Abstract
In eukaryotes, regulation of signaling mediators/effectors in the nucleus is one of the principal mechanisms that govern duration and strength of signaling. Smads are a family of structurally related intracellular proteins that serve as signaling effectors for transforming growth factor beta (TGF-β) and TGF-β-related proteins. Accumulating evidence demonstrates that Smads possess intrinsic nucleocytoplasmic shuttling capacity, which enables them to transmit TGF-β signals from cell membrane to nucleus. We recently identified two important steps in the termination of nuclear Smad signaling. The first step is initiated by a serine/threonine phosphatase PPM1A that dephosphorylates Smad2/3 in the nucleus, thereby shutting down signaling capacity of phosphorylated Smad2/3. The second step involves nuclear export of dephosphorylated Smad2/3 with the aid of nuclear protein RanBP3 to terminate Smad signaling. This chapter introduces methods for examining nuclear export of Smad2/3 in TGF-β signaling.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
Similar content being viewed by others
References
Feng XH, Derynck R (2005) Specificity and versatility in tgf-beta signaling through Smads. Annu Rev Cell Dev Biol 21:659–693
Massague J, Seoane J, Wotton D (2005) Smad transcription factors. Genes Dev 19:2783–2810
Lin X, Duan X, Liang YY et al (2006) PPM1A functions as a Smad phosphatase to terminate TGFbeta signaling. Cell 125:915–928
Watanabe M, Masuyama N, Fukuda M, Nishida E (2000) Regulation of intracellular dynamics of Smad4 by its leucine-rich nuclear export signal. EMBO Rep 1:176–182
Pierreux CE, Nicolas FJ, Hill CS (2000) Transforming growth factor beta-independent shuttling of Smad4 between the cytoplasm and nucleus. Mol Cell Biol 20:9041–9054
Inman GJ, Nicolas FJ, Hill CS (2002) Nucleocytoplasmic shuttling of Smads 2, 3, and 4 permits sensing of TGF-beta receptor activity. Mol Cell 10:283–294
Nicolas FJ, De Bosscher K, Schmierer B, Hill CS (2004) Analysis of Smad nucleocytoplasmic shuttling in living cells. J Cell Sci 117:4113–4125
Schmierer B, Hill CS (2005) Kinetic analysis of Smad nucleocytoplasmic shuttling reveals a mechanism for transforming growth factor beta-dependent nuclear accumulation of Smads. Mol Cell Biol 25:9845–9858
Xiao Z, Brownawell AM, Macara IG, Lodish HF (2003) A novel nuclear export signal in Smad1 is essential for its signaling activity. J Biol Chem 278:34245–34252
Kurisaki A, Kose S, Yoneda Y, Heldin CH, Moustakas A (2001) Transforming growth factor-beta induces nuclear import of Smad3 in an importin-beta1 and Ran-dependent manner. Mol Biol Cell 12:1079–1091
Xu L, Chen YG, Massague J (2000) The nuclear import function of Smad2 is masked by SARA and unmasked by TGFbeta-dependent phosphorylation. Nat Cell Biol 2:559–562
Xu L, Kang Y, Col S, Massague J (2002) Smad2 nucleocytoplasmic shuttling by nucleoporins CAN/Nup214 and Nup153 feeds TGFbeta signaling complexes in the cytoplasm and nucleus. Mol Cell 10:271–282
Coburn GA, Wiegand HL, Kang Y, Ho DN, Georgiadis MM, Cullen BR (2001) Using viral species specificity to define a critical protein/RNA interaction surface. Genes Dev 15:1194–1205
Cullen BR (2004) Assaying nuclear messenger RNA export in human cells. Methods Mol Biol 257:85–92
Dai F, Lin X, Chang C, Feng X-H (2009) Nuclear export of Smad2 and Smad3 by RanBP3. Dev Cell 16:345–357
Englmeier L, Fornerod M, Bischoff FR, Petosa C, Mattaj IW, Kutay U (2001) RanBP3 influences interactions between CRM1 and its nuclear protein export substrates. EMBO Rep 2:926–932
Wu JW, Hu M, Chai J et al (2001) Crystal structure of a phosphorylated Smad2. Recognition of phosphoserine by the MH2 domain and insights on Smad function in TGF-beta signaling. Mol Cell 8:1277–1289
Duan X, Liang YY, Feng XH, Lin X (2006) Protein serine/threonine phosphatase PPM1A dephosphorylates Smad1 in the bone morphogenetic protein signaling pathway. J Biol Chem 281:36526–36532
Feng XH, Derynck R (1996) Ligand-independent activation of transforming growth factor (TGF) beta signaling pathways by heteromeric cytoplasmic domains of TGF-beta receptors. J Biol Chem 271:13123–13129
Wieser R, Wrana JL, Massague J (1995) GS domain mutations that constitutively activate T beta R-I, the downstream signaling component in the TGF-beta receptor complex. EMBO J 14:2199–2208
Acknowledgments
We thank members of Feng and Lin labs for their contributions to the original research and helpful discussion. The described research is supported by NIH grants (R01AR053591 and R01CA108454 to X.-H.F., R01DK073932 to X.L.) and a Leukemia and Lymphoma Society Scholar Award (X.-H.F.).
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2010 Springer Science+Business Media, LLC
About this protocol
Cite this protocol
Dai, F., Duan, X., Liang, YY., Lin, X., Feng, XH. (2010). Coupling of Dephosphorylation and Nuclear Export of Smads in TGF-β Signaling. In: Higgins, P. (eds) Transcription Factors. Methods in Molecular Biology, vol 647. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-60761-738-9_7
Download citation
DOI: https://doi.org/10.1007/978-1-60761-738-9_7
Published:
Publisher Name: Humana Press, Totowa, NJ
Print ISBN: 978-1-60761-737-2
Online ISBN: 978-1-60761-738-9
eBook Packages: Springer Protocols