Requirement for the Dynein Light Chain km23-1 in a Smad2-dependent Transforming Growth Factor-β Signaling Pathway

Abstract

We have identified km23-1 as a novel transforming growth factor-␤ (TGF␤) receptor (T␤R)-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 TGF␤ but not in the absence, km23-1 was present in early endosomes with the T␤Rs. Further, confocal microscopy studies indicate that endogenous km23-1 was co-localized with endogenous Smad2 at early times after TGF␤ treatment, prior to Smad2 translocation to the nucleus. In addition, immunoprecipitation/blot analyses showed that TGF␤ 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 TGF␤ 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 TGF␤/Smad2-dependent ARE-Lux transcriptional activity, which was rescued by a km23-1 small interfering RNA-resistant construct. In contrast, a reduction in TGF␤/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 TGF␤-stimulated nuclear translocation of Smad2. Collectively, our findings indicate for the first time that a dynein light chain is required for a Smad2-dependent TGF␤ signaling pathway.

The TGFβ Receptor-interacting Protein km23-1/DYNLRB1 Plays an Adaptor Role in TGFβ1 Autoinduction via Its Association with Ras

“…Additional studies demonstrated that kinasenegative T␤Rs are required for km23-1 effects (23,27). km23-1 was also required for both Smad2-dependent and Smad-independent TGF␤ responses (23,27,32). In the current report, we describe a novel role for km23-1 in linking Ras activation by TGF␤ to TGF␤ autoinduction in TGF␤-sensitive epithelial cells.…”

“…km23-1 interacts with and co-localizes in early endosomes with T␤RII and Smad2 after TGF␤ treatment (23,32). Moreover, km23-1 undergoes rapid phosphorylation on serine residues after T␤R activation, in keeping with the kinase specificity of the T␤Rs (23).…”

Signaling Receptors for TGF-β Family Members

“…The motor protein dynein light chain km23-1 has been shown to bind TbRII and to be phosphorylated after TGF-b stimulation (Tang et al 2002). km23-1 promotes both Smad-dependent (Jin et al 2007a) and Smad-independent signaling. In contrast, the motor protein dynein light chain Tctex2b associates with the short cytoplasmic tail of endoglin in endothelial cells and with TbRII and betaglycan in endothelial and other cell types, providing negative regulation of TGF-b signaling (Meng et al 2006).…”

The regulation of TGFβ signal transduction

“…Smads are sequestered away from the receptors when bound to microtubules, from where they can be released, for example, by connexin 43 (GJ1), which competes with microtubules to bind Smads . Additional motor proteins, such as the dynein light chain km23-1 (DYNLRB1), also promote Smad traffic towards the nucleus of mammalian cells (Jin et al, 2007). The developmental roles of the connexin 43 and km23-1 mechanisms have not yet been specifically addressed.…”