We describe here the diversity of chloroplast proteins required for embryo development in Arabidopsis (Arabidopsis thaliana). Interfering with certain chloroplast functions has long been known to result in embryo lethality. What has not been reported before is a comprehensive screen for embryo-defective (emb) mutants altered in chloroplast proteins. From a collection of transposon and T-DNA insertion lines at the RIKEN chloroplast function database (http://rarge.psc.riken.jp/chloroplast/) that initially appeared to lack homozygotes and segregate for defective seeds, we identified 23 additional examples of EMB genes that likely encode chloroplast-localized proteins. Fourteen gene identities were confirmed with allelism tests involving duplicate mutant alleles. We then queried journal publications and the SeedGenes database (www.seedgenes.org) to establish a comprehensive dataset of 381 nuclear genes encoding chloroplast proteins of Arabidopsis associated with embryo-defective (119 genes), plant pigment (121 genes), gametophyte (three genes), and alternate (138 genes) phenotypes. Loci were ranked based on the level of certainty that the gene responsible for the phenotype had been identified and the protein product localized to chloroplasts. Embryo development is frequently arrested when amino acid, vitamin, or nucleotide biosynthesis is disrupted but proceeds when photosynthesis is compromised and when levels of chlorophyll, carotenoids, or terpenoids are reduced. Chloroplast translation is also required for embryo development, with genes encoding chloroplast ribosomal and pentatricopeptide repeat proteins well represented among EMB datasets. The chloroplast accD locus, which is necessary for fatty acid biosynthesis, is essential in Arabidopsis but not in Brassica napus or maize (Zea mays), where duplicated nuclear genes compensate for its absence or loss of function.

Her2 is overexpressed in 20% to 30% of breast tumors and correlates with reduced disease-free and overall patient survival. Trastuzumab, a humanized monoclonal antibody directed against Her2, represents the first Her2-targeted therapy, which decreases the risk of relapse and prolongs patient survival.

The molecular mechanisms by which mammalian receptor tyrosine kinases are negatively regulated remain largely unexplored. Previous genetic and biochemical studies indicate that Kekkon-1, a transmembrane protein containing leucine-rich repeats and an immunoglobulin-like domain in its extracellular region, acts as a feedback negative regulator of epidermal growth factor (EGF) receptor signaling in Drosophila melanogaster development. Here we tested whether the related human LRIG1 (also called Lig-1) protein can act as a negative regulator of EGF receptor and its relatives, ErbB2, ErbB3, and ErbB4. We observed that in co-transfected 293T cells, LRIG1 forms a complex with each of the ErbB receptors independent of growth factor binding. We further observed that co-expression of LRIG1 with EGF receptor suppresses cellular receptor levels, shortens receptor half-life, and enhances ligand-stimulated receptor ubiquitination. Finally, we observed that co-expression of LRIG1 suppresses EGF-stimulated transformation of NIH3T3 fibroblasts and that the inducible expression of LRIG1 in PC3 prostate tumor cells suppresses EGF-and neuregulin-1-stimulated cell cycle progression. Our observations indicate that LRIG1 is a negative regulator of the ErbB family of receptor tyrosine kinases and suggest that LRIG1-mediated receptor ubiquitination and degradation may contribute to the suppression of ErbB receptor function.The four members of the ErbB family of receptor tyrosine kinases (epidermal growth factor (EGF) 1 receptor, ErbB2, ErbB3, and ErbB4) play key roles in mediating the development of a variety of tissues, and the aberrant activation of these receptors contributes to the growth and progression of numerous tumor types (1, 2). Binding of EGF-like family ligands to ErbB receptors stimulates receptor dimerization, kinase activation, autophosphorylation, and the engagement of multiple intracellular growth signaling pathways. Although considerable effort over the past two decades has gone into understanding mechanisms by which ErbB receptors are activated and signals are propagated, our understanding of the variety of molecular mechanisms underlying the suppression of growth factor receptor activity remains in its infancy.Growth factor-stimulated receptor down-regulation, involving receptor internalization and the cbl-mediated ubiquitination and trafficking of receptors to lysosomes (3, 4), represents one mechanism for preventing hypersignaling by the ErbB receptors. However, whereas EGF receptor (ErbB1 or EGFR) efficiently couples to cbl following stimulation with its ligand EGF, the ErbB2, ErbB3, and ErbB4 receptors do not efficiently couple to cbl following stimulation with neuregulin-1 (NRG1) (5) and do not undergo efficient NRG1-stimulated down-regulation (6, 7). Hence, other negative regulatory mechanisms may play major roles in suppressing ErbB receptor activity.Studies from the fruit fly Drosophila melanogaster point to the existence of several classes of proteins that negatively regulate EGF receptor activity in flies (...

This report is the only prospective study to document the natural history of spondylolysis and spondylolisthesis from onset through more than 45 years of life in a population unselected for pain. Subjects with pars defects follow a clinical course similar to that of the general population. There appears to be a marked slowing of slip progression with each decade, and no subject has reached a 40% slip.

