Conjugation to Nedd8 instigates ubiquitylation and down-regulation of activated receptor tyrosine kinases

When appended to the epidermal growth factor receptor (EGFR), ubiquitin serves as a sorting signal for lysosomal degradation. Here we demonstrate that the ubiquitin ligase of EGFR, namely c-Cbl, also mediates receptor modification with the ubiquitin-like molecule Nedd8. EGF stimulates receptor neddylation, which enhances subsequent ubiquitylation, as well as sorting of EGFR for degradation. Multiple lysine residues, located within the tyrosine kinase domain of EGFR, serve as attachment sites for Nedd8. A set of clathrin coat-associated binders of ubiquitin also bind Nedd8, but they undergo ubiquitylation, not neddylation. We discuss the emerging versatility of the concerted action of ubiquitylation and neddylation in the process that desensitizes growth factor-activated receptor tyrosine kinases.     

Loss of ATRX, genome instability, and an altered DNA damage response are hallmarks of the alternative lengthening of telomeres pathway

The Alternative Lengthening of Telomeres (ALT) pathway is a telomerase-independent pathway for telomere maintenance that is active in a significant subset of human cancers and in vitro immortalized cell lines. ALT is thought to involve templated extension of telomeres through homologous recombination, but the genetic or epigenetic changes that unleash ALT are not known. Recently, mutations in the ATRX/DAXX chromatin remodeling complex and histone H3.3 were found to correlate with features of ALT in pancreatic neuroendocrine cancers, pediatric glioblastomas, and other tumors of the central nervous system, suggesting that these mutations might contribute to the activation of the ALT pathway in these cancers. We have taken a comprehensive approach to deciphering ALT by applying genomic, molecular biological, and cell biological approaches to a panel of 22 ALT cell lines, including cell lines derived in vitro. Here we show that loss of ATRX protein and mutations in the ATRX gene are hallmarks of ALT-immortalized cell lines. In addition, ALT is associated with extensive genome rearrangements, marked micronucleation, defects in the G2/M checkpoint, and altered double-strand break (DSB) repair. These attributes will facilitate the diagnosis and treatment of ALT positive human cancers.     

Why fly the extra mile? Using stress biomarkers to assess wintering habitat quality in migratory shorebirds

Migratory birds make decisions about how far to travel based on cost-benefit trade-offs. However, in many cases the net effect of these trade-offs is unclear. We sought to address this question by measuring feather corticosterone (CORT_f), leucocyte profile, avian malaria parasite prevalence and estimating fueling rates in three spatially segregated wintering populations of the migratory shorebird ruddy turnstone Arenaria interpres during their stay in the winter habitat. These birds fly from the high-Arctic breeding ground to Australia, but differ in that some decide to end their migration early (Broome, Western Australia), whereas others travel further to either South Australia or Tasmania. We hypothesized that the extra costs in birds migrating greater distances and overwintering in colder climates would be offset by benefits when reaching their destination. This would be evidenced by lower stress biomarkers in populations that travel further, owing to the expected benefits of greater resources and improved vitality. We show that avian malaria prevalence and physiological stress levels were lower in birds flying to South Australia and Tasmania than those overwintering in Broome. Furthermore, our modeling predicts that birds in the southernmost locations enjoy higher fueling rates. Our data are consistent with the interpretation that birds occupying more costly wintering locations in terms of higher migratory flight and thermoregulatory costs are compensated by better feeding conditions and lower blood parasite infections, which facilitates timely and speedy migration back to the breeding ground. These data contribute to our understanding of cost-benefit trade-offs in the decision making underlying migratory behaviour.     

Neuronal calcium sensor-1 and phosphatidylinositol 4-kinase beta regulate IgE receptor-triggered exocytosis in cultured mast cells

We examined the possible occurrence and function of neuronal Ca(2+) sensor 1 (NCS-1/frequenin) in the mast cell line rat basophilic leukemia, RBL-2H3. This protein has been implicated in the control of neurosecretion from dense core granules in neuronal cells as well as in the control of constitutive secretory pathways in both yeast and mammalian cells. We show that RBL-2H3 cells, secretory cells of the immune system, endogenously express the 22-kDa NCS-1 protein as well as an immune-related 50-kDa protein. Both proteins associate in vivo with phosphatidylinositol 4-kinase beta (PI4Kbeta) and colocalize with the enzyme in the Golgi region. We show further that overexpression of NCS-1 in RBL-2H3 cells stimulates the catalytic activity of PI4Kbeta, increases IgE receptor (FcepsilonRI)-triggered hydrolysis of phosphatidylinositol 4,5-bisphosphate (PI(4,5)P(2)), and stimulates FcepsilonRI-triggered, but not Ca(2+) ionophore-triggered, exocytosis. Conversely, expression of a kinase-dead mutant of PI4Kbeta reduces PI4Kbeta activity, decreases FcepsilonRI-stimulated phosphatidylinositol 4,5-bisphosphate hydrolysis, and blocks FcepsilonRI-triggered, but not Ca(2+) ionophore-triggered, exocytosis. Our results indicate that PI(4)P, produced by the Golgi-localized PI4Kbeta, is the rate-limiting factor in the synthesis of the pool of PI(4,5)P(2) that serves as substrate for the generation of lipid-derived second messengers in FcepsilonRI-triggered cells. We conclude that NCS-1 is involved in the control of regulated exocytosis in nonneural cells, where it contributes to stimulus-secretion coupling by interacting with PI4Kbeta and positive regulation of its activity.     

