JAMP, a Jun N-terminal kinase 1 (JNK1)-associated membrane protein, regulates duration of JNK activity

We report the identification and characterization of JAMP (JNK1 [Jun N-terminal kinase 1]-associated membrane protein), a predicted seven-transmembrane protein that is localized primarily within the plasma membrane and associates with JNK1 through its C-terminal domain. JAMP association with JNK1 outcompetes JNK1 association with mitogen-activated protein kinase phosphatase 5, resulting in increased and prolonged JNK1 activity following stress. Elevated expression of JAMP following UV or tunicamycin treatment results in sustained JNK activity and a higher level of JNK-dependent apoptosis. Inhibition of JAMP expression by RNA interference reduces the degree and duration of JNK activation and concomitantly the level of stress-induced apoptosis. Through its regulation of JNK1 activity, JAMP emerges as a membrane-anchored regulator of the duration of JNK1 activity in response to diverse stress stimuli.     

Diffusion of clusters of transmembrane proteins as a model of focal adhesion remodeling

Focal adhesions play a major role in maintaining the cell shape and motility, and in regulating numerous cellular processes. Observations suggest that the functioning of focal adhesions is possible due to their dynamic nature, yet the mechanisms that govern their motion are not well understood. This study addresses the process of focal adhesion remodeling using two distinct theoretical approaches. Namely, adhesion sites are modeled as clusters of integrins that are either bound to cytoskeletal elements or dissociated and temporarily free of any attachments. In the first approach effects of cluster size and permeability on the diffusion of mobile adhesion structures are studied using Brinkman’s effective medium approach. Diffusion coefficients calculated by this hydrodynamic model significantly decrease with the increase in contact area (the effective size of the focal adhesion). In the second approach focal adhesions are modeled as clusters of transmembrane proteins tightly connected to the cytoskeleton, but still capable of motion. The remodeling of these clusters is coupled to the deformation of the cytoskeleton by means of equating energies at the end states of a reversible elastodynamic interaction. Due to large uncertainty of the plasma membrane and the cytoskeleton properties, predicted diffusion coefficients vary within several orders of magnitude. However, a reasonable set of parameters for each model yields diffusion coefficients that compare favorably with those measured by single-particle tracking (SPT), fluorescence recovery after photobleaching (FRAP), and fluorescence digital imaging (FDI). The estimated Young’s modulus of the stress fibers is also in good agreement with measurements. To assess the relevance of the models to focal adhesion remodeling and to improve their predictions, further data on the morphology of focal adhesions and on properties of the plasma membrane and the cytoskeleton are required.     

Nickel enhances telomeric silencing in Saccharomyces cerevisiae

Certain nickel compounds including crystalline nickel sulfide (NiS) and subsulfide (Ni3S2) are potent human and animal carcinogens. In Chinese hamster embryo cells, an X-linked senescence gene was inactivated following nickel-induced DNA methylation. Nickel also induced the inactivation of the gpt reporter gene by chromatin condensation and a DNA methylation process in a transgenic gpt+ Chinese hamster cell line (G12), which is located near a heterochromatic region. To determine if nickel can cause gene silencing independently of DNA methylation, based only on the induction of changes in chromatin structure, we measured its effect on gene silencing in Saccharomyces cerevisiae. Growth of yeast in the presence of nickel chloride repressed a telomeric marker gene (URA3) and resulted in a stable epigenetic switch. This phenomenon was dependent on the number of cell doubling prior to selection and also on the distance of the marker gene from the end of the chromosome. The level of TPE (telomeric position effect) increased linearly with elevations of nickel concentration. Addition of magnesium inhibited this effect, but magnesium did not silence the reporter gene by itself. The level of silencing was also assessed following treatment with other transition metals: cobalt, copper and cadmium. In the sublethal range, cobalt induced similar effects as nickel, while copper and cadmium did not change the basal level of gene expression. Silencing by copper and cadmium were evident only at concentrations of those metals where the viability was very low.     

Molecular biology of nickel carcinogenesis

A review of the molecular mechanisms of nickel carcinogenesis has been compiled. This work is based upon approximately 20 years of research conducted in my laboratory. Molecular mechanisms of nickel carcinogenesis are considered from the pointofview of the uptake of nickel, both soluble and insoluble particles in cells, its dissolution and its effects on heterochromatin. Molecular mechanisms by which nickel induces gene silencing in cells by DNA hypermethylation in mammalian cells and by inhibiting histone acetylation in yeast cells are also discussed.     

Endogenous RNA cleavages at the ribosomal SRL site likely reflect miRNA (miR) mediated translational suppression

We previously suggested a mechanism whereby the RNA induced silencing complex (RISC) brings about a specific cleavage at the sarcin–ricin loop (SRL) of 28S ribosomal RNA thereby eliciting translational suppression. Here we experimentally show that endogenous cleavages take place at the SRL site, in both mammalian cells and in Caenorhabditis elegans. Furthermore we demonstrate that bulged and looped-out residues present in the imperfect miRNA–[mRNA target site] duplexes, are complementary to the SRL site. These results support, and are compatible with, our described mechanism whereby microRNAs mediate cleavage of the highly conserved 28S rRNA sarcin/ricin loop leading to translational suppression.     

RNF5, a RING finger protein that regulates cell motility by targeting paxillin ubiquitination and altered localization

RNF5 is a RING finger protein found to be important in the growth and development of Caenorhabditis elegans. The search for RNF5-associated proteins via a yeast two-hybrid screen identified a LIM-containing protein in C. elegans which shows homology with human paxillin. Here we demonstrate that the human homologue of RNF5 associates with the amino-terminal domain of paxillin, resulting in its ubiquitination. RNF5 requires intact RING and C-terminal domains to mediate paxillin ubiquitination. Whereas RNF5 mediates efficient ubiquitination of paxillin in vivo, protein extracts were required for in vitro ubiquitination, suggesting that additional modifications and/or an associated E3 ligase assist RNF5 targeting of paxillin ubiquitination. Mutant Ubc13 efficiently inhibits RNF5 ubiquitination, suggesting that RNF5 generates polychain ubiquitin of the K63 topology. Expression of RNF5 increases the cytoplasmic distribution of paxillin while decreasing its localization within focal adhesions, where it is primarily seen under normal growth. Concomitantly, RNF5 expression results in inhibition of cell motility. Via targeting of paxillin ubiquitination, which alters its localization, RNF5 emerges as a novel regulator of cell motility.