Scientists long to understand exactly how cells change from being highly polarized and ordered in a sheet-like arrangement, to losing this apical–basal polarity and becoming migratory. This process of epithelial–mesenchymal transition (EMT) is crucial during morphogenesis, but is also involved during the progression of some diseases. So the finding, reported in Science by a group led by Jeff Wrana, that transforming growth factor-β (TGFβ) promotes EMT by mediating an interaction between the polarity protein Par6, the E3 ubiquitin ligase Smurf1 and the cytoskeletal regulator RhoA will excite developmental biologists and cancer researchers alike.

TGFβ signals through its serine/threonine kinase transmembrane receptors TβRI and TβRII to cytosolic Smad proteins, which interact with DNA-binding partners in the nucleus to mediate transcription. Wrana's group explored a potential role for TGFβ-mediated non-transcriptional signalling in EMT. In a screen for TβRI binding partners they uncovered Par6, which regulates the assembly of tight junctions — a type of epithelial cell–cell junction — and the apical–basal polarity of epithelial cells. As TGFβ induces tight-junction loss during EMT, the authors delved deeper.

They showed that Par6 associated with TβRI independently of TGFβ or receptor kinase activity. Furthermore, in a confluent monolayer of mammary epithelial cells, cell-surface TβRI localized in a distinct band around the apical borders of cells, together with Par6 and the tight junction components zona occludens-1 (ZO-1) and occludin. Adding TGFβ induced TβRII to redistribute into tight junctions, probably by promoting a TβRI–TβRII complex.

Examining the possibility that Par6 might be a substrate of the TGFβ receptor complex, the authors found that Par6 was phosphorylated by TβRII, rather than TβRI, on Ser345, its penultimate residue. Mutation of this residue to an alanine (S345A) conferred resistance to TGFβ-induced tight junction dissolution and actin rearrangement, so phosphorylation of Par6 on Ser345 is important in TGFβ-induced EMT. Significantly, Par6S345A had no effect on TGFβ-stimulated Smad transcriptional activity or transcriptional induction.

The E3 ubiquitin ligase Smurf1 can mediate ubiquitylation and the subsequent degradation of RhoA, a Rho-family GTPase that regulates cell junction formation and polarity. So, could Smurf1 be involved in TGFβ-mediated EMT? The authors found endogenous Smurf1 to preferentially bind to Ser345-phosphorylated Par6 in a cell-binding assay. And when treated with TGFβ for 6 hours, endogenous Smurf1 became localized at cell–cell junctions, a response that was blocked by expressing Par6S345A. Overexpressing Smurf1 caused cells to lose their tight junctions and adopt a mesenchymal morphology, whereas small interfering RNA (siRNA) against Smurf1 inhibited TGFβ-induced EMT.

The authors saw a 30% reduction in the amount of RhoA after TGFβ treatment, which was not observed in the presence of Par6S345A or a proteasome inhibitor. Mutational analysis identified lysines 6 and 7 on RhoA as acceptors for Smurf1-transferred ubiquitin, and changing these two residues to arginines inhibited TGFβ-dependent EMT. The addition of a proteasome inhibitor prevented TGFβ-induced dissolution of tight junctions, whereas siRNA against RhoA promoted dissolution of tight junctions. TGFβ, through Par6 phosphorylation, therefore seems to induce Smurf1-dependent turnover of RhoA at tight junctions during EMT.


ORIGINAL RESEARCH PAPER Ozdamar, B. et al. Regulation of the polarity protein Par6 by TGFβ receptors controls epithelial cell plasticity. Science 307, 1603–1609 (2005)