Characterization of growth factor-induced serine phosphorylation of tumor necrosis factor-alpha converting enzyme and of an alternatively translated polypeptide

Tumor necrosis factor-alpha converting enzyme (TACE) is a prototype member of the adamalysin family of transmembrane metalloproteases that effects ectodomain cleavage and release of many transmembrane proteins, including transforming growth factor-alpha. Growth factors that act through tyrosine kinase receptors, as well as other stimuli, induce shedding through activation of the Erk mitogen-activated protein (MAP) kinase pathway without the need of new protein synthesis. How MAP kinase regulates shedding by TACE is not known. We now report that the cytoplasmic domain of TACE is phosphorylated in response to growth factor stimulation. We also identified a naturally expressed smaller polypeptide corresponding to most of the cytoplasmic domain of TACE. This protein, which we named SPRACT, is derived through alternative translation of the TACE-coding sequence and is, similarly to TACE, phosphorylated in response to growth factor and phorbol 12-myristate 13-acetate stimulation. Phosphoamino acid analysis revealed that growth factor-induced phosphorylation of TACE occurs only on serine and not on threonine or tyrosine. Tryptic mapping experiments coupled with site-directed mutagenesis identified Ser(819) as the major target of growth factor-induced phosphorylation, whereas Ser(791) undergoes dephosphorylation in response to growth factor stimulation. The phosphorylation of Ser(819), but not the dephosphorylation of Ser(791), depends on activation of the Erk MAP kinase pathway. Increased SPRACT expression or mutation of the TACE cytoplasmic domain to inactivate growth factor-induced phosphorylation did not detectably affect growth factor-induced shedding of transmembrane transforming growth factor-alpha by TACE. The roles of SPRACT and the cytoplasmic phosphorylation of TACE remain to be defined.     

Mitotic phosphorylation of the dynein light intermediate chain is mediated by cdc2 kinase

Cytoplasmic dynein, a large minus-end-directed microtubule motor, performs multiple functions during the cell cycle. In interphase, dynein moves membrane organelles, while in mitosis it moves chromosomes and helps to form the mitotic spindle. The cell-cycle regulation of dynein activity may be controlled, at least in part, by the phosphorylation of its light intermediate chains (DLIC), since a 10-fold increase in light intermediate chain phosphorylation correlates with a decrease in dynein-based membrane transport of similar magnitude in mitosis. In this study, we sought to identify the kinase responsible for this potentially important phosphorylation event. We show that bacterially-expressed chicken light intermediate chain (chDLIC) will undergo mitosis-specific phosphorylation when added to Xenopus egg extracts. Mutation of a conserved cdc2 kinase consensus site (Ser197) abolishes this phosphorylation event, and mass spectroscopy analysis confirms that the wild-type DLIC is stoichiometrically phosphorylated at this site when incubated with metaphase but not interphase extracts. We also show that purified cdc2 kinase phosphorylates purified DLICs at Ser197 in vitro and that Ser197 phosphorylation is dramatically reduced in metaphase extracts depleted of cdc2 kinase. These results indicate that cdc2 kinase directly phosphorylates dynein and thus may be an important regulator of dynein activity in the cell cycle.     

Widespread interspecific divergence in cis-regulation of transposable elements in the Arabidopsis genus

Transposable elements (TEs) are so abundant and variable that they count among the most important mutational sources in genomes. Nonetheless, little is known about the genetics of their variation in activity or silencing across closely related species. Here, we demonstrate that regulation of TE genes can differ dramatically between the two closely related Arabidopsis species A. thaliana and A. lyrata. In leaf and floral tissues of F1 interspecific hybrids, about 47% of TEs show allele-specific expression, with the A. lyrata copy being generally expressed at higher level. We confirm that TEs are generally expressed in A. lyrata but not in A. thaliana. Allele-specific differences in TE expression are associated with divergence in epigenetic modifications like DNA and histone methylation between species as well as with sequence divergence. Our data demonstrate that A. thaliana silences TEs much better than A. lyrata. For long terminal repeat retrotransposons, these differences are more pronounced for younger insertions. Interspecific differences in TE silencing may have a great impact on genome size changes.     

Experimental stress in inflammatory rheumatic diseases: a review of psychophysiological stress responses

Introduction  

Stressful events are thought to contribute to the aetiology, maintenance and exacerbation of rheumatic diseases. Given the growing interest in acute stress responses and disease, this review investigates the impact of real-life experimental psychosocial, cognitive, exercise and sensory stressors on autonomic, neuroendocrine and immune function in patients with inflammatory rheumatic diseases.     

