Enhanced phosphorylation of p53 by ATM in response to DNA damage.

The ATM protein, encoded by the gene responsible for the human genetic disorder ataxia telangiectasia (A-T), regulates several cellular responses to DNA breaks. ATM shares a phosphoinositide 3-kinase-related domain with several proteins, some of them protein kinases. A wortmannin-sensitive protein kinase activity was associated with endogenous or recombinant ATM and was abolished by structural ATM mutations. In vitro substrates included the translation repressor PHAS-I and the p53 protein. ATM phosphorylated p53 in vitro on a single residue, serine-15, which is phosphorylated in vivo in response to DNA damage. This activity was markedly enhanced within minutes after treatment of cells with a radiomimetic drug; the total amount of ATM remained unchanged. Various damage-induced responses may be activated by enhancement of the protein kinase activity of ATM.

Wiskott-Aldrich syndrome protein (WASp) is a binding partner for c-Src family protein-tyrosine kinases.

BACKGROUND: Receptor-mediated signal transduction requires the assembly of multimeric complexes of signalling proteins, and a number of conserved protein domains, such as the SH2, SH3 and PH domains, are involved in mediating protein-protein interactions in such complexes. The identification of binding partners for these domains has added considerably to our understanding of signal-transduction pathways, and the purpose of this work was to identify SH3-binding proteins in haematopoietic cells. RESULTS: We performed affinity-chromatography experiments with a panel of GST-SH3 fusion proteins (composed of glutathione-S-transferase appended to various SH3 domains) to search for SH3-binding proteins in a human megakaryocytic cell line. Protein microsequencing identified one of the SH3-binding proteins as WASp, the protein that is defective in Wiskott-Aldrich syndrome (WAS) and isolated X-linked thrombocytopenia. WASp bound preferentially in vitro to SH3 domains from c-Src family kinases, and analysis of proteins expressed in insect cells using a baculovirus vector demonstrated a specific interaction between WASp and the Fyn protein-tyrosine kinase. Finally, in vivo experiments showed that WASp and Fyn physically associate in human haematopoietic cells. CONCLUSIONS: Haematopoietic cells from individuals with WAS exhibit defects in cell morphology and signal transduction, including reduced proliferation and tyrosine phosphorylation in response to stimulatory factors. Members of the c Src family of protein-tyrosine kinases, including Fyn, are involved in a range of signalling pathways - such as those regulating cytoskeletal structure - in both haematopoietic and non-haematopoietic cells. Our data suggest that binding of Fyn to WASp may be a critical event in such signalling pathways in haematopoietic cells.

Interaction between Wiskott-Aldrich Syndrome protein (WASP) and the Fyn protein-tyrosine kinase.

Wiskott-Aldrich Syndrome (WAS) is a severe X-linked disorder characterised by immune deficiency, thrombocytopenia and eczema, resulting from abnormalities in a range of haematopoietic cell types. The protein that is defective in WAS, named WASP, appears to be involved in regulating changes in the cytoskeletal organisation of haematopoietic cells in response to external stimuli. In support of this idea, WASP has been found to be physically associated in haematopoietic cells in vivo with a number of SH3 domain-containing proteins involved in signal transduction, including the cytoplasmic protein-tyrosine kinase Fyn. Here, we have used a baculovirus expression system to explore the biochemical consequences of the interaction between WASP and Fyn. We find that the kinase activity of Fyn is stimulated as a result of binding to WASP, and that a cellular protein, which may be WASP itself, becomes phosphorylated on tyrosine as a result of the binding of WASP to Fyn.

ATM is required for IkappaB kinase (IKKk) activation in response to DNA double strand breaks.

Following challenge with proinflammatory stimuli or generation of DNA double strand breaks (DSBs), transcription factor NF-kappaB translocates from the cytoplasm to the nucleus to activate expression of target genes. In addition, NF-kappaB plays a key role in protecting cells from proapoptotic stimuli, including DSBs. Patients suffering from the genetic disorder ataxia-telangiectasia, caused by mutations in the ATM gene, are highly sensitive to inducers of DSBs, such as ionizing radiation. Similar hypersensitivity is displayed by cell lines derived from ataxia-telangiectasia patients or Atm knockout mice. The ATM protein, a member of the phosphatidylinositol 3-kinase (PI3K)-like family, is a multifunctional protein kinase whose activity is stimulated by DSBs. As both ATM and NF-kappaB deficiencies result in increased sensitivity to DSBs, we examined the role of ATM in NF-kappaB activation. We report that ATM is essential for NF-kappaB activation in response to DSBs but not proinflammatory stimuli, and this activity is mediated via the IkappaB kinase complex. DNA-dependent protein kinase, another member of the PI3K-like family, PI3K itself, and c-Abl, a nuclear tyrosine kinase, are not required for this response.

Evidence that the Wiskott-Aldrich syndrome protein may be involved in lymphoid cell signaling pathways.

Wiskott-Aldrich syndrome is an X-linked combined immunodeficiency affecting cells of several different hemopoietic lineages. The Wiskott-Aldrich syndrome protein (WASP), which has no homology with any other known protein families, is rich in proline motifs known to contribute to Src homology 3 binding sites. However, its function has not been determined. The Tec family of cytoplasmic tyrosine kinases, which include Btk (the X-linked agammaglobulinemia gene), Itk, and Tec, is thought to be involved in lymphoid cell signaling pathways. In this work, we show binding of WASP to the Src homology 3 domains of Btk, Itk, Tec, Grb2, and phospholipase C-gamma, which suggests a function for WASP in lymphoid cell signaling.

Characterization of ATM gene mutations in 66 ataxia telangiectasia families.

Ataxia telangiectasia (AT) is an autosomal recessive disease characterized by neurological and immunological symptoms, radiosensitivity and cancer predisposition. The gene mutated in AT, designated the ATM gene, encodes a large protein kinase with a PI-3 kinase-related domain. In this study, we investigated the mutational spectrum of the ATM gene in a cohort of AT patients living in Germany. We amplified and sequenced all 66 exons and the flanking untranslated regions from genomic DNA of 66 unrelated AT patients. We identified 46 different ATM mutations and 26 sequence polymorphisms and variants scattered throughout the gene. A total of 34 mutations have not been described in other populations. Seven mutations occurred in more than one family, but none of these accounted for more than five alleles in our patient group. The majority of the mutations were truncating, confirming that the absence of full-length ATM protein is the most common molecular basis of AT. Transcript analyses demonstrated single exon skipping as the consequence of most splice site substitutions, but a more complex pattern was observed for two mutations. Immunoblot studies of cell lines carrying ATM missense substitutions or in-frame deletions detected residual ATM protein in four cases. One of these mutations, a valine deletion proximal to the kinase domain, resulted in ATM protein levels >20% of normal in an AT lymphoblastoid cell line. In summary, our results survey and characterize a plethora of variations in the ATM gene identified by exon scanning sequencing and indicate a high diversity of mutations giving rise to AT in a non-isolated population.