A conserved amino-terminal Shc domain binds to phosphotyrosine motifs in activated receptors and phosphopeptides

AbstractBackground: Signal transduction by growth factor receptor protein-tyrosine kinases is generally initiated by autophosphorylation on tyrosine residues following ligand binding. Phosphotyrosines within activated receptors form binding sites for the Src homology 2 (SH2) domains of cytoplasmic signalling proteins. One such protein, Shc, is tyrosine phosphorylated in response to a large number of growth factors and cytokines. Phosphorylation of Shc on tyrosine residue Y317 allows binding to the SH2 domain of Grb2, and hence stimulation of the Ras pathway. Shc is therefore implicated as an adaptor protein able to couple normal and oncogenic protein-tyrosine kinases to Ras activation. Shc itself contains an SH2 domain at its carboxyl terminus, but the function of the amino-terminal half of the protein is unknown.Results We have found that the Shc amino-terminal region binds to a number of tyrosine-phosphorylated proteins in v-src-transformed cells. This domain also bound directly to the activated epidermal growth factor (EGF) receptor. A phosphotyrosine (pY)-containing peptide modeled after the Shc-binding site in polyoma middle T antigen (LLSNPTpYSVMRSK) was able to compete efficiently with the activated EGF receptor for binding to the Shc amino terminus. This competition was dependent on phosphorylation of the tyrosine residue within the peptide, and was abrogated by deletion of the leucine residue at position –5. The Shc amino-terminal domain also bound to the autophosphorylated nerve growth factor receptor (Trk), but bound significantly less well to a mutant receptor in which tyrosine Y490 in the receptor's Shc-binding site had been substituted by phenylalanine.Conclusion These data implicate the amino-terminal region of Shc in binding to activated receptors and other tyrosine-phosphorylated proteins. Binding appears to be specific for phosphorylated tyrosine residues within the sequence NPXpY, which is conserved in many Shc-binding sites. The Shc amino-terminal region bears only very limited sequence identity to known SH2 domains, suggesting that it represents a new class of phosphotyrosine-binding modules. Consistent with this view, the amino-terminal Shc domain is highly conserved in a Drosophila Shc homologue. Binding of Shc to activated receptors through its amino terminus could leave the carboxy-terminal SH2 domain free for other interactions. In this way, Shc may function as an adaptor protein to bring two tyrosine-phosphorylated proteins together

Finishing the euchromatic sequence of the human genome

The sequence of the human genome encodes the genetic instructions for human physiology, as well as rich information about human evolution. In 2001, the International Human Genome Sequencing Consortium reported a draft sequence of the euchromatic portion of the human genome. Since then, the international collaboration has worked to convert this draft into a genome sequence with high accuracy and nearly complete coverage. Here, we report the result of this finishing process. The current genome sequence (Build 35) contains 2.85 billion nucleotides interrupted by only 341 gaps. It covers ∼99% of the euchromatic genome and is accurate to an error rate of ∼1 event per 100,000 bases. Many of the remaining euchromatic gaps are associated with segmental duplications and will require focused work with new methods. The near-complete sequence, the first for a vertebrate, greatly improves the precision of biological analyses of the human genome including studies of gene number, birth and death. Notably, the human enome seems to encode only 20,000-25,000 protein-coding genes. The genome sequence reported here should serve as a firm foundation for biomedical research in the decades ahead

Cleft Palate, Moderate Lung Developmental Retardation and Early Postnatal Lethality in Mice Deficient in the Kir7.1 Inwardly Rectifying K+ Channel.

Kir7.1 is an inwardly rectifying K+ channel of the Kir superfamily encoded by the kcnj13 gene. Kir7.1 is present in epithelial tissues where it colocalizes with the Na+/K+-pump probably serving to recycle K+ taken up by the pump. Human mutations affecting Kir7.1 are associated with retinal degeneration diseases. We generated a mouse lacking Kir7.1 by ablation of the Kcnj13 gene. Homozygous mutant null mice die hours after birth and show cleft palate and moderate retardation in lung development. Kir7.1 is expressed in the epithelium covering the palatal processes at the time at which palate sealing takes place and our results suggest it might play an essential role in late palatogenesis. Our work also reveals a second unexpected role in the development and the physiology of the respiratory system, where Kir7.1 is expressed in epithelial cells all along the respiratory tree

Accelerated Mammary Tumor Development in Mutant Polyomavirus Middle T Transgenic Mice Expressing Elevated Levels of Either the Shc or Grb2 Adapter Protein

