By: Seth Palmer
The Hippo pathway — which regulates tissue growth by restricting cell proliferation and growth and promoting programmed cell death (apoptosis) — is an evolutionarily conserved signaling pathway known to be mutated in human cancer patients, making it an important starting point for scientists investigating tumor development.
Dr. Zhi-Chun Lai, a professor of biochemistry and molecular biology at Penn State, has for the better part of a decade focused his research primarily on the Hippo pathway.
"The Hippo kinase plays an important role in growth inhibition during animal development, but how Hippo activity is regulated has not been fully understood," says Dr. Lai. "To better investigate Hippo regulation in living cells, we have applied the Bimolecular Fluorescence Complementation (BiFC) technique to directly visualize protein-protein interaction in developing tissues."
The BiFC technique involves a splitting a fluorescent protein called Venus into two parts — Venus N-terminus (VN) and C-terminus (VC) — which alone cannot produce a fluorescent signal, but together will fluoresce whenever two molecules to which they are attached interact with one another.
By fusing VN and VC to the Hippo protein, Dr. Lai's lab has been able to observe via fluorescence the interaction between Hippo molecules during the activation of the Hippo pathway.
Significance and potential application
The importance of the Lai Lab's discovery lies in the potential for targeted exploitation of the Hippo pathway's growth-inhibiting — and thus tumor-suppressing — action.
Many current cancer treatments focus on the proteins (known as oncoproteins) encoded by oncogenes — genes with the potential to cause cancer when mutated or expressed at high levels — using drugs to decrease or "knock down" the activity of the oncoproteins, which can also block the function of normal proteins with similar structure, causing severe side effects and resulting in damage to normal cells.
But as the Hippo pathway is naturally growth-inhibiting, it can potentially be targeted with a reagent to amplify its activity in a given cell, organ, or tumor — yielding the same net effect as a knockdown treatment, and perhaps with less negative side effects.
Drosophila and the human Hippo pathway
The Hippo pathway is evolutionarily conserved — meaning that its function remains the same across a wide variety of species — and since the core components of the Hippo pathway are the same in both flies and humans, the Lai Lab conducts its studies on Drosophila (fruit flies).
While the core components of the Hippo pathway are fairly well studied, the upstream factors regulating the Hippo pathway are significantly less understood — and it is mainly these factors that the Lai Lab set out to study and decipher.
The central part of the Hippo pathway comprises a series of protein-protein interactions involving a kinase cascade — where protein kinases (enzymes) activate one another, in series, through a process known as phosphorylation (addition of a phosphate group) — which inhibits a growth-promoting protein named Yorkie (in fly)/YAP (in human) that acts with other molecules to regulate the expression of downstream target genes involved in cell proliferation and apoptosis.
The regulating factors upstream of the Hippo pathway, however, are much more diverse and, in many cases, partially redundant — operating as a complex network whose parts somewhat overlap in function; but they are known to be found in a given set of locations corresponding to specific stages of development — making them key to better understanding how the Hippo kinase is involved in tissue growth and organ size control during development.
Observations and conclusions
By analyzing the patterns of fluorescence in the BiFC assays, Dr. Lai's lab was able to determine that during activation of the Hippo pathway, Hippo kinase proteins are localized to the apical region of the cellular plasma membrane where they undergo a process known as homo-dimerization — the formation of a single complex from two identical molecules — followed by a trans-phosphorylation reaction (where each half of the homo-dimer activates the other half via phosphorylation) that is crucial to the Hippo pathway's activation.
In the process, the Lai Lab also found that three upstream regulators — the proteins known as Merlin, Expanded, and Kibra — appear to work together to increase both the efficiency of the Hippo dimerization process and the recruitment of Hippo proteins to the cellular plasma membrane.
"Based on these observations," says Dr. Lai, "it becomes clear that Hippo dimerization and its subcellular localization, regulated in-part by the Merlin-Expanded-Kibra complex, work together to regulate Hippo kinase activity."
About the researchers
"This project was accomplished by three talented graduate students from three different graduate programs at Penn State," said Dr. Lai, "and while Yaoting Deng was responsible for most of the experiments, she and Yurika Matsui and Yifan Zhang worked admirably as a team to advance our understanding of the mechanism of Hippo activation for growth inhibition and tumor suppression."
Dr. Lai is Professor of Biology, Biochemistry and Molecular Biology at Penn State, Chair of the Huck Institutes' Intercollege Graduate Degree Program (IGDP) in Cell and Developmental Biology, as well as a faculty member of the IGDPs in Genetics, Molecular Medicine, and Neuroscience, and a researcher in the Center for Cellular Dynamics.