Yki stability and activity are regulated by Ca2+-calpains axis in Drosophila.

Yorkie (Yki) is a key effector of the Hippo pathway that activates the expression of targets by associating with the transcription factor Scalloped (Sd). Various upstream signals, such as cell polarity and mechanical cues, control transcriptional programs by regulating Yki activity. Searching for Yki regulatory factors has far-reaching significance for studying the Hippo pathway in animal development and human diseases. In this study, we identify Calpain-A (CalpA) and Calpain-B (CalpB), two calcium (Ca2+)-dependent modulatory proteases of the calpain family, as critical regulators of Yki in Drosophila that interact with Yki respectively. Ca2+ induces Yki cleavage in a CalpA/CalpB-dependent manner, and the protease activity of CalpA/CalpB is pivotal for the cleavage. Furthermore, overexpression of CalpA or CalpB in Drosophila partially restores the large wing phenotype caused by Yki overexpression, and F98 of Yki is an important cleavage site by the Ca2+-calpains axis. Our study uncovers a unique mechanism whereby the Ca2+-calpain axis modulates Yki activity through protein cleavage.

C-terminal-mediated homodimerization of Expanded is critical for its ability to promote Hippo signalling in Drosophila.

Hippo signalling plays key role in tissue growth and homeostasis, and its dysregulation is implicated in various human diseases. Expanded (Ex) is an important upstream activator of Hippo signalling; however, how Ex activates Hippo signalling is still poorly understood. Here, we demonstrate that Ex forms a homodimer via C-terminal interaction, and that the ExC2 region (912-1164 aa) is sufficient and essential for Ex dimerization. Functional analysis shows that ExC2 is required for Ex to promote the phosphorylation and inactivation of Yki in Drosophila cells. Further in vivo analysis shows that ExC2 is important for Ex to control Drosophila tissue growth. Our study, thus, uncovers a novel mechanism whereby Ex homodimerization mediates its full activation to promote Hippo signalling in growth control.

Usp10 Modulates the Hippo Pathway by Deubiquitinating and Stabilizing the Transcriptional Coactivator Yorkie.

The Hippo signaling pathway is an evolutionarily conserved regulator that plays important roles in organ size control, homeostasis, and tumorigenesis. As the key effector of the Hippo pathway, Yorkie (Yki) binds to transcription factor Scalloped (Sd) and promotes the expression of target genes, leading to cell proliferation and inhibition of apoptosis. Thus, it is of great significance to understand the regulatory mechanism for Yki protein turnover. Here, we provide evidence that the deubiquitinating enzyme ubiquitin-specific protease 10 (Usp10) binds Yki to counteract Yki ubiquitination and stabilize Yki protein in Drosophila S2 cells. The results in Drosophila wing discs indicate that silence of Usp10 decreases the transcription of target genes of the Hippo pathway by reducing Yki protein. In vivo functional analysis ulteriorly showed that Usp10 upregulates the Yki activity in Drosophila eyes. These findings uncover Usp10 as a novel Hippo pathway modulator and provide a new insight into the regulation of Yki protein stability and activity.

The SCF ubiquitin ligase Slimb controls Nerfin-1 turnover in Drosophila.

The C2H2 type zinc-finger transcription factor Nerfin-1 expresses dominantly in Drosophila nervous system and plays an important role in early axon guidance decisions and preventing neurons dedifferentiation. Recently, increasing reports indicated that INSM1 (homologue to nerfin-1 in mammals) is a useful marker for prognosis of neuroendocrine tumors. The dynamic expression of Nerfin-1 is regulated post-transcriptionally by multiple microRNAs; however, its post-translational regulation is still unclear. Here we showed that the protein turnover of Nerfin-1 is regulated by Slimb, the substrate adaptor of SCFSlimb ubiquitin ligase complex. Mechanistically, Slimb associates with Nerfin-1 and promotes it ubiquitination and degradation in Drosophila S2R+ cells. Furthermore, we determined that the C-terminal half of Nerfin-1 (Nerfin-1CT) is required for its binding to Slimb. Genetic epistasis assays showed that Slimb misexpression antagonizes, while knock-down enhances the activity of Nerfin-1CT in Drosophila eyes. Our data revealed a new link to understand the underlying mechanism for Nerfin-1 turnover in post-translational level, and provided useful insights in animal development and disease treatment by manipulating the activity of Slimb and Nerfin-1.