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Molecular and Cellular Biology, November 2005, p. 9845-9858, Vol. 25, No. 22
0270-7306/05/$08.00+0     doi:10.1128/MCB.25.22.9845-9858.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

Kinetic Analysis of Smad Nucleocytoplasmic Shuttling Reveals a Mechanism for Transforming Growth Factor ß-Dependent Nuclear Accumulation of Smads{dagger}

Bernhard Schmierer and Caroline S. Hill*

Laboratory of Developmental Signalling, Cancer Research UK London Research Institute, 44 Lincoln's Inn Fields, London WC2A 3PX, United Kingdom

Received 26 May 2005/ Returned for modification 15 July 2005/ Accepted 25 August 2005

Upon transforming growth factor ß (TGF-ß) stimulation, Smads accumulate in the nucleus, where they regulate gene expression. Using fluorescence perturbation experiments on Smad2 and Smad4 fused to either enhanced green fluorescent protein or photoactivatable green fluorescent protein, we have studied the kinetics of Smad nucleocytoplasmic shuttling in a quantitative manner in vivo. We have obtained rate constants for import and export of Smad2 and show that the cytoplasmic localization of Smad2 in uninduced cells reflects its nuclear export being more rapid than import. We find that TGF-ß-induced nuclear accumulation of Smad2 is caused by a pronounced drop in the export rate of Smad2 from the nucleus, which is associated with a strong decrease in nuclear mobility of Smad2 and Smad4. TGF-ß-induced nuclear accumulation involves neither a release from cytoplasmic retention nor an increase in Smad2 import rate. Hence, TGF-ß-dependent nuclear accumulation of Smad2 is caused exclusively by selective nuclear trapping of phosphorylated, complexed Smad2. The proposed mechanism reconciles signal-dependent nuclear accumulation of Smad2 with its continuous nucleocytoplasmic cycling properties.

* Corresponding author. Mailing address: Cancer Research UK London Research Institute, 44 Lincoln's Inn Fields, London WC2A 3PX, United Kingdom. Phone: 44-20-7269-2941. Fax: 44-20-7269-3093. E-mail: Caroline.Hill{at}cancer.org.uk.

{dagger} Supplemental material for this article may be found at http://mcb.asm.org/.

Molecular and Cellular Biology, November 2005, p. 9845-9858, Vol. 25, No. 22
0022-538X/05/$08.00+0     doi:10.1128/MCB.25.22.9845-9858.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.



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