One fundamental problem in biology is the question `what mechanisms restrict the size of animal tissues and organs?' This question is not only relevant to development but also applies after maturation, when many tissues and organs, such as the liver, continuously grow and diminish in order to regenerate. Although much progress has been made in understanding the genetic programs that control cell growth, cell division and cell survival, little is known about the mechanisms that sense and limit tissue and organ size. However, new insight into the underlying program is provided by a recent Cell article; a team of American scientists led by Duojia Pan reveals that the Hippo signalling pathway universally controls size in metazoan organisms.
The Hippo signalling pathway has been worked out recently in Drosophila and is a protein phosphorylation cascade, initiated by the activation of the Hippo (Hpo) kinase. The pathway ultimately phosphorylates and inactivates a transcriptional co-activator called Yorkie (Yki). As Yki promotes cell growth, survival and proliferation, its inactivation results in suppression of growth. But precisely how the Hippo pathway regulates Yki activity wasn't clear.
To answer this question, Pan's team expressed components of the Hippo signalling cascade together with a tagged version of Yki in Drosophila cells so that they could track the protein's location after phosphorylation. They found that Yki is excluded from the nucleus when Hippo signalling induces Yki phosphorylation, preventing it from accessing its target genes in the nucleus and suppressing cell growth.
Knowing that 14-3-3 proteins, involved in protein re-localization, bind to motifs containing phosphorylated serine or threonine residues, the team scanned Yki's amino acid sequence for 14-3-3 binding motifs. They found a potential binding site including serine 168 (Ser168) as a candidate for phosphorylation. In a series of immunological experiments they demonstrated that this serine is the only residue that is phosphorylated by the Hippo cascade and that 14-3-3 only binds Yki after phosphorylation.
The role of Yki phosphorylation in growth suppression was finally confirmed by in vivo studies using transgenic flies that produce a mutant Yki where Ser168 is replaced by an alanine. The mutant flies showed excessive tissue growth, showing that the Ser168 phosphorylation of Yki is needed to suppress tissue growth.
Having established a growth-suppressive signalling pathway in Drosophila, Pan's team wanted to know whether the Hippo cascade also plays a role in higher animals. Finding a conserved serine in the mammalian Yki homologue YAP suggested that this might be the target of a mammalian Hippo pathway. The team went on to dissect its possible role in mice and showed that the Hippo pathway leads to growth suppression caused by phosphorylation-dependent exclusion of YAP from the nucleus. Directly testing the role of Hippo signalling in mammalian organ size control, the scientists over-expressed YAP in the liver of a transgenic mouse line, where they could control gene expression by administering an antibiotic, and found a marked overgrowth of the liver tissue after inducing YAP expression. Interestingly,this effect was reversible, suggesting that the Hippo pathway not only controls organ size during development but also maintains the balance between growth and diminution in mature organs. In line with its role in size control,they finally found that misregulation of the Hippo pathway in transgenic mice leads to tumorgenesis.
Elucidating the Hippo pathway in Drosophila and mice has brought exciting insights into a universal mechanism controlling organ growth and size homeostasis. However, while many questions remain, the most important one is discovering the identity of the signal molecules that activate the Hippo pathway.
