The Hippo pathway, a highly conserved signaling pathway from Drosophila to mammals, plays a key role in the transcriptional regulation of target genes in cell proliferation and apoptosis during organ development and tumorigenesis (Ma et al., 2019; Zheng and Pan, 2019). In Drosophila, the core of this pathway is a Hpo-Wts kinase cascade composed of two kinases: Hippo (Hpo) and Warts (Wts), and their corresponding activators Salvador (Sav) and Mob as Tumor suppressor (Mats). Upon intrinsic and/or extrinsic stimulation, the activated Hpo-Wts kinase cassette phosphorylates a transcriptional coactivator Yorkie (Yki), the major effector of the Hpo-Wts kinase cascade, causing Yki cytoplasmic retention by binding to protein 14-3-3 (Wu et al., 2003; Huang et al., 2005; Lai et al., 2005; Wu et al., 2008). Conversely, unphosphorylated Yki enters the nucleus and coordinates with transcription factors such as Scalloped (Sd) to promote the expression of target genes, including diap1 and cycE.

The nuclear localization and transcriptional activity of Yki are not only negatively regulated by phosphorylation via the core Hpo-Wts kinase cascade, but they are also modulated by direct protein-protein interaction. Expanded (Ex), a FERM domain-containing protein, and Hpo were reported to interact with Yki directly in a phosphorylation-independent manner, thus sequestering Yki nuclear activity in the cytoplasm (Badouel et al., 2009; Oh et al., 2009). Proteostasis is another critical way of regulating protein activity, and the proteasome, lysosome, and proteases/peptidases-mediated protein turnover are the three main pathways for proteostasis maintenance. Yki was reported to be degraded via lysosome in the cytoplasm and proteasome in the nucleus, which is controlled through the ubiquitination-deubiquitination system (Gao et al., 2019; Sun et al., 2019). However, whether peptidases regulate Yki stability remains to be elucidated.

Given the crucial role of Yki and its conserved homologs YAP/TAZ in animal development and human diseases, we overexpressed GFP-Yki in Drosophila S2 cells and isolated Yki protein by GFP-trap for mass spectrometry analysis to find potential novel regulators may modulate Yki activity. Intriguingly, we found that a member of the calpain family, Calpain-A (CalpA), is a new Yki-associating protein in two independent assays. Calpains are calcium-dependent, cytosolic proteases active at neutral pH conditions (Franco and Huttenlocher, 2005). Calpains exist in organisms ranging from bacteria to humans (Ono et al., 2016), and in Drosophila, there are four members containing CalpA, CalpB, CalpC, and CalpD. CalpA and CalpB contain three conserved calpain active sites, and mutating one or three sites to Ala (A) can destroy their enzyme activities to varying degrees (Friedrich et al., 2004). Calpains perform biological functions by cleaving their substrates in a limited manner. For example, calpains can specifically cleave glutamate receptor IIA, affecting nerve conduction at the neuromuscular junction in Drosophila (Metwally et al., 2019). In addition, CalpA binds to Cactus (NF-kappa-B inhibitor alpha/IkB-alpha in mammals) and cleaves it, making it unable to respond to the Toll signaling pathway, thereby affecting the immune response (Fontenele et al., 2013). There is a genetic interaction between CalpB and the adhesion protein complex If, Mys, and Rhea, which regulates the migration of boundary cells in the Drosophila egg chamber (Kokai et al., 2012).

In this study, we reported that the Ca2+-CalpA/CalpB (CalpA/B) signal axis is a new regulator of Yki protein stability. Both members of the calpain family, CalpA and CalpB interact with Yki. Ca2+ in the cytoplasm promotes Yki cleavage through CalpA and CalpB, and simultaneously knocking down CalpA and CalpB suppresses Ca2+-mediated Yki cleavage. CalpA and CalpB accelerate the cleavage of Yki in a calpain protease-dependent fashion. Furthermore, Ca2+, CalpA, and CalpB regulate Yki activity in Drosophila. Lastly, Bioinformatics analysis and immunoblotting experiments revealed that Yki-F98, located in the Sd-binding domain, is the key cleavage site recognized by calpains. Therefore, our findings uncover a novel mechanism by which the Ca2+-CalpA/B axis cleaves Yki and represses its Sd-binding activity in Drosophila.