Appropriate cell number and organ size in a multicellular organism are determined by coordinated cell growth, proliferation, and apoptosis. Disruption of these processes can cause cancer. Recent studies have identified the Large tumor suppressor (Lats)/Warts (Wts) protein kinase as a key component of a pathway that controls the coordination between cell proliferation and apoptosis. Growth inhibitory functions are described for a Mob superfamily protein, termed Mats (Mob as tumor suppressor), in Drosophila. Loss of Mats function results in increased cell proliferation, defective apoptosis, and induction of tissue overgrowth. Mats and Wts function in a common pathway. Mats physically associates with Wts to stimulate the catalytic activity of the Wts kinase. A human Mats ortholog (Mats1) can rescue the lethality associated with loss of Mats function in Drosophila. Since Mats1 is mutated in human tumors, Mats-mediated growth inhibition and tumor suppression is likely conserved in humans (Lai, 2005).

Individual Mob family proteins also interact with Tricornered (Trc), the Drosophila Ndr (Nuclear Dbf2-related) serine/threonine protein kinase that is required for the normal morphogenesis of a variety of polarized outgrowths including epidermal hairs, bristles, arista laterals, and dendrites. In yeast the Trc homolog Cbk1 needs to bind Mob2 to activate the RAM pathway. Genetic and biochemical data is provided that Drosophila Trc interacts with and is activated by Drosophila Dmob proteins, specifically Mats and Dmob2 (FlyBase terms the gene Dmob2 Mob1). Evidence is also provided that Drosophila Mob proteins interact with the related Warts/Lats kinase, which functions as a tumor suppressor in flies and mammals. In trc mutants the overall pattern of denticles is partly disorganized and many denticles are split. Split denticles are infrequent in wild-type larvae. The denticle pattern of mats mutant larvae is also disorganized and contains many split denticles. Interestingly, the overgrowth tumor phenotype that results from mutations in Dmob1 (mats) is only seen in genetic mosaics and not when the entire animal is mutant. Unlike in yeast, in Drosophila individual Mob proteins interact with multiple kinases and individual NDR family kinases interact with multiple Mob proteins; in particular, Mats interacts physically and genetically with trc and mats phenotype resembles that of trc. Notably, trc:mats double mutant larvae do not have a more severe phenotype than the single mutants. This lack of additivity argues that trc and mats function in a common pathway during denticle development. These observations also suggest that mats functions with both Trc and Wts (He, 2005b).

During normal development of multicellular organisms, appropriate cell number and organ size are determined by coordinated cell growth, cell proliferation, and apoptosis. Disruption or malfunction of these processes can cause diseases such as cancer. Using model organisms such as Drosophila melanogaster, genetic studies have helped identify novel molecules and pathways that are critical for regulating these processes. In particular, a pathway that involves Hippo (Hpo), Salvador (Sav)/Shar-pei, and Large tumor suppressor (Lats)/Warts (Wts) proteins has been shown to play a crucial role in tissue growth and cell number control (reviewed by Hay, 2003: Rothenberg, 2003; Ryoo, 2003; Lai, 2005 and references therein).

A critical role of hpo pathway in the linkage of cell proliferation and apoptosis was first elucidated through genetic studies, because hpo mutations result in increased tissue growth and impaired apoptosis. Like hpo, clones of sav or wts mutant cells acquire growth advantage compared to their wild-type neighboring cells and display reduced apoptosis. The hpo gene encodes a protein kinase highly related to mammalian Mst1 and Mst2 proteins. Hpo associates with and phosphorylates Sav scaffold protein, and association with Sav promotes Wts phosphorylation by Hpo. The Hpo-Sav-Wts pathway has been shown to regulate cell proliferation by targeting key cell cycle regulators such as Cyclin E; Cyclin E expression was elevated in the absence of hpo, sav, or wts function. Moreover, Hpo directly phosphorylates an apoptosis inhibitor DIAP1 and may regulate DIAP1 levels through degradation. Hpo may also negatively regulate diap1 at the transcriptional level. Thus, the Hpo-Sav-Lats pathway functions to coordinate cell proliferation and cell death by regulating the levels of key molecules required for cell cycle and apoptosis control. As an important component of this newly discovered pathway, wts encodes a serine/threonine protein kinase related to the NDR and Dbf2 kinases (reviewed by Tamaskovic, 2003). In particular, the putative kinase activity of Wts has been shown to be required to inhibit cell proliferation and induce apoptosis. However, it is not clear how the catalytic activity of Wts protein kinase can be directly regulated for growth inhibition and apoptosis promotion (Lai, 2005 and references therein).

