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Research

1. Proliferative signaling is tightly regulated by ubiquitination

As explained in the MISSION section, growth factors regulate cell proliferation in animals. When growth factors bind to their specific receptors on the cell membrane, the receptors are activated, and intracellular signaling occurs. In response to these signals, cells promote the cell cycle, replicate their DNA, and undergo mitosis. Excessive proliferative signaling can lead to tumor and cancer formation. Normal cells prevent this via various mechanisms, including “ligand-dependent receptor endocytosis.” In this mechanism, growth factors (ligands) bind to and thereby activate growth factor receptors, which are then internalized into intracellular vesicles (Fig. 1). The receptors then pass through the endosomes and are finally degraded by hydrolytic enzymes in the lysosomes. This mechanism reduces the number of receptors on the cell membrane and weakens the proliferative signals to some extent. When we first started our laboratory, we focused our research on ligand-dependent receptor endocytosis.

Ubiquitination is a process in which a small protein, called ubiquitin, binds covalently to other proteins and affects them in various ways depending on their functions. In the case of receptor proteins, ubiquitination serves to trigger their endocytosis (Fig. 2). Generally, the enzymes that link ubiquitin molecules to target proteins are called ubiquitin ligases. For example, the ubiquitin ligase designated as c-Cbl carries out the ubiquitination of growth factor receptors. These ubiquitinated receptors are then recognized by ubiquitin-binding proteins, such as Eps15 and Epsin. These proteins in turn contribute to the endocytic machinery that forms invaginations of the cell membrane around the receptors, creating intracellular vesicles. These vesicles then fuse to form endosomes. Subsequently, the receptors in the endosomes are recognized by the endosomal sorting complex required for transport-0 (ESCRT-0), which is made up of the ubiquitin-binding proteins designated as Hrs and STAM. We have investigated the function of this ESCRT-0 in detail and found that it is involved in the transportation of the receptors to the inner vesicles of endosomes (Komada & Kitamura, Mol. Cell. Biol. 1995; Komada et al., J. Biol. Chem. 1997; Komada & Soriano, Genes Dev. 1999; Mizuno et al., Mol. Biol. Cell 2003; Morino et al., Exp. Cell Res. 2004; Mizuno et al., J. Biochem. 2004; Komada & Kitamura, J. Biochem. 2005). Subsequent fusion of the endosomes with lysosomes results in the degradation of the receptors in the inner vesicles.

We also found another protein that interacts with ESCRT-0; namely, USP8, which acts as a deubiquitinating enzyme to remove the ubiquitin molecules from the receptors. The deubiquitination process promotes receptor recycling to the plasma membrane (Mizuno et al., Mol. Biol. Cell 2005; Mizuno et al., Traffic 2006; Komada, Curr. Drug Discov. Technol. 2008; Mukai et al., EMBO J. 2010). Collectively, the balance between the ubiquitination and deubiquitination processes regulates the availability of the receptors, thereby appropriately controlling the intensity of the proliferative signal (Fig. 2).

 

2. Abnormalities in ubiquitination lead to the development of neoplastic diseases

We predicted that an abnormality in the regulation of ubiquitination would result in excessive proliferative signaling, leading to the formation of tumors and cancer. Later, we discovered that mutations in the USP8 gene frequently occurred in the pituitary tumors that caused Cushing’s disease, a severe condition that is still incurable. The mutation rate of the USP8 gene in these tumors was found to be over 40%.

In Cushing’s disease, the pituitary tumor secretes an excessive amount of adrenocorticotropic hormone (ACTH), causing the adrenal glands to secrete an excessive amount of glucocorticoids in response similarly. Excessive glucocorticoids in the body can cause various symptoms, such as moon face, central obesity, diabetes, and hypertension, and the mortality of untreated patients is very high (Fig. 3). Unfortunately, there is no specific medicine to treat the disease; instead, the primary treatment is the surgical removal of the pituitary tumors, which requires a high level of skill.

We collaborated with a research group in Germany that had conducted a genetic analysis of this tumor. We found that gene mutations caused the excessive activation of USP8 and excessive proliferative signaling (Reincke et al., Nat. Genet. 2015; Perez-Rivas et al., J. Clin. Endocrinol. Metab. 2015; Theodoropoulou et al., Eur. J. Endocrinol. 2015; Hayashi et al., Eur. J. Endocrinol. 2016). Furthermore, the mechanism underlying the abnormal secretion of ACTH, a characteristic of Cushing’s disease tumors, is becoming more evident. We have also started translational research to apply our findings to develop the world’s first therapeutic drug for this disease. Thus, we are working with a sense of mission to bridge basic research results to clinical practice.

