Regulation of Hippo Pathway Kinases MST1/2 by DLG5 and Other Signaling Factors

The Hippo signal transduction pathway plays critical roles in regulating development, organ size, and cancer progression, and is broadly conserved across metazoans. Mammalian Sterile 20-like Kinase 12 (MST1/2) are mammalian homologues of the Drosophila Hippo kinase, and along with downstream Large Tumor Suppressor Kinase 12 (LATS1/2), constitute the core kinases of the Hippo pathway. How MST1/2 are regulated, and in particular, what mechanisms connect them to upstream signaling components is a key question in the field. To address this, I employed affinity purification coupled to mass spectrometry to identify novel interacting proteins with important regulatory roles for MST1/2 and the Hippo pathway. I examined 25 different bait proteins related to the pathway, and identified 274 high confidence protein-protein interactions (PPI). The most intriguing PPI I identified was between MST1/2 and Discs Large Homolog 5 (DLG5), a widely conserved, large, multi-domain containing protein, which plays a role in maintenance of epithelial cell polarity. Using human, mouse, and fly model systems, fellow collaborators and I investigated the potential functional connections between DLG5, MST1/2, and other components of the Hippo pathway. We found that DLG5 acts as an inhibitor of Hippo signaling, in vivo and ex vivo. Mechanistically, I demonstrate that DLG5 binds directly to both MST1/2 and MAP/Microtubule Affinity-Regulating Kinase 3 (MARK3) and promotes their association. I found that MARK3 promotes hyperphosphorylation of MST1/2 and inhibits MST1/2 kinase activity. I propose a model wherein DLG5 recruits MARK3 to inhibit MST1/2 and the Hippo pathway. Although additional details of MARK3 regulation of MST1/2 await exploration, I have characterized DLG5 as a new and conserved component of the Hippo pathway and a negative regulator of MST1/2, providing the first example of a scaffold protein that directly links MST1/2 to other regulatory kinases and cell polarity factors.Ph.D