The Hippo signaling pathway is a key regulator of cell proliferation, apoptosis, and organ growth. Warts (LATS1/LATS2 in mammals) is a central kinase that phosphorylates and inhibits Yorkie (YAP/TAZ in mammals), thereby functioning as a tumor suppressor. Warts activation is promoted by apical protein complexes containing the upstream regulators Expanded, Kibra, and Merlin, which facilitate phosphorylation of Warts within its kinase domain and suppression of proliferation. In contrast, the Ajuba family protein Jub (AJUBA, LIMD1, and WTIP in mammals) acts as a junctional regulator that modulates Warts activity in response to cell–cell adhesion and cytoskeletal tension. Under conditions of high cytoskeletal tension, Jub inhibits Warts activity, thereby promoting Yorkie-dependent transcriptional programs. Despite these advances, the precise molecular mechanisms by which Jub regulates Warts remain incompletely understood.In this dissertation, we investigated how Jub regulates Warts in Drosophila and explored whether similar mechanisms operate in mammalian cells. Previous studies reported the colocalization of Jub and Warts at adherens junctions in a punctate pattern, raising the possibility that their interaction may involve liquid–liquid phase separation. Upon expression in Drosophila Schneider 2 (S2) cells, Jub formed circular enrichments displaying properties characteristic of biomolecular condensates, including dynamic fusion events and recovery after photobleaching. Although Warts contains an intrinsically disordered N-terminal region similar to that of Jub, it did not coalesce on its own but was efficiently recruited into condensates by Jub. Warts was also detected in condensates together with the upstream Hippo pathway activators Expanded and Kibra, suggesting that these structures may represent sites where both inhibitory and activating regulators converge to control Warts activity.We further found that Jub induces robust phosphorylation of the N-terminal region of Warts, which modulates its recruitment by upstream activators. Expanded and Kibra exhibited significantly stronger colocalization with non-phosphorylatable Warts compared to phosphomimetic Warts. Functional Drosophila wing growth assay, together with analysis of the Yorkie transcriptional reporter ex-lacZ, demonstrated that non-phosphorylatable Warts displays increased activity relative to phosphomimetic Warts. Screening of Drosophila proline-directed kinases identified Minibrain and Homeodomain-interacting protein kinase as mediators of Jub-dependent Warts phosphorylation.To examine whether similar regulatory mechanisms operate in mammals, we analyzed the behavior of LATS kinases in HEK293 cells. LIMD1 recruited both LATS1 and LATS2 into condensates, although recruitment and N-terminal phosphorylation were more prominent for LATS1 than for LATS2. Consistent with this observation, non-phosphorylatable LATS1 resulted in increased cytoplasmic retention of YAP, indicating enhanced LATS1 activity. Together, this work uncovers a dual mechanism in which Ajuba family proteins control Hippo pathway output by organizing Warts/LATS kinases into condensates and modulating their N-terminal phosphorylation. This mechanism appears to be at least partially conserved between Drosophila and mammals, although further work will be required to definitively establish its role in mammalian systems.
Elmira Kirichenko (Thu,) studied this question.