No abstract

Despite numerous recent advances in our understanding of the molecular mechanisms underlying receptor tyrosine kinase downregulation and degradation in response to growth factor binding, relatively little is known about ligand-independent receptor tyrosine kinase degradation mechanisms. In a screen for proteins that might regulate the trafficking or localization of the ErbB3 receptor, we have identified a tripartite or RBCC (RING, B-box, coiled-coil) protein that interacts with the cytoplasmic tail of the receptor in an activation-independent manner. We have named this protein Nrdp1 for neuregulin receptor degradation protein-1. Northern blotting reveals ubiquitous distribution of Nrdp1 in human adult tissues, but message is particularly prominent in heart, brain, and skeletal muscle. Nrdp1 interacts specifically with the neuregulin receptors ErbB3 and ErbB4 and not with epidermal growth factor receptor or ErbB2. When coexpressed in COS7 cells, Nrdp1 mediates the redistribution of ErbB3 from the cell surface to intracellular compartments and induces the suppression of ErbB3 and ErbB4 receptor levels but not epidermal growth factor receptor or ErbB2 levels. A putative dominant-negative form of Nrdp1 potentiates neuregulin-stimulated Erk1͞2 activity in transfected MCF7 breast tumor cells. Our observations suggest that Nrdp1 may act to regulate steady-state cell surface neuregulin receptor levels, thereby influencing the efficiency of neuregulin signaling.

Nrdp1 is a RING finger-containing E3 ubiquitin ligase that physically interacts with and regulates steadystate cellular levels of the ErbB3 and ErbB4 receptor tyrosine kinases and has been implicated in the degradation of the inhibitor-of-apoptosis protein BRUCE. Here we demonstrate that the Nrdp1 protein undergoes efficient proteasome-dependent degradation and that mutations in its RING finger domain that disrupt ubiquitin ligase activity enhance stability. These observations suggest that Nrdp1 self-ubiquitination and stability could play an important role in regulating the activity of this protein. Using affinity chromatography, we identified the deubiquitinating enzyme USP8 (also called Ubpy) as a protein that physically interacts with Nrdp1. Nrdp1 and USP8 could be coimmunoprecipitated, and in transfected cells USP8 specifically bound to Nrdp1 but not cbl, a RING finger E3 ligase involved in ligand-stimulated epidermal growth factor receptor down-regulation. The USP8 rhodanese and catalytic domains mediated Nrdp1 binding. USP8 markedly enhanced the stability of Nrdp1, and a point mutant that disrupts USP8 catalytic activity destabilized endogenous Nrdp1. Our results indicate that Nrdp1 is a specific target for the USP8 deubiquitinating enzyme and are consistent with a model where USP8 augments Nrdp1 activity by mediating its stabilization.

The molecular mechanisms underlying epidermal growth factor (EGF) receptor tyrosine kinase downregulation in response to growth factor binding are coming into focus and involve cbl-mediated receptor ubiquitination followed by lysosomal degradation. However, mechanisms underlying the ligand-stimulated degradation of the related receptor tyrosine kinases of the ErbB family do not involve cbl and remain unexplored. Previous studies have demonstrated that the E3 ubiquitin ligase Nrdp1 contributes to the maintenance of steady-state ErbB3 levels by mediating its growth factor-independent degradation. Here we demonstrate that treatment of cells with the ErbB3 ligand neuregulin-1 (NRG1) stabilizes the deubiquitinating enzyme USP8, which in turn stabilizes Nrdp1. The catalytic activity of USP8 is required for NRG1-induced Nrdp1 stabilization. We provide evidence that Akt-mediated phosphorylation of USP8 threonine residue T907 contributes to USP8 stability. Finally, we demonstrate that Nrdp1 or USP8 knockdown suppresses NRG1-induced ErbB3 ubiquitination and degradation in MCF7 breast cancer cells. We conclude that an NRG1-induced protein stability cascade involving USP8 and Nrdp1 mediates the down-regulation of ErbB3. Our observations raise the possibility that the ligand-induced augmentation of pathways involved in the maintenance of basal levels of receptor tyrosine kinases can contribute to ligand-stimulated down-regulation.The ErbB family of receptor tyrosine kinases (RTKs) consists of four members (epidermal growth factor [EGF] receptor, ErbB2, ErbB3, and ErbB4) that play essential roles in a variety of developmental processes (7,8,15,49). Years of accumulating evidence also implicate the aberrant activation of ErbB receptors in the malignancy of various human tumors. The overexpression of EGF receptor, ErbB2, and ErbB3 has been observed in numerous solid tumor types and correlates with a high degree of receptor activation (21). For example, amplification of the erbB2 gene is observed in 25 to 30% of breast cancer patients, and overexpression of the product correlates with an earlier relapse and poor prognosis (46,47). ErbB2 is a validated target for therapeutic intervention, and a number of antibody and small-molecule agents are either already in clinical use or under development for the treatment of patients whose tumors overexpress ErbB2 (35).The members of the ErbB receptor family undergo a network of homo-and heterodimerization events as part of their signaling mechanism. Particularly noteworthy is a strong propensity of ErbB2 to heterodimerize with and activate ErbB3 (3, 9, 37, 42). Since ErbB3 lacks intrinsic tyrosine kinase activity (16) and no diffusible ligand that binds to ErbB2 has been described, these two receptors must necessarily collaborate in propagating signals in response to growth factors such as the ErbB3 ligand neuregulin-1 (NRG1) ( 9, 48). In vitro, ErbB2 and ErbB3 synergize in promoting the growth and transformation of cultured fibroblasts (2, 10) and the proliferation of breast tumor cells (...