Polar expression of ErbB-2/HER2 in epithelia. Bimodal regulation by Lin-7

ErbB-2/HER2 drives epithelial malignancies by forming heterodimers with growth factor receptors. The primordial invertebrate receptor is sorted to the basolateral epithelial surface by binding of the PDZ domain of Lin-7 to the receptor's tail. We show that all four human ErbBs are basolaterally expressed, even when the tail motif is absent. Mutagenesis of hLin-7 unveiled a second domain, KID, that binds to the kinase region of ErbBs. The PDZ interaction mediates stabilization of ErbB-2 at the basolateral surface. On the other hand, binding of KID is involved in initial delivery to the basolateral surface, and in its absence, unprocessed ErbB-2 molecules are diverted to the apical surface. Hence, distinct domains of Lin-7 regulate receptor delivery to and maintenance at the basolateral surface of epithelia.     

Loss of periodicity in breeding success of waders links to changes in lemming cycles in Arctic ecosystems

Lemming population cycles in the Arctic have an important impact on the Arctic food web, indirectly also affecting breeding success in Arctic‐nesting birds through shared predators. Over the last two decades lemming cycles have changed in amplitude and even disappeared in parts of the Arctic. To examine the large scale effect of these recent changes we re‐analysed published data from the East Atlantic Flyway (EAF), where a relationship between lemming cycles and wader breeding success was earlier found, and new data on breeding success of waders in the East Asian–Australasian Flyway (EAAF). We found that 1) any long‐term periodicities in wader breeding success existed only until the year 2000 in the EAAF and until the 1980s in the EAF; 2) studying these patterns at a smaller spatial scale, where the Siberian–Alaskan breeding grounds were divided into five geographical units largely based on landscape features, breeding success of waders from the EAAF was not correlated to an index of predation pressure, but positively correlated to Arctic summer temperatures in some species. We argue that fading out of lemming cycles in some parts of the Arctic is responsible for faltering periodicity in wader breeding success along both flyways. These changed conditions have not yet resulted in any marked changing trends in breeding success across years, and declining numbers of waders along the EAAF are therefore more likely a result of changing conditions at stop‐over and wintering sites.     

Phosphorylation of carboxyl-terminal tyrosines modulates the specificity of Sprouty-2 inhibition of different signaling pathways

Sprouty proteins are evolutionarily conserved negative feedback regulators of multiple receptor tyrosine kinases. Mammalian versions of these proteins differentially regulate signaling induced by the fibroblast and the epidermal growth factors (FGF and EGF, respectively). Herein we show that, although both growth factors elevate expression of Sprouty-2, FGF- and not EGF-induced activation of the Erk/MAPK pathway is inhibited by Sprouty-2. Attenuation of FGF-signaling is accompanied by the induction of Sprouty-2 phosphorylation on the amino-terminal as well as carboxyl-terminal tyrosine residues, which are less effectively modified upon EGF treatment. Mutagenesis of carboxyl-terminal tyrosines, especially a newly identified phosphorylation site, tyrosine 227, impaired the inhibitory activity of Sprouty-2. These results attribute a novel role for carboxyl-terminal tyrosine residues and yet unidentified phosphotyrosine-binding proteins in the differential regulation of Sprouty-2 activity.     

Hypoxia of endothelial cells leads to MMP-2-dependent survival and death

Exposure of endothelial cells (ECs) to hypoxia has separately been shown to induce their angiogenesis or death. Matrix metalloproteinase (MMP)-2 is associated with EC angiogenesis, although recent studies also implicate this molecule in EC death. We studied the effect of hypoxia in the absence or presence of TNF- (characteristic of the inflammatory microenvironment accompanying hypoxia) on MMP-2 expression and its role in angiogenesis (proliferation, migration, and tube formation) and in the death of primary human umbilical vein endothelial cells (HUVECs). Hypoxia alone (24–48 h in 0.3% O₂ in the hypoxic chamber) and furthermore, when combined with TNF-, significantly enhanced MMP-2 expression and activity. Hypoxia also led to a reduction in membrane type 1 MMP (MT1-MMP) and tissue inhibitor of metalloproteinase-2 mRNA and protein while enhancing the expression of _v3 integrin and the cytoskeletal protein phosphopaxillin. Moreover, hypoxia led to colocalization of _v3 and MMP-2, but not MT1-MMP, with phosphopaxillin in ECs. These results suggest MT1-MMP-independent activation of MMP-2 during hypoxia and support interactions between the ECM, integrins, and the cytoskeleton in hypoxia-induced MMP-2-related functions. Hypoxia enhanced EC migration in an MMP-2-dependent manner while leading to a reduction of cell number via their apoptosis, which was also dependent on MMP-2. In addition, hypoxia caused an aberrant tubelike formation on Matrigel that appeared to be unaffected by MMP-2. The hypoxia-induced, MMP-2-dependent migration of ECs is in accordance with the proangiogenic role ascribed to MMP-2, while the involvement of this protease in the hypoxia-related death of ECs supports an additional apoptotic role for this protease. Hence, in the hypoxic microenvironment, MMP-2 appears to have a dual autocrine role in determining the fate of ECs. gelatinase activity; angiogenesis; apoptosis; tumor necrosis factor-