Kinesin-Mediated Transport of Smad2 Is Required for Signaling in Response to TGF-β Ligands

Abstract

During vertebrate development, Activin/Nodal-related ligands signal through Smad2, leading to its activation and accumulation in the nucleus. Here, we demonstrate that Smad2 constantly shuttles between the cytoplasm and nucleus both in early Xenopus embryo explants and in living zebrafish embryos, providing a mechanism whereby the intracellular components of the pathway constantly monitor receptor activity. We have gone on to demonstrate that an intact microtubule network and kinesin ATPase activity are required for Smad2 phosphorylation and nuclear accumulation in response to Activin/Nodal in early vertebrate embryos and TGF-beta in mammalian cells. The kinesin involved is kinesin-1, and Smad2 interacts with the kinesin-1 light chain subunit. Interfering with kinesin activity in Xenopus and zebrafish embryos phenocopies loss of Nodal signaling. Our results reveal that kinesin-mediated transport of Smad2 along microtubules to the receptors is an essential step in ligand-induced Smad2 activation.

Regulation of the Anaphase-promoting Complex–Separase Cascade by Transforming Growth Factor-β Modulates Mitotic Progression in Bone Marrow Stromal Cells

“…Uncontrolled elevated CDK1 inhibits chromatid separation via phosphorylation of separase. Demonstration of TGF-␤-mediated mitotic regulation unravels a previous mystery in which mitotic regulator is down-regulated in response to TGF-␤ signaling (Choi et al, 1999;Wan et al, 2001) and further explains the presence of activated Smad2/3 detected during mitosis (Batut et al, 2007), which advances our understanding of TGF-␤ biology.…”

“…Recent studies have suggested a potential involvement of the Smad cascade in regulating the cellular mitotic apparatus (Batut et al, 2007). To examine the effect of TGF-␤ signaling in the regulation of mitosis, we determined the effect of the presence of activated Smad3 on mitosis by biochemical and immunocytochemical analyses.…”

Nucleocytoplasmic shuttling of Smad proteins

“…The same study used a heterokaryon assay to demonstrate the shuttling ability of Smad2 and to map the export activity to a region comprising the linker and MH2 domain. Fluorescence perturbation and photoactivation experiments with GFP-fusions of Smad2 have, more recently, provided direct visual proof of the continuous exchange between the nuclear and the cytoplasmic pools of Smad2 both in tissue culture cells, in tissue explants of Xenopus laevis embryos, and in whole living zebrafish embryos [21][22][23]. Thus there is compelling evidence that Smad2 and 4 continuously shuttle between the cytoplasm and the nucleus in unstimulated cells.…”

“…The most likely explanation is that continuous nucleocytoplasmic shuttling of Smads, even in actively signaling cells, which are in a state of maximal nuclear Smad accumulation, connects events at the plasma membrane with the nucleus. This notion has now been directly demonstrated by photobleaching and photoactivation experiments using GFP fusions of Smad2 and 4 [21][22][23].…”

Distinct Xenopus Nodal ligands sequentially induce mesendoderm and control gastrulation movements in parallel to the Wnt/PCP pathway

“…For western blotting, embryos were snap-frozen at stage 10 and processed as described (Batut et al, 2007). We used rabbit anti-pSmad2 (Cell Signaling, 1/1000), mouse anti-Smad2 (BD Biosciences, 1/1000) and mouse anti--tubulin (Sigma, 1/10,000) antibodies.…”