 

 

3. Ubiquitination is involved in the regulation of various cellular functions

In the ubiquitination reaction, the C-terminal carboxyl group of ubiquitin is linked to the amino group of the lysine residue (or N-terminal methionine residue) of the target protein by a peptide bond. In turn, the next ubiquitin can be linked to one of the seven lysine residues (or N-terminal methionine residues) of the previously linked ubiquitin. In this way, a “ubiquitin chain” can be formed through repeated reactions. In addition, it is possible to form chains with different linkage types, depending on the residue to which the next ubiquitin is linked. Thus, although most cellular proteins undergo ubiquitination, the structures of the ubiquitin chains are diverse because of the different number of ubiquitin molecules in the chain and the different linkage types formed.

Researchers were awarded the 2004 Nobel Prize in Chemistry to reveal that ubiquitination mediates the degradation of target proteins. Since then, other researchers have found one after another that ubiquitination has non-degradative functions as well. The underlying mechanism is becoming clear; ubiquitin chains with different structures bind to different ubiquitin-binding proteins, resulting in different molecular outputs.

Our research on receptor endocytosis had led us to enter the field of ubiquitin research. We have been investigating the functions of various ubiquitin ligases, ubiquitin-binding proteins, and deubiquitinating enzymes. We have shown that ubiquitination is involved in the regulation of a wide variety of cellular events, including insulin/insulin-like growth factor signaling (Fukushima et al., Nat. Commun. 2015; Fukushima et al., BBRC 2017), the formation of intracellular organelles such as stress granules and nucleoli (Endo et al., J. Cell Sci. 2009; Endo et al., J. Biol. Chem. 2009; Xie et al., J. Cell Sci. 2018), and the intracellular trafficking of collagen (Kawaguchi et al., BBRC 2018) (Fig. 4).

 

4. Discovery of a new protein that regulates cell proliferation in the placenta and mammary glands, tissues unique to mammals

In parallel, we have focused on some protein kinases and investigated their roles in regulating proliferative signals. One of them is Nik-related kinase (Nrk), which is expressed exclusively in the placenta and mammary glands, which are unique mammalian tissues. Nrk-deficient mice exhibit placental hyperplasia and frequently develop breast cancer, suggesting that Nrk plays a role in preventing excessive cell proliferation in the placenta and mammary glands (Denda et al., J. Biol. Chem. 2011; Yanagawa et al., Am. J. Pathol. 2016; Naito et al., FEBS Lett. 2020) (Fig. 5). We are now clarifying the detailed molecular mechanisms by which Nrk suppresses proliferative signals. Furthermore, our evolutionary analyses suggest that Nrk had undergone rapid molecular evolution during early mammalian evolution and may have acquired the function of suppressing proliferative signals.

The placenta and mammary glands develop rapidly during pregnancy and lactation, respectively. Nrk seems to have evolved as a mammalian-specific growth regulatory protein to control cell proliferation in these rapidly developing tissues appropriately. Mouse experiments have revealed that Nrk suppresses the development of breast cancer. However, further studies are needed to determine whether the same is true in humans. Assuming that Nrk suppresses the onset of breast cancer in humans, we may contribute to breast cancer treatment by developing substances that mimic the effects of this kinase.

 

5. Future research

Proliferative signals fluctuate depending on various environmental factors. For example, cells can adapt to nutrient deficiency by inhibiting proliferative signals, given that this environmental stress condition is not suitable for proliferation.

Proliferative signals also fluctuate depending on the developmental stage and physiological conditions in which that the cells are. For example, during lactation, various hormones induce proliferative signals in the mammary cells, causing mammary gland development. However, after lactation, the hormones decrease, the proliferative signals weaken, and the mammary glands regress. This mechanism allows cell proliferation to occur at the right time and in the right place.

On a long time scale, the regulatory mechanisms of proliferative signals seem to have evolved and been optimized for the lifestyle of each organism. A representative case is the molecular evolution of Nrk during early mammalian evolution.

Thus, the proliferative signals of animal cells fluctuate at various spatiotemporal scales. However, these signals' exact molecular mechanisms and biological significance are issues that remain to be clearly defined. We are trying to solve these issues using cell biology-, biochemistry-, molecular biology approaches, and the latest technologies such as proteomics, cell imaging, and bioinformatics.