# Introduction

Many pathways have been evolved from metazoans, or animals, that regulate organ as well as organism size. The main pathway that is described here is the Salvador-Warts-Hippo pathway, known more commonly as Hippo. Scientists have done research on the Hippo pathway for the past 8 years and have noticed a complex network of signals and levels of modularity. There is a large amount of the WW module in the core kinase of Hippo, the components of upstream regulation as well as the nuclear proteins involved in the downstream movement. This WW domain can help predict new pathways and their components.

# History and Description of the Hippo Pathway

The Hippo pathway, abbreviated as Hpo pathway, was discovered about a decade ago. From observing Drosophila tissues that had mutations, scientists discovered that there was a set of genes that could be isolated where the mutated tissues could grow to larger sizes. The proteins that encode these genes were included in two kinases, the Hpo and Warts (Wts), as well as a protein involved in scaffolding known as Salvador (Sav). These three proteins interacted with one another and made up the core composition of the Hippo pathway that will be later referred to as Hpo kinase cassette. The pathway also included a nuclear effector called Yorkie, a downstream transcription factor called Scalloped, and upstream regulators such as receptors of membrane spans. The Hpo pathway is mostly found in flies and mammals. Hpo pathways have also been researched in the fields of homeostasis, stem cells and cancer.
Even when it was first discovered up until today, the Hpo pathway has been quite the topic of research. The Hpo pathway has been determined as a tumor suppressor pathway that can restrict the growth of cancer cells and promote apoptosis or cell death of these unwanted cells. Hpo kinase cassette can be regulated by many upstream regulators or proteins. An example of an upstream regulator protein is the complex of membrane span receptors, Ft and Ds. The external areas of these proteins can be exerted onto cells that are next to the proteins and the cell and protein can interact. Once a downstream signal is activated, the protein is stabilized and the cell becomes localized. The Ft protein can be cleaved into two fragments with one fragment homodimerizing and the other fragment heterodimerizing. Ft, however, opposes a myosin protein that activates Yorkie, the nuclear effector and destabilizes the Wts (Warts protein) which is one of the major components of the Hippo pathway. There are other proteins that can act similarly to Ft and Ds. These are known as Kibra, Ex and Merlin (Mer) which make up the KEM complex. Kibra is involved mainly in the WW domain, which is a domain that consists of two tryptophans that can bind peptides that are abundant in proline. On the other hand, Ex and Mer are involved in the FERM domain, which is involved in localizing proteins to the plasma membrane. These proteins, Kibra, Ex and Mer can be localized in the apical junction of flies (Drosophila) and their epithelial cells. The proteins can also interact with one another, as suspected. The KEM complex, however, can use part of the Hpo pathway to activate its proteins towards the apical membrane of the fly. KEM complex proteins can also interact or signal proteins that are involved in the upstream pathway of Hpo such as Ft and Ds.
Ultimately, the Hpo pathway has four core proteins: Hpo, Wts, Sav and Mats and are considered the conserved Hpo kinase cassette. Hpo and Wts are kinases that are attracted to serine and threonine. Their enzymes can be activated by phosphorylation. Also, autophosphorylation can activate Hpo which then phosphorylates Wts, Sav and Mats in their respective order. Sav is a protein that can scaffold or assemble Hpo and Wts together in order to facilitate the process of phosphorylation of Wts by Hpo. This Hpo core kinase cassette is conserved in eukaryotes ranging from yeast to humans.
Hpo pathways depend on their nuclear effectors which are different for flies and humans. For flies, the effector is Yorkie, but for humans the effectors are YAP (Yes kinase-associated protein) and TAZ (transcriptional coactivator with PDZ-binding, also known as WW domain containing transcription regulator (WWTR1)). Yorkie can be phosphorylated by activating Wts in flies; meanwhile YAP can be phosphorylated by LATS 1/2 which are large tumor suppressor kinases. Because Yorkie and YAP are transcriptional co-activators, they can be inhibited by mediated cytoplasmic retention when they are phosphorylated. LATS 1/2 phosphorylation of YAP begins at the serine end of the molecule and recruits a ligase that triggers the degradation of YAP. When Yorkie is not in the cytoplasm, it can activate transcription factors such as Sd, Homothorasx (Hth), and Teashirt (Tsh). In humans, YAP can activate Sd orthologues in the TEA domain and the internal region of a mammalian homolog of avian erythroblastosis virus oncogene receptor.

# Other Regulators of the Hippo Pathway

The Merlin (Mer) protein is one regulator that can shuttle from the cytoplasm to the nucleus and Mer can then exert signals that suppress proliferation of cells. This can have a function in the KEM complex. The Hpo pathway in humans was suspected to possibly be regulated by Merlin just as expected in flies. However, there was no regulation by Merlin in humans and the MST2 signal was never present in the extra cellular signal regulated kinase pathway. MST2, derived from Merlin, might not act as a tumor suppressor but just as a positive regulator of proliferation unlike the Hpo proteins.

# Modularity

Modularity means that homologous structures are being reused by individuals as well as species. A module is not only a structure but can also be a process or pathway that is characterized by internal integration. The WW domain mediates protein interactions and can be used by components of the Hpo pathway.

# WW domain

This domain is quite small, and probably one of the smallest modular protein domains existing. The domain has a pattern of imperfect repitition of 38 amino acids in a spliced isomeric form of YAP called YAP2. These 38 amino acids were added by the process of splicing to YAP, which already had a copy of the semiconserved sequence. This sequence showed that there were two conserved tryptophans that were about 22 amino acids apart. There have been approximately one hundred WW domains identified in humans. This domain can fold as a compact beta sheet and is stable when there are no ligands or cofactors. Some ligands can be present causing instability. These ligands were proline rich. Furthermore, there is an upstream complex that is made of kibra and merlin in humans but known as KEM in flies that can integrate signals that have not been categorized into the Hpo kinase cassette. There are many modular protein domains that can determine the Hpo pathway and its components.

# Reference

Sudol, M. and Harvey, KF. Modularity in Hippo signaling pathway. Trends Biochem Sci. 2010, Nov. 35(11):627-33. Epub 2010 Jul 2. Review.