Cbl-mediated ubiquitinylation is required for lysosomal sorting of epidermal growth factor receptor but is dispensable for endocytosis

Ligand-induced down-regulation controls the signaling potency of the epidermal growth factor receptor (EGFR/ErbB1). Overexpression studies have identified Cbl-mediated ubiquitinylation of EGFR as a mechanism of ligand-induced EGFR down-regulation. However, the role of endogenous Cbl in EGFR down-regulation and the precise step in the endocytic pathway regulated by Cbl remain unclear. Using Cbl-/- mouse embryonic fibroblast cell lines, we demonstrate that endogenous Cbl is essential for ligand-induced ubiquitinylation and efficient degradation of EGFR. Further analyses using Chinese hamster ovary cells with a temperature-sensitive defect in ubiquitinylation confirm a crucial role of the ubiquitin machinery in Cbl-mediated EGFR degradation. However, internalization into early endosomes did not require Cbl function or an intact ubiquitin pathway. Confocal immunolocalization studies indicated that Cbl-dependent ubiquitinylation plays a critical role at the early endosome to late endosome/lysosome sorting step of EGFR down-regulation. These findings establish Cbl as the major endogenous ubiquitin ligase responsible for EGFR degradation, and show that the critical role of Cbl-mediated ubiquitinylation is at the level of endosomal sorting, rather than at the level of internalization.     

Widespread occurrence of Mycobacterium tuberculosis DNA from 18th-19th century Hungarians

A large number (265) of burials from 1731-1838 were discovered in sealed crypts of the Dominican Church, Vác, Hungary in 1994. Many bodies were naturally mummified, so that both soft tissues and bones were available. Contemporary archives enabled the determination of age at death, and the identification of family groups. In some cases, symptoms before death were described and, occasionally, occupation. Initial radiological examination of a small number of individuals had indicated calcified lung lesions and demonstrable acid-fast bacteria suggestive of tuberculosis infection. Tuberculosis was endemic in 18th-19th century Europe, so human remains should contain detectable Mycobacterium tuberculosis complex (MTB) DNA, enabling comparisons with modern isolates. Therefore, a comprehensive examination of 168 individuals for the presence of MTB DNA was undertaken. Specific DNA amplification methods for MTB showed that 55% of individuals were positive and that the incidence varied according to age at death and sampling site in the body. Radiographs were obtained from 27 individuals and revealed an association between gross pathology and the presence of MTB DNA. There was an inverse relationship between PCR positivity and MTB target sequence size. In some cases, the preservation of MTB DNA was excellent, and several target gene sequences could be detected from the same sample. This information, combined with MTB DNA sequencing data and molecular typing techniques, will enable us to study the past epidemiology of TB infection, and extends the timeframe for studying changes in molecular fingerprints.     

Microstructural variation in conodont enamel is a functional adaptation

Recognition that conodonts were the earliest vertebrate group to experiment with skeletal biomineralization provides a window in which to study the origin and early evolution of this developmental system. It has been contended that the conodont skeleton comprised a classic suite of vertebrate hard tissues, while others suggest that conodont hard tissues represent divergent specializations within the early diversification of vertebrate hard tissues, supporting a view that the hard tissues of conodonts, particularly enamel, exhibit a range of microstructural variation beyond that seen in vertebrates. New evidence reveals that, although variable, conodont enamel microstructure is consistent between homologous portions of homologous dentitions. Although there is a correlation between morphology and microstructure, this belies a stronger correlation between the commonality of microstructure and dental function. The enamel of conodonts evolved in response to changes in dental function and differentiation of the microstructural layer into a number of enamel types and can be linked to dental occlusion, heterodonty, a permanent dentition, enamel thickness and, probably above all, the small size of the dental elements.     

Identification of tyrosine residues in constitutively activated fibroblast growth factor receptor 3 involved in mitogenesis, Stat activation, and phosphatidylinositol 3-kinase activation

Fibroblast growth factor receptor 3 (FGFR3) mutations are frequently involved in human developmental disorders and cancer. Activation of FGFR3, through mutation or ligand stimulation, results in autophosphorylation of multiple tyrosine residues within the intracellular domain. To assess the importance of the six conserved tyrosine residues within the intracellular domain of FGFR3 for signaling, derivatives were constructed containing an N-terminal myristylation signal for plasma membrane localization and a point mutation (K650E) that confers constitutive kinase activation. A derivative containing all conserved tyrosine residues stimulates cellular transformation and activation of several FGFR3 signaling pathways. Substitution of all nonactivation loop tyrosine residues with phenylalanine rendered this FGFR3 construct inactive, despite the presence of the activating K650E mutation. Addition of a single tyrosine residue, Y724, restored its ability to stimulate cellular transformation, phosphatidylinositol 3-kinase activation, and phosphorylation of Shp2, MAPK, Stat1, and Stat3. These results demonstrate a critical role for Y724 in the activation of multiple signaling pathways by constitutively activated mutants of FGFR3.     

Recreating a functional ancestral archosaur visual pigment

The ancestors of the archosaurs, a major branch of the diapsid reptiles, originated more than 240 MYA near the dawn of the Triassic Period. We used maximum likelihood phylogenetic ancestral reconstruction methods and explored different models of evolution for inferring the amino acid sequence of a putative ancestral archosaur visual pigment. Three different types of maximum likelihood models were used: nucleotide-based, amino acid-based, and codon-based models. Where possible, within each type of model, likelihood ratio tests were used to determine which model best fit the data. Ancestral reconstructions of the ancestral archosaur node using the best-fitting models of each type were found to be in agreement, except for three amino acid residues at which one reconstruction differed from the other two. To determine if these ancestral pigments would be functionally active, the corresponding genes were chemically synthesized and then expressed in a mammalian cell line in tissue culture. The expressed artificial genes were all found to bind to 11-cis-retinal to yield stable photoactive pigments with lambda(max) values of about 508 nm, which is slightly redshifted relative to that of extant vertebrate pigments. The ancestral archosaur pigments also activated the retinal G protein transducin, as measured in a fluorescence assay. Our results show that ancestral genes from ancient organisms can be reconstructed de novo and tested for function using a combination of phylogenetic and biochemical methods.     

Initial activation of cyclin-B1-cdc2 kinase requires phosphorylation of cyclin B1

At the G₂/M transition of the cell cycle, the cdc25c phosphatase dephosphorylates inhibitory residues of cdc2, and cyclin-B–cdc2 kinase (MPF) is activated. Phosphorylation of cyclin B1 induces its nuclear accumulation, and, since cdc25c is also believed to accumulate and activate shortly before G₂/M in the nucleus, it has been proposed that this induces cyclin-B1–cdc2 kinase activation. We demonstrate that cyclin B1 phosphorylation has another essential function in vivo: it is required for cdc25c and MPF activation, which does not require nuclear accumulation of cyclin B1, and occurs in the cytoplasm.