Requirement of a dynein light chain in TGFbeta/Smad3 signaling.
We have previously reported that the dynein light chain (DLC) km23-1 is required for Smad2-dependent TGFbeta signaling. Here we describe another member of the km23/DYNLRB/LC7/robl family of DLCs, termed km23-2, which is also involved in TGFbeta signaling. We show not only that TGFbeta stimulates the interaction of km23-2 (DYNLRB2) with TGFbeta receptor II (TbetaRII) but also that TGFbeta regulates the interaction between km23-2 and endogenous TbetaRII in vivo. In addition, TGFbeta treatment causes km23-2 phosphorylation, whereas a kinase-deficient form of TbetaRII prevents km23-2 phosphorylation. In contrast to the km23-1 isoform, blockade of km23-2 expression using small interfering RNAs (siRNAs) decreased key TGFbeta/Smad3-specific responses, including the induction of both plasminogen activator inhibitor-1 (PAI-1) gene expression and p21 protein expression. Blockade of km23-1 expression had no effect on these two major TGFbeta/Smad3 responses under similar conditions. Further, km23-2 was required for TGFbeta stimulation of Smad3-dependent Smad-binding element (SBE)2-Luc transcriptional activity, but not for TGFbeta stimulation of Smad2-dependent activin responsive element (ARE)-Lux transcriptional activity. In order to assess the mechanisms underlying the preferential stimulation of Smad3- versus Smad2-specific TGFbeta responses, immunoprecipitation (IP)/blot analyses were performed, which demonstrate that TGFbeta stimulated preferential complex formation of km23-2 with Smad3, relative to Smad2. Collectively, our findings indicate that km23-2 is required for Smad3-dependent TGFbeta signaling. More importantly, we demonstrate that km23-2 has functions in TGFbeta signaling that are distinct from those for km23-1. This is the first report to describe a differential requirement for unique isoforms of a specific DLC family in Smad-specific TGFbeta signaling.
Jin
Qunyan
Q
Department of Biochemistry and Molecular Biology, Pennsylvania State University College of Medicine, Hershey, Pennsylvania 17033, USA.
Gao
Guofeng
G
Mulder
Kathleen M
KM
D000818:Animals; D002460:Cell Line; D050759:Cyclin-Dependent Kinase Inhibitor p21 / Q000235:genetics / Q000378:metabolism; D056445:Cytoplasmic Dyneins; D004398:Dyneins / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D015870:Gene Expression / Q000235:genetics; D006801:Humans; D008907:Mink; D009363:Neoplasm Proteins / Q000502:physiology*; D010766:Phosphorylation / Q000187:drug effects; D017395:Plasminogen Activator Inhibitor 1 / Q000235:genetics / Q000378:metabolism; D011485:Protein Binding / Q000187:drug effects / Q000502:physiology; D020033:Protein Isoforms / Q000235:genetics / Q000378:metabolism; D017346:Protein Serine-Threonine Kinases / Q000235:genetics / Q000378:metabolism; D034741:RNA, Small Interfering / Q000235:genetics; D000077293:Receptor, Transforming Growth Factor-beta Type I; D000077294:Receptor, Transforming Growth Factor-beta Type II; D018125:Receptors, Transforming Growth Factor beta / Q000235:genetics / Q000378:metabolism; D015398:Signal Transduction / Q000502:physiology*; D051899:Smad2 Protein / Q000235:genetics / Q000378:metabolism; D051900:Smad3 Protein / Q000235:genetics / Q000378:metabolism*; D014162:Transfection; D016212:Transforming Growth Factor beta / Q000494:pharmacology / Q000502:physiology*
C493956:CDKN1A protein, human; D050759:Cyclin-Dependent Kinase Inhibitor p21; C504797:DYNLRB1 protein, human; D009363:Neoplasm Proteins; D017395:Plasminogen Activator Inhibitor 1; D020033:Protein Isoforms; D034741:RNA, Small Interfering; D018125:Receptors, Transforming Growth Factor beta; C051321:SERPINE1 protein, human; C497782:SMAD2 protein, human; C497790:SMAD3 protein, human; D051899:Smad2 Protein; D051900:Smad3 Protein; D016212:Transforming Growth Factor beta; D017346:Protein Serine-Threonine Kinases; D000077293:Receptor, Transforming Growth Factor-beta Type I; D000077294:Receptor, Transforming Growth Factor-beta Type II; D056445:Cytoplasmic Dyneins; C449283:DYNLRB2 protein, human; D004398:Dyneins