A distinct nuclear localization signal in the N terminus of Smad 3 determines its ligand-induced nuclear translocation.
Smad proteins are intracellular mediators of transforming growth factor beta (TGF-beta) and related cytokines and undergo ligand-induced nuclear translocation. Here we describe the identification of a nuclear localization signal (NLS) in the N-terminal region of Smad 3, the major Smad protein involved in TGF-beta signaling. An NLS-like basic motif (Lys(40)-Lys-Leu-Lys-Lys(44)), conserved among all pathway-specific Smad proteins, not only is responsible for constitutive nuclear localization of the isolated Smad 3 MH1 domain but also is crucial for Smad 3 nuclear import in response to ligand. Mutations in this motif completely abolished TGF-beta-induced nuclear translocation but had no impact on ligand-induced phosphorylation of Smad 3, complex formation with Smad 4, or specific binding to DNA. Hence Smad 3 proteins with NLS mutations are dominant-negative inhibitors of TGF-beta-induced transcriptional activation. Smad 4, which cannot translocate into the nucleus in the absence of Smad 3 or another pathway-specific Smad, contains a Glu in place of the last Lys in this motif. Smad 3 harboring the same mutation (K44E) does not undergo ligand-induced nuclear import. Conversely, the isolated Smad 4 MH1 domain does not accumulate in the nucleus but becomes nuclear enriched when Glu(49) is replaced with Lys. We propose that this highly conserved five-residue NLS motif determines ligand-induced nuclear translocation of all pathway-specific Smads.
Xiao
Z
Z
Whitehead Institute for Biomedical Research, Nine Cambridge Center, Cambridge, MA 02142, USA.
Liu
X
X
Henis
Y I
YI
Lodish
H F
HF
D030201:Activin Receptors, Type I; D000595:Amino Acid Sequence; D001665:Binding Sites; D001692:Biological Transport; D002451:Cell Compartmentation; D002467:Cell Nucleus / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D019913:Nuclear Localization Signals; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism; D000077293:Receptor, Transforming Growth Factor-beta Type I; D018125:Receptors, Transforming Growth Factor beta / Q000378:metabolism; D015398:Signal Transduction; D051900:Smad3 Protein; D015534:Trans-Activators / Q000235:genetics / Q000378:metabolism*; D015533:Transcriptional Activation; D016212:Transforming Growth Factor beta / Q000378:metabolism*
D004268:DNA-Binding Proteins; D019913:Nuclear Localization Signals; D018125:Receptors, Transforming Growth Factor beta; D051900:Smad3 Protein; D015534:Trans-Activators; D016212:Transforming Growth Factor beta; D017346:Protein Serine-Threonine Kinases; D030201:Activin Receptors, Type I; D000077293:Receptor, Transforming Growth Factor-beta Type I