Abstract
Trafficking of Smad proteins between the cytoplasm and nucleus is a critical component of transforming growth factor beta (TGF-beta) signal transduction. Smad4 translocates into the nucleus either in response to TGF-beta stimulation or when its nuclear export is blocked by leptomycin B (LMB). We demonstrate that both TGF-beta-induced and basal state spontaneous nuclear import of Smad4 require importin 7 and 8 (Imp7,8). Our data suggest that in the nuclear import of Smad4, the role of Imp8 is irreplaceable by Imp7, and that Smads preferentially bind Imp8. Interestingly, in contrast to its mammalian counterpart Smad4, Drosophila Medea appears to utilize different mechanisms for TGF-beta-induced or basal state nuclear accumulation, with the latter independent of Msk (Drosophila Imp7/8) function. In addition, overexpression of Imp8 alone was sufficient to cause an increased concentration of Smad1, 3 and 4 in the nucleus, but had very limited effects on Smad2. These observations suggest selective involvement of Imp8/Msk in nuclear import of different Smads under different conditions.
Highlights
Largely present in the nucleus at the basal state and reportedly undergoes nucleus-to-cytoplasm translocation in response to TGF-
TGF- treatment led to a decrease in the cytoplasm/nucleus ratio of Smad4 concentration reflecting signalinduced nuclear accumulation of Smad4 (Fig. 1A)
We found, as reported previously, Subcellular Distribution of Smad7 Is Not Affected by Levels of that upon co-expression of Mad and the TGF- receptor Imp7 and 8—When overexpressed in 293T and HeLa cells, kinases Punt and Thickvien (Tkv), Medea was present predom- Smad7 was present in both the cytoplasm and nucleus with a inantly in the nucleus of 68% of transfected cells
Summary
Cell Culture, Transfection, and Cytokine Treatment—293T and HeLa cell culture were maintained in Dulbecco’s Modified Eagle’s media (DME) supplemented with 10% fetal bovine serum, penicillin (100 units/ml), and streptomycin (100 units/ ml) (all from Invitrogen). S2Rϩ cells were grown at 25 °C in Schneider’s Drosophila Medium with 10% fetal bovine serum, penicillin (100 units/ml), and streptomycin (100 units/ml, all from Invitrogen). The cell pellet was resuspended in ice-cold buffer containing 20 mM HEPES pH 7.4, 250 mM sucrose, 5 mM MgCl2, 10 mM KCl, 1 mM EDTA, 1 mM EGTA, 100 g/ml digitonin (Calbiochem), 2 mM dithiothreitol, and supplemented with protease inhibitors. Fluorescence microscopy images captured under the same exposure conditions were analyzed using Image J to quantify per unit area staining intensity in the cytoplasm and nucleus (ϳ20 positively stained cells from multiple fields). Protein-Protein Interaction—293T cells with or without prior TGF- treatment (100 pM, 1 h) were harvested and lysed for 20 min at 4 °C in a buffer containing 20 mM HEPES, pH 7.4, 200 mM NaCl, 1% Triton X-100, 5% glycerol, 2 mM dithiothreitol, and protease inhibitors. After incubation for 6 h, the beads were pelleted and washed 3ϫ in the same buffer used during immunoprecipitation
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