The Hippo signaling pathway regulates tissue growth and cell fate, and its dysregulation can induce tumorigenesis. When Hippo is activated by cell-cell contact, extracellular signals, or cell polarity among others, the large tumor suppressor 1 (LATS1) kinase catalyzes inhibitory phosphorylation of the transcriptional coactivator Yes-associated protein (YAP) to maintain YAP in the cytoplasm or promote its degradation. Separately, calmodulin is a Ca2+-dependent protein that modulates the activity of target proteins and regulates several signaling cascades; however, its potential role in the Hippo pathway has not been identified. Here, using diverse experimental approaches, including in vitro binding analyses, kinase assays, RT-PCR, and confocal microscopy, we reveal that calmodulin promotes Hippo signaling. We show that purified YAP and LATS1 bind directly to calmodulin and form a Ca2+-dependent ternary complex in vitro. Importantly, Ca2+/calmodulin directly stimulated the activity of LATS1 kinase. In cultured mammalian cells, we demonstrated that endogenous YAP and LATS1 coimmunoprecipitate with endogenous calmodulin. In cells with activated Hippo signaling, we show that calmodulin antagonism significantly (i) decreases YAP phosphorylation, (ii) increases expression of two Hippo target genes (connective tissue growth factor [CTGF] and cysteine-rich angiogenic inducer 61 [CYR61]) that regulate cell proliferation and tumor progression, and (iii) enhances the interaction of YAP with its major transcription factor, thereby facilitating transcription of target genes. Collectively, our data demonstrate that calmodulin activates the Hippo kinase cascade and inhibits YAP activity via a direct interaction with LATS1 and YAP, thereby uncovering previously unidentified crosstalk between the Ca2+/calmodulin and Hippo signaling pathways.
Published by Elsevier Inc.

Given the functional attributes of Doublecortin-like kinase 1 (DCLK1) in tumor growth, invasion, metastasis, cell motility, and tumor stemness, it is emerging as a therapeutic target in gastrointestinal cancers. Although a series of specific or nonspecific ATP-competitive inhibitors were identified against DCLK1, different types of scaffolds that can be utilized for the development of highly selective inhibitors or structural understanding of binding specificities of the compounds remain limited. Here, we present our work to repurpose a Janus kinase 1 inhibitor, ruxolitinib as a DCLK1 inhibitor, showing micromolar binding affinity and inhibitory activity. Furthermore, to gain an insight into its interaction mode with DCLK1, a crystal structure of the ruxolitinib-complexed DCLK1 has been determined and analyzed. Ruxolitinib as a nonspecific DCLK1 inhibitor characterized in this work is anticipated to provide a starting point for the structure-guided discovery of selective DCLK1 inhibitors.

Hippo signaling is an evolutionarily conserved pathway that restricts growth and regeneration predominantly by suppressing the activity of the transcriptional coactivator Yap. Using a high-throughput phenotypic screen, we identified a potent and non-toxic activator of Yap. In vitro kinase assays show that the compound acts as an ATP-competitive inhibitor of Lats kinases-the core enzymes in Hippo signaling. The substance prevents Yap phosphorylation and induces proliferation of supporting cells in the murine inner ear, murine cardiomyocytes, and human Müller glia in retinal organoids. RNA sequencing indicates that the inhibitor reversibly activates the expression of transcriptional Yap targets: upon withdrawal, a subset of supporting-cell progeny exits the cell cycle and upregulates genes characteristic of sensory hair cells. Our results suggest that the pharmacological inhibition of Lats kinases may promote initial stages of the proliferative regeneration of hair cells, a process thought to be permanently suppressed in the adult mammalian inner ear.

Elucidating signaling driven by lemur tyrosine kinase 3 (LMTK3) could help drug development. Here, we solve the crystal structure of LMTK3 kinase domain to 2.1Å resolution, determine its consensus motif and phosphoproteome, unveiling in vitro and in vivo LMTK3 substrates. Via high-throughput homogeneous time-resolved fluorescence screen coupled with biochemical, cellular, and biophysical assays, we identify a potent LMTK3 small-molecule inhibitor (C28). Functional and mechanistic studies reveal LMTK3 is a heat shock protein 90 (HSP90) client protein, requiring HSP90 for folding and stability, while C28 promotes proteasome-mediated degradation of LMTK3. Pharmacologic inhibition of LMTK3 decreases proliferation of cancer cell lines in the NCI-60 panel, with a concomitant increase in apoptosis in breast cancer cells, recapitulating effects of LMTK3 gene silencing. Furthermore, LMTK3 inhibition reduces growth of xenograft and transgenic breast cancer mouse models without displaying systemic toxicity at effective doses. Our data reinforce LMTK3 as a druggable target for cancer therapy.
Copyright © 2020 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works. Distributed under a Creative Commons Attribution NonCommercial License 4.0 (CC BY-NC).

Resistance to drought stress is fundamental to plant survival and development. Abscisic acid (ABA) is one of the major hormones involved in different types of abiotic and biotic stress responses. ABA intracellular signaling has been extensively explored in Arabidopsis thaliana and occurs via a phosphorylation cascade mediated by three related protein kinases, denominated SnRK2s (SNF1-related protein kinases). However, the role of ABA signaling and the biochemistry of SnRK2 in crop plants remains underexplored. Considering the importance of the ABA hormone in abiotic stress tolerance, here we investigated the regulatory mechanism of sugarcane SnRK2s-known as stress/ABA-activated protein kinases (SAPKs). The crystal structure of ScSAPK10 revealed the characteristic SnRK2 family architecture, in which the regulatory SnRK2 box interacts with the kinase domain αC helix. To study sugarcane SnRK2 regulation, we produced a series of mutants for the protein regulatory domains SnRK2 box and ABA box. Mutations in ScSAPK8 SnRK2 box aimed at perturbing its interaction with the protein kinase domain reduced protein kinase activity in vitro. On the other hand, mutations to ScSAPK ABA box did not impact protein kinase activity but did alter the protein autophosphorylation pattern. Taken together, our results demonstrate that both SnRK2 and ABA boxes might play a role in sugarcane SnRK2 function.
Copyright © 2019 Righetto, Sriranganadane, Halabelian, Chiodi, Elkins, Massirer, Gileadi, Menossi and Couñago.