Orkhon Tsogtbaatar, ID: 20088110 April 18, 2012 Hippo pathway Orkhon Tsogtbaatar, ID: 20088110 April 18, 2012

Hippo pathway discovered as a signal pathway that regulates organ size in Drosophila melanogaster. known as the Salvador/Warts/Hippo (SWH) pathway Its key signaling component — the protein kinase Hippo (Hpo). Mutations in this gene lead to tissue overgrowth, or “hippopotamus”-like phenotype Adult fly Mice liver at 2 months

Hippo pathway Composed of three components: 1) cell surface upstream regulators (including cell adhesion molecules and cell polarity complexes; 2) a kinase cascade (comprising two Ser-Thr kinases with regulators and adaptors) 3) a downstream target, a transcription coactivator (transcription of cell proliferation-promoting and anti-apoptotic genes). J. Biochem. 2011; 149(4):361-379

Fundamental questions What mechanisms are used to regulate growth to form organs of particular size? What mechanisms are used to tell the proliferating cells that the certain organs has reached its original intended size and that it is time to exit the cell cycle?

During the period of 1995-2005 several groups carried out genetic screens in fruit flies using tissue-specific genetic mosaics, to identify mutants that specifically affected growth. One of the first tumor-suppressor genes identified by such screens was named large tumor suppressor (lats) by Gerald Rubin’s group.

Warts/large tumor suppressor (wts/lats) lats mutant phenotypes

Warts/large tumor suppressor (wts/lats) A kinase of the nuclear Dbf-2-related (NDR) family

Loss of hpo results in tissue overgrowth

hpo regulates cell proliferation and apoptosis pupal eye 3rd instar eye disc

hpo encodes a protein kinase Has caspase cleavage site

Genetic interaction among hpo, sav, and wts GMR-hpo+wts 100% early pupal lethal

Hpo/Sav interaction promotes Wts phosphorylation Sav facilitates the phosphorylation of Wts by Hpo Hpo binds and phosphorylates Sav

Hpo - Sav - Wts Regulate cell proliferation By targeting key cell cycle regulators such as Cyclin E Regulate cell proliferation

mats required to restrict cell proliferation and promote apoptosis Mutation in mats cause cell overproliferation and tumor growth mats required to restrict cell proliferation and promote apoptosis

mats genetically interacts with wts ※ Mats and Wts synergistically interact to control cell proliferation and apoptosis

Hpo kinase cascade ※ Loss-of-function mutations in any of these genes result in overgrowth due to increased cell proliferation and growth, and reduced cell death. All found as drosophila tumor suppressors. Loss-of-function mutations in any of these four genes result in overgrowth due to increased cell proliferation and growth, and reduced cell death. Hpo and Wts are both Ser/Thr kinases that are activated by phosphorylation, and act sequentially within the pathway. Sav binds to both Hpo and Wts, and is thought to act as a scaffolding protein that links them together. Mats is an essential co-factor for Wts. Although multiple substrates for both Hpo and Wts kinases have been identified, the crucial target of Hpo is Wts, and the crucial target of Wts is a transcriptional co-activator protein, Yorkie (Yki). Yki functions as an oncogene, as it promotes cell proliferation and growth, and inhibits cell death.

Overexpression of yki drives outgrowth Yorkie was identified in a biochemical screen (yeast-two-hybrid) for Warts interacting proteins. Overexpression of yki drives outgrowth

Overexpression of yki promotes cell proliferation and inhibits apoptosis Activation of yki leads to increased cell proliferation and decreased apoptosis Activation of yki leads to increased transcription of diap1 and CycE In wild type eye discs, cells posterior to the MF undergo a synchronous second mitotic wave that can be revealed as a band of BrdU-positive cells. Few BrdU-positive cells are found posterior to the SMW, in yki-overexpressing clones, cells fail to unddergo cell-cycle arrest posterior to the SMW and continue cell cycles. Cell death is absent in yki-overexpressing clones, not in WT cells Cell-autonomous increase in DIAP protein (cell death inhibitor) levels in yki-overexpressing cells

The Hpo signaling pathway antagonizes the transcriptional coactivator activity of Yki

yki is genetically epistatic to hpo, sav, and wts ※ Yki is a target of Wts in Hpo pathway

Upstream regulators

※ merlin and expanded regulate tissue size

Merlin and Expanded act upstream of Hippo and Warts

fat (ft) encodes atypical cadherin, a transmembrane protein that is known to control cell proliferation ※ ft restricts organ size by modulating activity of the Sav/Wts/Hpo pathway

The Sav/Wts/Hpo pathway 14-3-3

Schematic model of the Hippo signaling network (Diap1, bantam, cycE, dmyc…) Cell cycle promoting and anti-apoptotic gene transcription

The hippo signaling network in Drosophila and Mammals normal > yki > YAP

Summary The pathway negatively regulates the coactivator to restrict cell proliferation and to promote cell death. Thus, the pathway prevents tissue overgrowth and tumourigenesis. A dysfunction of the pathway is frequently detected in human cancers and correlates with poor prognosis. Recent works indicated that the Hippo pathway plays an important role in tissue homoeostasis through the regulation of stem cells, cell differentiation and tissue regeneration.

Future Study How the activity of the Hpo kinase is controlled by upstream regullators? How the levels of Wts protein are controlleed by Dachs and Zyx? How all of the different inputs into the pathway are related to each other? How these diverse inputs are integrated to achieve appropriate levels of Yki activity? How the Hpo pathway is integrated with other growth control pathways? The Hippo Pathway Regulates Wnt/β-Catenin Signaling (2010) This work identifies a cytoplasmic role for the transcriptional coactivator TAZ TAZ, a Hippo pathway mediator, binds Dishevelled and inhibits Wnt signaling β-catenin is mislocalized in polycystic kidneys of TAZ null mice Disrupting the Hippo pathway in cell lines and flies modulates Wnt signaling

Thank you for attention!

Drosophila melanogaster, a Fruit Fly