The Drosophila compound eye was used to address how cell proliferation and apoptosis are coordinately regulated for the determination of cell number and organ size during development. Drosophila eye development has been extensively studied, which greatly facilitates functional analysis of genes and pathways involved in fundamental biological processes such as cell proliferation and apoptosis. This study describes tumor suppressor functions for a Mob superfamily protein Mats. Since the first Mob gene mob1 (Mps one binder 1) was identified in yeast (Luca, 1998), over 130 members in the Mob supergene family have been found in all major kingdoms ranging from protists to animals. However, functions of most mob genes remain poorly understood. Loss of mats function leads to increased cell proliferation and dramatic tumor growth in Drosophila. The results also suggest that mats is required to facilitate apoptosis during early eye development. Importantly, mats synergistically interacts with wts and appears to function with wts in a common pathway. Mats associates with Wts and functions as an activating subunit of the Wts protein kinase. It was found that a human Mats ortholog, Mats1, can functionally replace Drosophila Mats to suppress tumor and lethal phenotypes induced by mats mutations, and Mats1 loss-of-function mutations appear to occur in both human and mouse tumor cells. These results suggest that Mats-mediated growth inhibition and tumor suppression may be conserved in mammals such as humans (Lai, 2005).

As a unique group of the Mob superfamily, Mats orthologs exist in both plants and animals. Since Mats proteins are highly conserved, their function may be conserved across species. In support of this, human Mats1 was found to functionally substitute for mats in Drosophila. Importantly, loss-of-function mutations in Mats1 have been identified in a human skin cancer and a mouse breast tumor, suggesting that mammalian Mats genes may indeed act as tumor suppressors. Further molecular analysis of mammalian Mats genes from tumor tissues will be needed to test this hypothesis. On the basis of these data, it is speculated that all mats genes from animals and plants may negatively regulate cell number and tissue growth by restricting cell proliferation and promoting apoptosis (Lai, 2005).

Tumor suppressors normally act as inhibitors of cell proliferation or activators of apoptosis and use a variety of mechanisms in tissue growth suppression. This work provides evidence that mats functions to restrict cell proliferation and promote apoptosis in Drosophila. In this regard, functions of mats are similar to those of hpo, sav, and wts. Like hpo, sav, and wts, mats negatively regulates expression of CycE and DIAP1, two key regulators involved in cell cycle or apoptosis control. However, the overgrowth phenotypes of mats mutants appear to be stronger than those of hpo, sav, and wts and therefore cannot be explained simply by increased expression of Cyclin E and loss of apoptosis. It is suspected that mats might use other mechanisms to regulate cell number and organ size. For instance, Mats may negatively regulate cell cycle regulators such as Cdc25 protein phosphatase that are required for the G2-M transition. Since yeast Mob1 is able to form a complex with Mps1 (Mono polar spindle 1) kinase, Mats may also play a role in the spindle assembly checkpoint by acting together with Mps1. Mps1 has been previously shown to be involved in the spindle assembly checkpoint in yeast, and Mps1 is also implicated in this process in vertebrate cells. Involvement of Mats in the spindle assembly checkpoint would help explain the dramatic overgrowth phenotypes of mats mutants. Clearly, further investigations are needed to test these hypotheses (Lai, 2005 and references therein).

Consistent with a model that Mats functions as a critical component of the Hpo-Sav-Wts pathway, the data show that Mats associates with Wts to form a protein complex. Supporting this, crystal structure analysis of human Mats1/Mob1A reveals that several evolutionarily conserved acidic residues are exposed on the surface to provide a strong electrostatic potential for mediating protein-protein interactions (Stavridi, 2003). Based on this finding, Mats binding regions are expected to be basic and indeed such regions do exist in Wts family proteins. It remains to be addressed as to how exactly Mats interacts with Wts and whether the Mats-Wts complex can be associated with Hpo and Sav. Excitingly, it was found that Mats functions as an activating subunit to stimulate Wts kinase activity. In this way, Wts activation can be effectively controlled by the availability of Mats protein through differential distribution of Mats in different tissues, cells, or subcellular locations. With Mats acting as an activator of Wts kinase, the relationship between Mats and Wts mimics that of Cyclin and Cyclin-dependent kinases, which are essential for cell cycle control (Lai, 2005).

