The F-box protein Ppa is a common regulator of core EMT factors Twist, Snail, Slug, and Sip1

Abstract

A small group of core transcription factors, including Twist, Snail, Slug, and Sip1, control epithelial-mesenchymal transitions (EMTs) during both embryonic development and tumor metastasis. However, little is known about how these factors are coordinately regulated to mediate the requisite behavioral and fate changes. It was recently shown that a key mechanism for regulating Snail proteins is by modulating their stability. In this paper, we report that the stability of Twist is also regulated by the ubiquitin-proteasome system. We found that the same E3 ubiquitin ligase known to regulate Snail family proteins, Partner of paired (Ppa), also controlled Twist stability and did so in a manner dependent on the Twist WR-rich domain. Surprisingly, Ppa could also target the third core EMT regulatory factor Sip1 for proteasomal degradation. Together, these results indicate that despite the structural diversity of the core transcriptional regulatory factors implicated in EMT, a common mechanism has evolved for controlling their stability and therefore their function.

Figure 1.

Twist is an unstable protein. (A) Embryos were injected with mRNA encoding Twist, cultured to stage 8, and treated with CHX to prevent further protein synthesis. Western analysis demonstrates Twist protein instability. Actin serves as a loading control. (B) Twist levels decrease rapidly over developmental time. Embryos injected with Twist mRNA were collected at blastula, gastrula, neurula, and tailbud stages (left to right), and protein levels were analyzed via Western blotting. Twist is undetectable by early migrating neural crest stages.

Figure 2.

The WR domain is targeted by ubiquitin and renders Twist unstable. (A) Polyubiquitinated forms of wild-type Twist were immunoprecipitated (IP) from lysates of embryos coinjected with epitope-tagged forms of Twist and ubiquitin. A ladder of polyubiquitinated Twist isoforms is noted when Twist and ubiquitin are coexpressed. IgG bands are indicated by an asterisk. IB, immunoblotted. (B) A schematic illustrating the Twist deletion constructs used in these experiments. (C) Polyubiquitinated forms of wild-type (WT) Twist and Twist C terminus (Cterm), but not Twist N terminus (Nterm) or Twist ΔWR, were immunoprecipitated from lysates of embryos coinjected with Twist deletion constructs and ubiquitin. IgG bands are indicated by asterisks. (D) Embryos were injected with wild-type Twist or Twist ΔWR mRNA, cultured to stage 8, and treated with CHX to block further protein synthesis. Western analysis shows that Twist ΔWR is highly stable compared with wild-type Twist. (E) Deletion of the Twist WR domain stabilizes Twist. Embryos injected with mRNA encoding wild-type Twist or Twist ΔWR were collected at blastula, gastrula, neurula, and tailbud stages, and protein expression levels were analyzed via Western blotting. Twist ΔWR is significantly more stable than wild-type Twist. Actin is used as a control.

Figure 3.

Twist interacts with the E3 ubiquitin ligase Ppa via the WR domain. (A) Snail and Twist were immunoprecipitated (IP) from lysates of embryos coinjected with myc-tagged forms of Snail, Twist, or Sox10 and Flag-tagged Ppa using an α-Flag antibody. Immunoprecipitates were resolved by SDS-PAGE, and Ppa-bound Snail and Twist were detected by α-Myc Western blotting. Sox10 does not immunoprecipitate with Ppa. IgG bands are indicated by an asterisk. IB, immunoblotted. (B) A schematic illustrating the Twist deletion and E12-WR fusion constructs. AD denotes the activation domains within E12 protein. WT, wild type. (C) The Twist WR domain is both necessary and sufficient for Ppa interaction. Both wild-type Twist and the E12-WR domain fusion protein were immunoprecipitated from lysates coinjected with either epitope-tagged forms of wild-type Twist or E12-WR and Ppa using the α-Flag antibody. Interacting proteins were detected by α-Myc Western blotting. E12 does not interact with Ppa, whereas the fusion protein strongly interacts. Deleting the WR domain eliminates interaction between Twist and Ppa. IgG bands are indicated by an asterisk. (D) Comparison of Xenopus Slug and Twist sequences required for Ppa interaction. The underlined residues denote amino acids required for Ppa–Slug interaction as determined in Vernon and LaBonne (2006).

Figure 4.

Ppa is an endogenous regulator of Twist stability. (A) Embryos injected with Twist alone or together with Ppa were treated with CHX at stage 8 and collected at the time points indicated. Twist protein is significantly destabilized by coexpression of Ppa. Actin is used as a loading control. (B) Embryos were coinjected with Twist and control or Ppa MO, treated with CHX at stage 8, and collected at the time points indicated. The loss of Ppa mediated by the Ppa MO significantly stabilizes Twist.

Figure 5.

Ppa and the UPS also regulate another core EMT factor, Sip1. (A) Sip1 was immunoprecipitated (IP) from lysates of embryos coinjected with epitope-tagged forms of Sip1 and Ppa or ubiquitin using α-Flag antibody, and interactions were detected by α-Myc Western blotting. IgG bands are indicated by an asterisk. IB, immunoblotted. (B) Embryos injected with Sip1 alone or together with Ppa were treated with CHX at stage 8 and collected at the time points indicated. Sip1 protein is significantly destabilized by coexpression of Ppa. Actin is used as a control. (C) A schematic illustrating the diversity in protein structure among the core EMT transcriptional factors. HD, homeodomain-like sequence; SBD, Smad-binding domain; ZnF, Zinc finger domain. (D) A model highlighting Ppa as a common control mechanism for the structurally diverse set of core EMT regulatory factors Snail, Slug, Sip1, and Twist. Multiple distinct signaling pathways converge on this common set of factors, but in the neural crest, Ppa serves as a common mechanism for UPS targeting. RTK, receptor tyrosine kinase.