Requirement for the dynein light chain km23-1 in a Smad2-dependent transforming growth factor-beta signaling pathway.
We have identified km23-1 as a novel transforming growth factor-beta (TGFbeta) receptor (TbetaR)-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 TGFbeta but not in the absence, km23-1 was present in early endosomes with the TbetaRs. Further, confocal microscopy studies indicate that endogenous km23-1 was co-localized with endogenous Smad2 at early times after TGFbeta treatment, prior to Smad2 translocation to the nucleus. In addition, immunoprecipitation/blot analyses showed that TGFbeta 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 TGFbeta 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 TGFbeta/Smad2-dependent ARE-Lux transcriptional activity, which was rescued by a km23-1 small interfering RNA-resistant construct. In contrast, a reduction in TGFbeta/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 TGFbeta-stimulated nuclear translocation of Smad2. Collectively, our findings indicate for the first time that a dynein light chain is required for a Smad2-dependent TGFbeta signaling pathway.
Jin
Qunyan
Q
Department of Pharmacology, Pennsylvania State University College of Medicine, Hershey, Pennsylvania 17033, USA.
Ding
Wei
W
Mulder
Kathleen M
KM
D021581:Active Transport, Cell Nucleus / Q000187:drug effects / Q000502:physiology; D000818:Animals; D000906:Antibodies; D002352:Carrier Proteins / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D056445:Cytoplasmic Dyneins; D004285:Dogs; D029721:Drosophila Proteins / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D004398:Dyneins / Q000235:genetics* / Q000276:immunology; D015870:Gene Expression / Q000502:physiology; D006801:Humans; D007668:Kidney / Q000166:cytology; D008168:Lung / Q000166:cytology; D008907:Mink; D034741:RNA, Small Interfering / Q000494:pharmacology; D011817:Rabbits; D015398:Signal Transduction / Q000187:drug effects / Q000502:physiology*; D051899:Smad2 Protein / Q000378:metabolism*; D016212:Transforming Growth Factor beta / Q000378:metabolism* / Q000494:pharmacology
D000906:Antibodies; D002352:Carrier Proteins; C504797:DYNLRB1 protein, human; D029721:Drosophila Proteins; D034741:RNA, Small Interfering; D051899:Smad2 Protein; D016212:Transforming Growth Factor beta; D056445:Cytoplasmic Dyneins; D004398:Dyneins