In large organisms - such as humans - cells must be able to acquire information from the environment, as well as communicate with each other inside organs and tissues. This level of communication is an impressive feat in fully developed organisms when the size and cell number in most tissues are fairly static. Even more astoundingly, during embryonic development this communication must be timed perfectly among cells that are rapidly dividing and differentiating. As these communication systems are so complex biologists often study them in a ‘reductionist’ system. Isolating single cells or even a handful of unique proteins has allowed researchers to identify key members of cell signaling pathways. This approach has yielded a strong foundation for biology allowing researchers to make sense of more complex systems. Here at Fred Hutch, researchers in the Vasioukhin Lab (Human Biology Division) in collaboration with scientists from the University of Toronto used genetically engineered mouse models, cells in culture, and purified proteins to uncover a new regulator of Hippo signaling, a pathway that regulates organ size and protects us from cancer by controlling cellular proliferation and death. While direct transducers of Hippo signaling have been well characterized previously, many important players in this signaling pathway are still being discovered. In particular Hippo signaling is known to be regulated by cellular polarity; however, the specific molecular mechanisms these pathways were not well understood. In a recent Genes and Development publication the Vasioukhin laboratory identified DLG5 protein as a linker between Hippo signaling and cell polarity proteins PAR-1a/b/c. The study demonstrated that DLG5 silences Hippo pathway suggesting that overabundance of DLG5 may function as a potential oncogenic driver in cancers overexpressing DLG5.
Vasioukhin laboratory is interested in signaling pathways connected to cell polarity and cell adhesion and they have previously identified DLG5 as a cell polarity protein. This group previously reported that a mouse model of DLG5 loss presented with developmental defects – particularly in the brain. These mutant mice develop brain hydrocephalus, synaptogenic defects, and are smaller than normal siblings. The small animal size suggested DLG5 was affecting a signaling pathway that controls cellular proliferation and organ size. To identify this pathway, scientists isolated DLG5 from human cells such that any interacting protein complexes would also be purified. Using mass spectrometry, researchers found that Hippo kinases MST1/2 bind to DLG5. In addition, these experiments revealed a novel interaction between DLG5 and important cell polarity proteins PAR-1a/b/c. While finding of these novel physical interactions was exciting, it did not reveal whether DLG5 was altering the Hippo pathway. To characterize signaling changes, researchers measured levels of MST1/2 and LATS1/2 phosphorylation in Dlg5-mutant and normal tissues. Phosphorylation of amino acids 183/180 on MST1/2 serve as readouts of MST1/2 activity as explained by Dr. Valeri Vasioukhin, “This is an activation loop phosphorylation event that can be both auto-phosphorylation and phosphorylation by other kinases.” Once MST kinases are active, they phosphorylate LATS1/2 kinases, which in turn phosphorylate YAP/TAZ transcriptional activators and silence YAP/TAZ-dependent transcription. The authors found that when cells lack DLG5 both MST and LATS kinases are hyper-active and silence Hippo transcriptional targets. These findings suggested that brain defects in DLG5-null mice may develop due to unconstrained MST signaling. Thus, if both DLG5 and MST1/2 were missing brain development should be closer to normal. While two ‘wrongs’ rarely make a ‘right’ – double deletion of Dlg5 and Mst1/2 genes did in fact rescue most brain defects observed in DLG5-null mice. This genetic evidence strongly supported biochemical findings, and suggested that DLG5 negatively regulates Hippo signaling during normal mammalian development. Moreover, it is likely that DLG5-mediated regulation of Hippo signaling is widespread and is not a unique activity in brain development. “We believe that this signaling pathway is conserved in many tissues and organisms. While we concentrated on developing mouse brain and neural progenitor cells, we also looked at skin and liver. In addition, we analyzed dlg5 in Drosophila [flies]. We saw similar phenotypes in these model systems.” said Dr. Vasioukhin.
To determine the molecular mechanisms responsible for DLG5-mediated negative regulation of Hippo signaling, researchers used cultured cells and purified proteins to find the regions of DLG5 that interact with MST1/2. Small fragments of the DLG5 protein were expressed in human cells and analyzed for their ability to bind MST1/2. This revealed that the PDZ3 domain was required for MST1/2 binding but was not sufficient to silence hippo signaling. This is consistent with DLG5 serving as a scaffold rather than a direct regulator. Along these lines, the previous mass spectrometry results had also identified MARK2/3 as DLG5 interacting protein. In fact, Dr. Vasioukhin said, “We found that Dlg5 links MST1/2 with MARK1/2/3 kinases and this interaction inhibits MST1/2. This is likely via inhibitory phosphorylation of Mst1/2 by MARKs.”
This work was an excellent demonstration of how different scientific models work together to increase our understanding of biology. Only by forming a strong understanding of basic science can we begin to characterize complex organisms. Moreover, it is paramount we understand normal biology in order to increase our therapeutic toolbox when it comes to treating human disease, which according to Dr. Vasioukhin may be a future development from these findings, “DLG5 is frequently overexpressed in human cancers. It will be interesting to analyze whether this is an important tumor driving event. For example, we found that DLG5 is overexpressed in 5% of human renal clear cell carcinomas and its overexpression is significantly associated with tumor recurrence. Based on our findings, we predict that overexpression of DLG5 should inactivate Hippo signaling and activate YAP/TAZ, and this could be a potent tumor promoting mechanism. These tumors may be potentially sensitive to inhibitors of YAP/TAZ, while tumors utilizing different signaling mechanisms may be quite resistant.”
Kwan J, Sczaniecka A, Arash EH, Nguyen L, Chen CC, Ratkovic S, Klezovitch O, Attisano L, McNeill H, Emili A, Vasioukhin V. 2016. DLG5 connects cell polarity and hippo signaling protein networks by linking PAR-1 with MST1/2. Genes and Development, 30:1-16.
Funding for this research was provided by the National Cancer Institute and National Institute of General Medical Sciences (NIH).