Identification of three novel Smad binding proteins involved in cell polarity.
A yeast two-hybrid screen was utilized to identify novel Smad 3 binding proteins expressed in developing mouse orofacial tissue. Three proteins (Erbin, Par-3, and Dishevelled) were identified that share several similar structural and functional characteristics. Each contains at least one PDZ domain and all have been demonstrated to play a role in the establishment and maintenance of cell polarity. In GST (glutathione S-transferase) pull-down assays, Erbin, Par-3, and Dishevelled bound strongly to the isolated MH2 domain of Smad 3, with weaker binding to a full-length Smad 3 protein. Failure of Erbin, Par-3, and Dishevelled to bind to a Smad 3 mutant protein that was missing the MH2 domain confirms that the binding site resides within the MH2 domain. Erbin, Par-3, and Dishevelled also interacted with the MH2 domains of other Smads, suggesting broad Smad binding specificity. Dishevelled and Erbin mutant proteins, in which the PDZ domain was removed, still retained their ability to bind Smad 3, albeit with lower affinity. While transforming growth factor beta (TGFbeta) has been suggested to alter cell polarity through a Smad-independent mechanism involving activation of members of the RhoA family of GTP binding proteins, the observation that Smads can directly interact with proteins involved in cell polarity, as shown in the present report, suggests an additional means by which TGFbeta could alter cell polarity via a Smad-dependent signaling mechanism.
Warner
Dennis R
DR
University of Louisville Birth Defects Center, Department of Molecular, Cellular, and Craniofacial Biology, University of Louisville School of Dentistry, 501 South Preston Street, Suite 301, Louisville, KY 40292, USA. drwarn01@gwise.louisville.edu
Pisano
M Michele
MM
Roberts
Emily A
EA
Greene
Robert M
RM
D048868:Adaptor Proteins, Signal Transducing; D000818:Animals; D002352:Carrier Proteins / Q000378:metabolism / Q000502:physiology*; D015815:Cell Adhesion Molecules; D018797:Cell Cycle Proteins; D016764:Cell Polarity; D004268:DNA-Binding Proteins / Q000378:metabolism*; D000072261:Dishevelled Proteins; D005260:Female; D047908:Intracellular Signaling Peptides and Proteins; D008297:Male; D051379:Mice; D008813:Mice, Inbred ICR; D010750:Phosphoproteins / Q000378:metabolism / Q000502:physiology*; D051900:Smad3 Protein; D015534:Trans-Activators / Q000378:metabolism*; D020798:Two-Hybrid System Techniques
D048868:Adaptor Proteins, Signal Transducing; D002352:Carrier Proteins; D015815:Cell Adhesion Molecules; D018797:Cell Cycle Proteins; D004268:DNA-Binding Proteins; D000072261:Dishevelled Proteins; C498804:Erbb2ip protein, mouse; D047908:Intracellular Signaling Peptides and Proteins; C115084:Pard3 protein, mouse; D010750:Phosphoproteins; D051900:Smad3 Protein; C497791:Smad3 protein, mouse; D015534:Trans-Activators