The human mind and body respond to stress1, a state of perceived threat to homeostasis, by activating the sympathetic nervous system and secreting the catecholamines adrenaline and noradrenaline in the ‘fight-or-flight’ response. The stress response is generally transient because its accompanying effects (for example, immunosuppression, growth inhibition and enhanced catabolism) can be harmful in the long term2. When chronic, the stress response can be associated with disease symptoms such as peptic ulcers or cardiovascular disorders3, and epidemiological studies strongly indicate that chronic stress leads to DNA damage4,5. This stress-induced DNA damage may promote ageing6, tumorigenesis4,7, neuropsychiatric conditions8,9 and miscarriages10. However, the mechanisms by which these DNA-damage events occur in response to stress are unknown. The stress hormone adrenaline stimulates β2-adrenoreceptors that are expressed throughout the body, including in germline cells and zygotic embryos11. Activated β2-adrenoreceptors promote Gs-protein-dependent activation of protein kinase A (PKA), followed by the recruitment of β-arrestins, which desensitize G-protein signalling and function as signal transducers in their own right12. Here we elucidate a molecular mechanism by which β-adrenergic catecholamines, acting through both Gs-PKA and β-arrestin-mediated signalling pathways, trigger DNA damage and suppress p53 levels respectively, thus synergistically leading to the accumulation of DNA damage. In mice and in human cell lines, β-arrestin-1 (ARRB1), activated via β2-adrenoreceptors, facilitates AKT-mediated activation of MDM2 and also promotes MDM2 binding to, and degradation of, p53, by acting as a molecular scaffold. Catecholamine-induced DNA damage is abrogated in Arrb1-knockout (Arrb1−/−) mice, which show pre served p53 levels in both the thymus, an organ that responds prominently to acute or chronic stress1, and in the testes, in which paternal stress may affect the offspring’s genome. Our results highlight the emerging role of ARRB1 as an E3-ligase adaptor in the nucleus, and reveal how DNA damage may accumulate in response to chronic stress.

One of the earliest events in the process of cell motility is the massive generation of free actin barbed ends, which elongate to form filaments adjacent to the plasma membrane at the tip of the leading edge. Both cofilin and Arp2/3 complex have been proposed to contribute to barbed end formation during cell motility. Attempts to assess the functions of cofilin and Arp 2/3 complex in vivo indicate that both cofilin and Arp2/3 complex contribute to actin polymerization: cofilin by severing and Arp2/3 by nucleating and branching. In order to determine if the activities of cofilin and Arp2/3 complex interact, we employed a light microscope-based assay to visualize actin polymerization directly in the presence of both proteins. The results indicate that cofilin generates barbed ends to increase the mass of freshly polymerized F-actin but does not directly affect the activity of Arp2/3 complex. However, while ADP, ADP-Pi, and newly polymerized ATP-filaments are all capable of supporting Arp2/3-mediated branching, newly polymerized F-actin supports most of the Arp2/3-induced branch formation. The results suggest that, in vivo, cofilin contributes to barbed end formation by inducing the initial increase in the number of barbed ends leading to increased ATP-F-actin, which in turn supports higher levels of dendritic nucleation by active Arp2/3 complex.

