Crk-associated substrate lymphocyte type regulates transforming growth factor-beta signaling by inhibiting Smad6 and Smad7.
Crk-associated substrate lymphocyte type (Cas-L) is a 105 kDa docking protein with diverse functional properties, including regulation of cell division, proliferation, migration and adhesion. Cas-L is also involved in beta1 integrin- or antigen receptor-mediated signaling in B and T cells. In the present study, we demonstrate that Cas-L potentiates transforming growth factor-beta (TGF-beta) signaling pathway by interacting with Smad6 and Smad7. Immunoprecipitation experiments reveal that single domain deletion of full-length Cas-L completely abolishes its docking function with Smad6 and Smad7, suggesting that the natural structure of Cas-L is necessary for its association with Smad6 and Smad7. On the other hand, both N-terminal and C-terminal deletion mutants of Smad6 and Smad7 still retain their docking ability to Cas-L, suggesting that Smad6 and Smad7 possess several binding motifs to Cas-L. Moreover, Cas-L interaction with Mad-homology (MH)2 domain, but not with MH1 domain of Smad6 or Smad7, ameliorates TGF-beta-induced signaling pathway. Finally, depletion of Cas-L by small-interfering RNA oligo attenuates TGF-beta-induced growth inhibition of Huh-7 cells, with a concomitant reduction in phosphorylation of Smad2 and Smad3. These results strongly suggest that Cas-L is a potential regulator of TGF-beta signaling pathway.
Inamoto
S
S
Division of Clinical Immunology, Advanced Clinical Research Center, Institute of Medical Science, The University of Tokyo, Tokyo, Japan.
Iwata
S
S
Inamoto
T
T
Nomura
S
S
Sasaki
T
T
Urasaki
Y
Y
Hosono
O
O
Kawasaki
H
H
Tanaka
H
H
Dang
N H
NH
Morimoto
C
C
D030201:Activin Receptors, Type I / Q000378:metabolism; D048868:Adaptor Proteins, Signal Transducing / Q000235:genetics / Q000378:metabolism*; D002460:Cell Line; D049109:Cell Proliferation; D006801:Humans; D010750:Phosphoproteins / Q000235:genetics / Q000378:metabolism*; D011485:Protein Binding; D017346:Protein Serine-Threonine Kinases; D034741:RNA, Small Interfering / Q000235:genetics; D000077293:Receptor, Transforming Growth Factor-beta Type I; D018125:Receptors, Transforming Growth Factor beta / Q000378:metabolism; D015398:Signal Transduction; D051897:Smad Proteins, Inhibitory / Q000378:metabolism; D051905:Smad6 Protein / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism; D051906:Smad7 Protein / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism; D014158:Transcription, Genetic / Q000235:genetics; D016212:Transforming Growth Factor beta / Q000235:genetics / Q000378:metabolism*
D048868:Adaptor Proteins, Signal Transducing; C099942:NEDD9 protein, human; D010750:Phosphoproteins; D034741:RNA, Small Interfering; D018125:Receptors, Transforming Growth Factor beta; D051897:Smad Proteins, Inhibitory; D051905:Smad6 Protein; D051906:Smad7 Protein; D016212:Transforming Growth Factor beta; D017346:Protein Serine-Threonine Kinases; D030201:Activin Receptors, Type I; D000077293:Receptor, Transforming Growth Factor-beta Type I