The human oligodendrocyte proteome

Myelination of the CNS is performed by oligodendrocytes (OLs), which have been implicated in brain disorders, such as multiple sclerosis and schizophrenia. We have used the human oligodendroglial cell line MO3.13 to establish an OL reference proteome database. Proteins were prefractionationated by SDS‐PAGE and after in‐gel digestion subjected to nanoflow LC‐MS analysis. Approximately 11 600 unique peptides were identified and, after stringent filtering, resulted in 2290 proteins representing nine distinct biological processes and various molecular classes and functions. OL‐specific proteins, such as myelin basic protein (MBP) and 2′,3′‐cyclic nucleotide 3′‐phosphodiesterase (CNP), as well as other proteins involved in multiple sclerosis and schizophrenia were also identified and are discussed. Proteins of this dataset have also been classified according to their chromosomal origin for providing useful data to the Chromosome‐centric Human Proteome Project (C‐HPP). Given the importance of OLs in the etiology of demyelinating and oligodendrogial disorders, the MO3.13 proteome database is a valuable data resource. The MS proteomics data have been deposited to the ProteomeXchange with identifier PXD000263 ( http://proteomecentral.proteomexchange.org/dataset/PXD000263 ).     

The lymphoid transcription factor LyF-1 is encoded by specific, alternatively spliced mRNAs derived from the Ikaros gene.

The lymphocyte-specific DNA-binding protein LyF-1 interacts with a critical control element in the terminal deoxynucleotidyltransferase (TdT) promoter as well as with the promoters for other genes expressed during early stages of B- and T-cell development. We have purified LyF-1 and have obtained a partial amino acid sequence from proteolytic peptides. The amino acid sequence suggests that LyF-1 is a zinc finger protein encoded by the Ikaros gene, which previously was implicated in T-cell development. Recombinant Ikaros expressed in Escherichia coli bound to the TdT promoter, and antisera directed against the recombinant protein specifically blocked the DNA-binding activity of LyF-1 in crude extracts. Further analysis revealed that at least six distinct mRNAs are derived from the Ikaros/LyF-1 gene by alternative splicing. Only two of the isoforms possess the N-terminal zinc finger domain that is necessary and sufficient for TdT promoter binding. Although both of these isoforms bound to similar sequences in the TdT, lambda 5, VpreB, and lck promoters, one isoform contains an additional zinc finger that resulted in altered recognition of some binding sites. At least four of the Ikaros/LyF-1 isoforms were detectable in extracts from B- and T-cell lines, with the relative amounts of the isoforms varying considerably. These data reveal that the LyF-1 protein is encoded by specific mRNAs derived from the alternatively-spliced Ikaros gene, suggesting that this gene may be important for the early stages of both B- and T-lymphocyte development.     

Membrane Ig cross-linking regulates phosphatidylinositol 3-kinase in B lymphocytes.

Cross-linking of the B cell AgR results in activation of mature B cells and tolerization of immature B cells. The initial signaling events stimulated by membrane immunoglobulin (mIg) cross-linking are tyrosine phosphorylation of a number of proteins. Among the targets of mIg-induced tyrosine phosphorylation are the tyrosine kinases encoded by the lyn, blk, fyn, and syk genes, the mIg-associated proteins MB-1 and Ig-beta, phospholipase C-gamma 1 and -gamma 2, as well as many unidentified proteins. In this report we show that mIg cross-linking also regulates phosphatidylinositol 3-kinase (PtdIns 3-kinase), an enzyme that phosphorylates inositol phospholipids and plays a key role in mediating the effects of tyrosine kinases on growth control in fibroblasts. Cross-linking mIg on B lymphocytes greatly increased the amount of PtdIns 3-kinase activity which could be immunoprecipitated with anti-phosphotyrosine (anti-tyr(P) antibodies. This response was observed after mIg cross-linking in mIgM- and mIgG-bearing B cell lines and after cross-linking either mIgM or mIgD in murine splenic B cells. Thus, regulation of PtdIns 3-kinase is a common feature of signaling by several different isotypes of mIg. This response was rapid and peaked 2 to 3 min after the addition of anti-Ig antibodies. The anti-Ig-stimulated increase in PtdIns 3-kinase activity associated with anti-Tyr(P) immunoprecipitates could reflect increased tyrosine phosphorylation of PtdIns 3-kinase, increased activity of the enzyme, or both. In favor of the first possibility, the tyrosine kinase inhibitor herbimycin A blocked the increase in ant-Tyr(P)-immunoprecipitated PtdIns 3-kinase activity as well as the anti-Ig-induced tyrosine phosphorylation. Moreover, this response was not secondary to phospholipase C activation but rather seemed to be a direct consequence of mIg-induced tyrosine phosphorylation. Activation of the phosphoinositide pathway by a transfected M1 muscarinic acetylcholine receptor expressed in WEHI-231 B lymphoma cells did not increase the amount of PtdIns 3-kinase activity which could be precipitated with anti-Tyr(P) antibodies. Similarly, inhibition of the phosphoinositide pathway did not abrogate the ability of mIg cross-linking to stimulate this response. Thus, mIg-induced tyrosine phosphorylation regulates PtdIns 3-kinase, an important mediator of growth control in fibroblasts and potentially an important regulatory component in B cells as well.     

pROC: an open-source package for R and S+ to analyze and compare ROC curves

Background  

Receiver operating characteristic (ROC) curves are useful tools to evaluate classifiers in biomedical and bioinformatics applications. However, conclusions are often reached through inconsistent use or insufficient statistical analysis. To support researchers in their ROC curves analysis we developed pROC, a package for R and S+ that contains a set of tools displaying, analyzing, smoothing and comparing ROC curves in a user-friendly, object-oriented and flexible interface.