). These results support the idea that genotoxic stress can regulate differentiation, and identify a new biological function for DNA damage-activated signaling network.

Posttranslational modification by small ubiquitin-like modifier (SUMO) conjugation regulates the subnuclear localization of several proteins; however, SUMO modification has not been directly linked to nuclear export. The ETS (E-Twenty-Six) family member TEL (ETV6) is a transcriptional repressor that can inhibit Rasdependent colony growth in soft agar and induce cellular aggregation of Ras-transformed cells. TEL is frequently disrupted by chromosomal translocations such as the t(12;21), which is associated with nearly one-fourth of pediatric B cell acute lymphoblastic leukemia. In the vast majority of t(12;21)-containing cases, the second allele of TEL is deleted, suggesting that inactivation of TEL contributes to the disease. Although TEL functions in the nucleus as a DNA-binding transcriptional repressor, it has also been detected in the cytoplasm. Here we demonstrate that TEL is actively exported from the nucleus in a leptomycin B-sensitive manner. TEL is posttranslationally modified by sumoylation at lysine 99 within a highly conserved domain (the ''pointed'' domain). Mutation of the sumo-acceptor lysine or mutations within the pointed domain that affect sumoylation impair nuclear export of TEL. Mutation of lysine 99 also results in an increase in TEL transcriptional repression, presumably because of decreased nuclear export. We propose that the ability of TEL to repress transcription and suppress growth is regulated by sumoylation and nuclear export.T he function of posttranslational modification by ligation of a small ubiquitin-like modifier (SUMO) to a target protein appears to be diverse and substrate specific. RanGAP1 was the first protein shown to be modified by addition of SUMO (1-3). Unmodified RanGAP1 is diffusely cytoplasmic, whereas sumoylation targets RanGAP1 to nuclear pore complexes. Sumoylation also directs targeted proteins such as promyelocytic leukemia and the homeodomain-interacting protein kinase 2 to distinct subnuclear domains (nuclear bodies or speckles; refs. 4 and 5). SUMO modification appears to activate the heat shock transcription factors 1 and 2 (6, 7), whereas sumoylation may negatively regulate c-JUN and c-MYB activity (8, 9). Sumoylation also affects protein stability if the modified lysine is also used for ubiquitination. Both inhibitor of NF-B␣ and murine double minute 2 are targeted for degradation by ubiquitination, but competition for the target lysine by SUMO stabilized these factors (10-12). Like ubiquitin, SUMO conjugation and deconjugation is a very dynamic process. Sumoylation studies are limited because of the rapid removal of the modification by SUMO-specific isopeptidases in cellular extracts. Thus, for each of these examples, the functional consequences of sumoylation were derived from conservative substitution of the modified lysine of the target protein.Like ubiquitination, sumoylation is a three-step process involving an E1-activating enzyme heterodimer Aos͞Uba2, the E2-conjugating enzyme Ubc9 and substrate-specific E3 ligases (for recent reviews, see refs. 13 ...

IL-17A is a pro-inflammatory cytokine that has been implicated in autoimmune and inflammatory diseases. Monoclonal antibodies inhibiting IL-17A signaling have demonstrated remarkable efficacy, but an oral therapy is still lacking. A high affinity IL-17A peptide antagonist (HAP) of 15 residues was identified through phage-display screening followed by saturation mutagenesis optimization and amino acid substitutions. HAP binds specifically to IL-17A and inhibits the interaction of the cytokine with its receptor, IL-17RA. Tested in primary human cells, HAP blocked the production of multiple inflammatory cytokines. Crystal structure studies revealed that two HAP molecules bind to one IL-17A dimer symmetrically. The N-terminal portions of HAP form a β-strand that inserts between two IL-17A monomers while the C-terminal section forms an α helix that directly blocks IL-17RA from binding to the same region of IL-17A. This mode of inhibition suggests opportunities for developing peptide antagonists against this challenging target.