No abstract

The cortical endoplasmic reticulum (ER) in tobacco (Nicotiana tabacum) epidermal cells is a network of tubules and cisternae undergoing dramatic rearrangements. Reticulons are integral membrane proteins involved in shaping ER tubules. Here, we characterized the localization, topology, effect, and interactions of five Arabidopsis thaliana reticulons (RTNs), isoforms 1-4 and 13, in the cortical ER. Our results indicate that RTNLB13 and RTNLB1-4 colocate to and constrict the tubular ER membrane. All five RTNs preferentially accumulate on ER tubules and are excluded from ER cisternae. All isoforms share the same transmembrane topology, with N and C termini facing the cytosol and four transmembrane domains. We show by Fö rster resonance energy transfer and fluorescence lifetime imaging microscopy that several RTNs have the capacity to interact with themselves and each other, and we suggest that oligomerization is responsible for their residence in the ER membrane. We also show that a complete reticulon homology domain is required for both RTN residence in high-curvature ER membranes and ER tubule constriction, yet it is not necessary for homotypic interactions.

We have previously observed that Sp1, a ubiquitous zinc finger transcription factor, is phosphorylated during terminal differentiation in the whole animal, and this results in decreased DNA binding activity (Leggett, R. W., Armstrong, S. A., Barry, D., and Mueller, C. R. (1995) J. Biol. Chem. 270, 25879 -25884). In this study, we demonstrate that casein kinase II (CKII) is able to phosphorylate the C terminus of Sp1 and results in a decrease in DNA binding activity. This suggests that CKII may be responsible for the observed regulation of Sp1. Mutation of a consensus CKII site at amino acid 579, within the second zinc finger, eliminates phosphorylation of this site and the CKII-mediated inhibition of Sp1 binding. Phosphopeptide analysis confirms the presence of a CKII site at Thr-579 as well as additional sites within the C terminus. No gross changes in CKII subunit levels were seen during de-differentiation associated with liver regeneration. The serine/threonine phosphatase PP1 was identified as the endogenous liver nuclear protein able to dephosphorylate Sp1 but again no gross changes in activity were observed in the regenerating liver. Okadaic acid treatment of K562 cells increases Sp1 phosphorylation and inhibits its DNA binding activity suggesting that steady state levels of Sp1 phosphorylation are established by a balance between kinase and phosphatase activities.Sp1 was originally characterized as a GC box binding protein (1) recognizing the consensus sequence GGGCGG. Its DNA binding domain consists of three C 2 H 2 zinc fingers (2), and a series of four domains required for transcriptional activity of Sp1 have been characterized (3). Two of these domains, A and B, correspond to glutamine-rich regions (4 -6) that interact with the transcriptional machinery by binding to TAF II 110 (7) and are needed for transcriptional synergy to occur (8). Domain C contains a region of high charge and functions only weakly as an independent transactivation domain (4). Domain D is required for synergistic activation in conjunction with the A and B domains and may be involved in the formation of higher order homomeric complexes (8). The zinc fingers and domain D may also be involved in the interaction of Sp1 with other proteins as they are required for binding to proteins such as YY1 (9), GATA-1 (10), and adenovirus E1A (11). Sp1 is a member of a small multi-gene family, with Sp2 and Sp3 being ubiquitously expressed (12, 13), whereas the expression of Sp4 may be limited to the brain (13). Sp3 recognizes the same DNA sequences as Sp1 and may act as a repressor of Sp1-mediated activation (14).Sp1 has traditionally been considered to be a constitutive transcription factor and has been implicated in the regulation of a wide variety of housekeeping genes and genes involved in growth regulation (15). It is becoming increasingly clear that Sp1 binding and transactivation is regulated by a variety of stimuli. The retinoblastoma gene product appears to be able to modulate Sp1-mediated transactivation (16 -18) possibly through the ...

Stress exposure has been proposed to contribute to the etiology of breast cancer. However, the validity of this assertion and the possible mechanisms involved are not well established. Epidemiologic studies differ in their assessment of the relative contribution of stress to breast cancer risk, while physiological studies propose a clear connection but lack the knowledge of intracellular pathways involved. The present review aims to consolidate the findings from different fields of research (including epidemiology, physiology, and molecular biology) in order to present a comprehensive picture of what we know to date about the role of stress in breast cancer development.