The Grb2 and Shc adapter proteins play critical roles in coupling activated growth factor receptors to several cellular signaling pathways. To assess the role of these molecules in mammary epithelial development and tumorigenesis, we have generated transgenic mice which individually express the Grb2 and Shc proteins in the mammary epithelium. Although mammary epithelial cell-specific expression of Grb2 or Shc accelerated ductal morphogenesis, mammary tumors were rarely observed in these strains. To explore the potential role of these adapter proteins in mammary tumorigenesis, mice coexpressing either Shc or Grb2 and a mutant form of polyomavirus middle T (PyV mT) antigen in the mammary epithelium were generated. Coexpression of either Shc or Grb2 with the mutant PyV mT antigen resulted in a dramatic acceleration of mammary tumorigenesis compared to parental mutant PyV mT strain. The increased rate of tumor formation observed in these mice was correlated with activation of the epidermal growth factor receptor family and mitogen-activated protein kinase pathway. These observations suggest that elevated levels of the Grb2 or Shc adapter protein can accelerate mammary tumor progression by sensitizing the mammary epithelial cell to growth factor receptor signaling

Basolateral expression of Kir7.1 channel in the epithelium of the airways.

<p>Immunohistochemical detection of Kir7.1 channel in trachea (left) and bronchiole (right) in adult <i>Kcnj13</i>^+/+, newborn <i>Kcnj13</i>^+/+ or newborn <i>Kcnj13</i>^-/- mice. Tissue sections were treated with anti-Kir7.1 antibody (1:15,000). Kir7.1 expression was restricted to the basolateral membrane of airway epithelium in adult and newborn <i>Kcnj13</i>^+/+ mice. Staining in <i>Kcnj13</i>^-/- tissues shows complete absence of specific immunoreactive signal. Nuclei were counterstained with Fast Red. Scale bar represents 50 μm.</p

Pulmonary abnormalities in embryonic lungs from <i>Kcnj13</i>^-/- mice.

<p>a. Hematoxylin and eosin stained lung sections taken at various gestational stages as indicated. Morphological differences in KO lungs were observed at E18.5 and P0. Null mutant mice show a lower air space and thicker walls at lung terminal sacs compared to WT and heterozygous mice. No differences were visible between <i>Kcnj13</i>^+/+ and <i>Kcnj13</i>^+/- genotypes. Scale bars represent 100 μm. b. Morphometric analysis of terminal sac spaces in lungs at various gestational stages. Significant reduction in spaces was observed in Kir7.1 deficient mice from E18.5 onwards. Results are expressed as mean ± S.E.M, # p<0.05 and * p<0.01 for the difference with WT by ANOVA. c. Graphical representation of newborn lung flotation test. Grey sections of columns correspond to percent of floating lungs, with black being the percent sinking lungs.</p

Morphology and body weights of <i>Kcnj13</i> null mutant mice.

<p>a. Analysis of Kir7.1 expression in WT, heterozygous and null mutant mice; cyclophilin A (Cyc1) is used as constitutively expressed control gene. b. Gross morphology of WT, and heterozygous and homozygous <i>Kcnj13</i> null mutant newborn pups. c. Body weight vs. embryonic stage for WT (circles), <i>Kcnj13</i>^<i>+/-</i> (triangles), and <i>Kcnj13</i>^-/- (squares) embryos. Results are expressed as mean ± S.E.M. of the following numbers of embryos: 12.5 dpc: WT 3, <i>Kcnj13</i>^<i>+/-</i> 9, <i>Kcnj13</i>^-/- 4; 13.5 dpc: WT 9, <i>Kcnj13</i>^<i>+/-</i> 14, <i>Kcnj13</i>^-/- 2; n 14.5 dpc: WT 3, <i>Kcnj13</i>^<i>+/-</i> 11, <i>Kcnj13</i>^-/- 6; 15.5 dpc: WT 7, <i>Kcnj13</i>^<i>+/-</i> 20, <i>Kcnj13</i>^-/- 14; 16.5 dpc: WT 7, <i>Kcnj13</i>^<i>+/-</i> 5, <i>Kcnj13</i>^-/- 4; 17.5 dpc:WT 5, <i>Kcnj13</i>^<i>+/-</i> 12, <i>Kcnj13</i>^-/- 6; 18.5 dpc: WT 4, <i>Kcnj13</i>^<i>+/-</i> 5, <i>Kcnj13</i>^-/- 6; P0: WT 7, <i>Kcnj13</i>^<i>+/-</i> 15, <i>Kcnj13</i>^-/- 10. * p< 0.001; ** p <0.05 for the differences between <i>Kcnj13</i>^-/- and <i>Kcnj13</i>^<i>+/+</i> data (ANOVA).</p