Identification of three novel Smad binding proteins involved in cell polarity

Abstract

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.

Bone morphogenetic proteins regulate neural tube closure by interacting with the apicobasal polarity pathway

“…Since PAR3, PAR6 and aPKC complex with pSMAD5/8 in a BMP-dependent manner, pSMAD5/8 must also be associated with apical junctions when the cell receives BMP signaling (Fig. 8A) (Warner et al, 2003). In the absence of canonical BMP signaling, the phosphorylation of SMAD proteins is severely attenuated, reducing interactions with the apical polarity complex (Fig.…”

ERBIN is a new SARA-interacting protein: competition between SARA and SMAD2 and SMAD3 for binding to ERBIN

“…ERBIN has been implicated in TGFb signalling through its interaction with both non-phosphorylated and phosphorylated SMAD2/3 (Warner et al, 2003;Dai et al, 2007). ERBIN overexpression inhibited TGFb-dependent transcription, whereas siRNA against ERBIN stimulated ligand-induced responses.…”

“…4) and found that a 25-amino-acid domain (amino acids 1241-1265) of ERBIN is responsible for the interaction with SARA. Because these 25 amino acids were included in a longer 110-amino-acid stretch (1172-1282) that has been shown to interact with SMAD2/3 (Warner et al, 2003;Dai et al, 2007), we further defined the interaction domain of SMAD2 and SMAD3 on ERBIN. Much to our surprise, the SMAD2 and SARA-interacting domains of ERBIN were identical, being contained between amino acids 1241 and 1265.…”

“…Among several interacting proteins, we have focused on the protein ERBIN, a protein that interacts with ERBB2 (also known as HER2) and is involved in the localisation and signalling of ERBB2 in epithelia (Borg et al, 2000). Importantly, ERBIN has previously been shown to interact with SMAD proteins (Warner et al, 2003;Dai et al, 2007). Here, we dissect the domains of SARA and ERBIN responsible for their interaction, we define ERBIN as a protein that can be recruited to the early endocytic compartment by SARA and we address the functional consequences of the ERBIN-SARA interaction in TGFb and activin A signalling.…”

Protéines LAP : de nouvelles clés de voûte de l’architecture épithéliale

“…Des travaux récents ont démontré l'implication de Erbin dans la régulation négative de la voie Ras-MAPK activée par ErbB2 en interagissant avec la forme activée de Ras [26]. Erbin interagit également avec les protéines Smad, maillons incontournables de la signalisation du TGFβR impliqué dans la transition épithéliale-mésenchyme [27]. Erbin n'est pas seulement impliquée dans des réseaux moléculaires de signalisation, mais est également au coeur d'intéressants complexes protéiques participant à l'adhérence cellulaire.…”