Ca2؉ signals regulate cell proliferation, but the spatial and temporal specificity of these signals is unknown. Here we use selective buffers of nucleoplasmic or cytoplasmic Ca 2؉ to determine that cell proliferation depends upon Ca 2؉ signals within the nucleus rather than in the cytoplasm. Nuclear Ca 2؉ signals stimulate cell growth rather than inhibit apoptosis and specifically permit cells to advance through early prophase. Selective buffering of nuclear but not cytoplasmic Ca 2؉ signals also impairs growth of tumors in vivo. These findings reveal a major physiological and potential pathophysiological role for nucleoplasmic Ca 2؉ signals and suggest that this information can be used to design novel therapeutic strategies to regulate conditions of abnormal cell growth. Ca2ϩ is a ubiquitous second messenger that mediates a wide range of cellular responses such as contraction, fluid and electrolyte secretion, exocytosis, gene transcription, and apoptosis (1). This ability to simultaneously control multiple processes occurs by careful modulation of Ca 2ϩ signals, not only over time but in different subcellular regions as well (2). For example, polarized Ca 2ϩ waves direct apical secretion in epithelia (3), whereas presynaptic increases in Ca 2ϩ trigger neurotransmitter release (4) and mitochondrial increases in Ca 2ϩ regulate apoptosis (5, 6). Ca 2ϩ signals also can be regulated independently in the nucleoplasm relative to the cytoplasm (7,8), but the physiological significance of this aspect of spatial control is not entirely understood. Nucleoplasmic Ca 2ϩ signals have distinct effects on activation of transcription factors (9, 10) and kinases (11, 12), but it is not known whether nuclear Ca 2ϩ signals also regulate more global aspects of cell function. Because cell proliferation (13,14) and progression through the cell cycle (15, 16) are Ca 2ϩ -dependent, we investigated the relative roles of nuclear and cytoplasmic Ca 2ϩ on cell growth. EXPERIMENTAL PROCEDURESMaterials, Reagents, and Cell Lines-SKHep1, HepG2, and HEK-293 cell lines were obtained from the American Type Culture Collection (Manassas, VA) and were used for all experiments. The cells were grown at 37°C with 5% CO 2 :95% O 2 in Dulbecco's modified Eagle's medium supplemented with 1% penicillin-streptomycin and 10% heat-inactivated fetal bovine serum, all from Invitrogen. The cells were grown on glass coverslips overnight in the absence of serum before infection with each parvalbumin (PV) 2 construct. Generation of Parvalbumin Constructs and AdenoviralInfection-Constructs encoding red fluorescence protein (DsRed) from Clontech (Mountain View, CA) and targeted PV proteins (PV-NLS, PV-NES, and PV-NLS-CD) were PCR-amplified and subcloned into pShuttle-CMV (kindly provided by Bert Vogelstein, Johns Hopkins) by restriction digestion with XhoI and XbaI to generate pShuttle-CMV-PVNLS-DSR, pShuttle-CMV-PVNES-DSR, and pShuttle-CMV-PVNLS-CD-DSR. Recombinant adenoviruses were generated by transformation of pShuttle-CMV-PV-NLS-DSR into AdEasier-1 cells, a deri...

The targeting of mRNA and local protein synthesis is important for the generation and maintenance of cell polarity. As part of the translational machinery as well as an actin/ microtubule-binding protein, elongation factor 1␣ (EF1␣) is a candidate linker between the protein translation apparatus and the cytoskeleton. We demonstrate in this work that EF1␣ colocalizes with ␤-actin mRNA and F-actin in protrusions of chicken embryo fibroblasts and binds directly to F-actin and ␤-actin mRNA simultaneously in vitro in actin cosedimentation and enzyme-linked immunosorbent assays. To investigate the role of EF1␣ in mRNA targeting, we mapped the two actin-binding sites on EF1␣ at high resolution and defined one site at the N-terminal 49 residues of domain I and the other at the C-terminal 54 residues of domain III. In vitro actin-binding assays and localization in vivo of recombinant full-length EF1␣ and its various truncates demonstrated that the C terminus of domain III was the dominant actin-binding site both in vitro and in vivo. We propose that the EF1␣-F-actin complex is the scaffold that is important for ␤-actin mRNA anchoring. Disruption of this complex would lead to delocalization of the mRNA. This hypothesis was tested by using two dominant negative polypeptides: the actin-binding domain III of EF1␣ and the EF1␣-binding site of yeast Bni1p, a protein that inhibits EF1␣ binding to F-actin and also is required for yeast mRNA localization. We demonstrate that either domain III of EF1␣ or the EF1␣-binding site of Bni1p inhibits EF1␣ binding to ␤-actin mRNA in vitro and causes delocalization of ␤-actin mRNA in chicken embryo fibroblasts. Taken together, these results implicate EF1␣ in the anchoring of ␤-actin mRNA to the protrusion in crawling cells.

Most eukaryotic cells rely on localized actin polymerization to generate and sustain the protrusion activity necessary for cell movement [1, 2]. Such protrusions are often in the form of a flat lamellipod with a leading edge composed of a dense network of actin filaments [3, 4]. The Arp2/3 complex localizes within that network in vivo [3, 4] and nucleates actin polymerization and generates a branched network of actin filaments in vitro [5-7]. The complex has thus been proposed to generate the actin network at the leading edge of crawling cells in vivo [3, 4, 8]. However, the relative contributions of nucleation and branching to protrusive force are still unknown. We prepared antibodies to the p34 subunit of the Arp2/3 complex that selectively inhibit side binding of the complex to F-actin. We demonstrate that side binding is required for efficient nucleation and branching by the Arp2/3 complex in vitro. However, microinjection of these antibodies into cells specifically inhibits lamellipod extension without affecting the EGF-stimulated appearance of free barbed ends in situ. These results indicate that while the side binding activity of the Arp2/3 complex is required for nucleation in vitro and for protrusive force in vivo, it is not required for EGF-stimulated increases in free barbed ends in vivo. This suggests that the branching activity of the Arp2/3 complex is essential for lamellipod extension, while the generation of nucleation sites for actin polymerization is not sufficient.