Requirement for the dynein light chain km23-1 in a Smad2-dependent transforming growth factor-β signaling pathway

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Abstract

We have identified km23-1 as a novel transforming growth factor-β (TGFβ) receptor (TβR)-interacting protein that is also a light chain of the motor protein dynein (dynein light chain). Herein, we demonstrate by sucrose gradient analyses that, in the presence of TGFβ but not in the absence, km23-1 was present in early endosomes with the TβRs. Further, confocal microscopy studies indicate that endogenous km23-1 was co-localized with endogenous Smad2 at early times after TGFβ treatment, prior to Smad2 translocation to the nucleus. In addition, immunoprecipitation/blot analyses showed that TGFβ regulated the interaction between endogenous km23-1 and endogenous Smad2 in vivo. Blockade of km23-1 using a small interfering RNA approach resulted in a reduction in both total intracellular Smad2 levels and in nuclear levels of phosphorylated Smad2 after TGFβ treatment. This decrease was reversed by lactacystin, a specific inhibitor of the 26 S proteasome, suggesting that knockdown of km23-1 causes proteasomal degradation of phosphorylated (i.e. activated) Smad2. Blockade of km23-1 also resulted in a reduction in TGFβ/Smad2-dependent ARE-Lux transcriptional activity, which was rescued by a km23-1 small interfering RNA-resistant construct. In contrast, a reduction in TGFβ/Smad3-dependent SBE2-Luc transcriptional activity did not occur under similar conditions. Furthermore, overexpression of the dynactin subunit dynamitin, which is known to disrupt dynein-mediated intracellular transport, blocked TGFβ-stimulated nuclear translocation of Smad2. Collectively, our findings indicate for the first time that a dynein light chain is required for a Smad2-dependent TGFβ signaling pathway.

Original languageEnglish (US)
Pages (from-to)19122-19132
Number of pages11
JournalJournal of Biological Chemistry
Volume282
Issue number26
DOIs
StatePublished - Jun 29 2007

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All Science Journal Classification (ASJC) codes

  • Biochemistry
  • Molecular Biology
  • Cell Biology

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