How does Mats association lead to Wts activation? In a model, association with Mats may allow Wts to undergo an allosteric conformational change critical for Wts activation or to simply relieve an autoinhibition of Wts. Interestingly, the N-terminal region of Wts was shown to be able to associate with its C-terminal kinase domain through intramolecular binding, and this interaction may be inhibitory for the Wts kinase activity. Thus, association with Mats may activate Wts by disrupting this intramolecular binding within Wts. In the case of human Ndr kinase, an autoinhibitory sequence has been identified and binding of the hMats1/hMob1A protein induces a release of this autoinhibition (Bichsel, 2004). In another model, Mats association may allow the Mats-Wts complex to recruit additional coactivators or to prevent coinhibitors from being recruited in order for Wts to be activated. Clearly, any model of Wts activation would have to consider the effect of Wts phosphorylation. (1)Wts has been shown to be phosphorylated in a cell cycle-dependent manner. Because Wts kinase activity can be increased through treatment of phosphatase inhibitors, phosphorylation appears to be critical for Wts kinase activity. (2) The Drosophila homolog of C-terminal Src kinase (dCsk) gene has been shown to genetically interact with wts to inhibit cell proliferation, and dCsk phosphorylates Wts in vitro. (3) Human Wts2 is a phosphorylation target of Aurora-A kinase, and this phosphorylation plays a role in regulating centrosomal localization of hWts2. (4) Hpo can directly target Wts for phosphorylation, and this event is facilitated by Sav. At present, it is unclear how Mats may affect Wts phosphorylation by Hpo or how Mats-Wts complex may be regulated by Hpo through phosphorylation. In yeast, Mob1 is essential for the phosphorylation of Dbf2 kinase by an upstream kinase Cdc15. Further studies on Wts phosphorylation are expected to provide a better understanding of how Wts is regulated (Lai, 2005).

While functions of most Mob superfamily proteins are still poorly understood, this work on Mats supports that a common feature of Mats proteins is to function as coactivators of protein kinases such as Wts. Identification and functional studies of Mats have revealed a mechanism for the control of Wts tumor suppressor activity. Because Mats-mediated growth inhibition and tumor suppression appear to be evolutionarily conserved, it extends the understanding of tissue growth and cell number control during development and tumorigenesis and raises the possibility that Mats-dependent growth inhibition may have important implications for the understanding and treatment of human cancers (Lai, 2005).

Trc, Dmob, and Fry Function in a Common Signaling Pathway

Previous genetic data pointed out the importance of tricornered (trc) and furry (fry), encoding a large conserved protein with multiple isoforms, for the morphogenesis of polarized cellular extension. Based on homology to yeast regulatory pathways involving homologs of trc, furry and mob, it seemed likely that one or more of the Drosophila mob genes would function along with trc and fry. Evidence was found supporting this hypothesis but the results are complicated by both pleiotropy and redundancy. This was illustrated most clearly in experiments with mats. Mutations in mats displayed phenotypes that were typical of both trc (split denticles and multiple hair cells) and of wts/lats (tumors, bulged cells, advanced hair differentiation). Mats can also interact with both Trc and Wts as detected using the yeast two-hybrid system. These observations stand in contrast to the situation in yeast, in which individual mob genes show specificity for individual Ndr family members. Further evidence for redundancy comes from the gene dosage interactions seen between trc and the other Dmobs (He, 2005b).

Evidence for a direct physical interaction has been reported for Ndr and Mob family members from yeast, flies and mammals (Colman-Lerner, 2001; Mah, 2001; Weiss, 2002; Hou, 2003; Bichsel, 2004; Lai, 2005). Previous yeast two-hybrid experiments showed evidence for a physical interaction between Trc and Mats (Giot, 2003) and Mats and Wts (Lai, 2005). The results of this study extended these observations by showing a similar interaction between Trc and Dmob2 by both two hybrid and coimmunoprecipitation experiments and that Wts and Dmob2 interact in the two hybrid system. Residues known to be important for the interaction between yeast Mob1 and Dbf2 are also important for the interaction between Trc and Dmob2. The conservation of many of these residues in Dmob3 and Dmob4 suggests that these proteins will also interact with Trc. The genetic interactions seen between trc and Dmobs suggest that the binding of Dmobs to Trc is essential for in vivo function and activation of the protein. Consistent with this hypothesis it was found that Dmob2 and Trc colocalized to growing hairs in pupal wing cells (He, 2005b).

The observation that for two phenotypes (pupal wing cell cross section and time of hair initiation) mats and wts clone cells share a similar phenotype that is the opposite of trc is intriguing and needs to be reconciled with the positive gene dose interactions seen between mats and trc for the sensitized multiple wing hair cell assay and the similar denticle phenotype. Given this complexity it seems unlikely that a single simple mechanism is involved. Because Mats appears to function along with both Trc and Wts, some of the complexity may reflect interactions between these two kinase modules. Cells mutant for mats could have both modules inactive, although it is possible that the degree of possible mats redundancy might not be equivalent for the two modules. In principle these two modules could function in parallel or one could be upstream of the other. The observation that mats and wts clones have increased Fry accumulation in hairs is consistent with mats/wts being upstream of trc/fry/mats(mob). Because increased Fry accumulation in hairs is also seen in trc mutant cells, this hypothesis is also consistent with the positive gene dosage interactions. However, a different explanation is needed to explain the observation that with regard to cell size and the timing of hair initiation the mats/wts phenotype is opposite to that of trc/fry. If both modules are considered to be equally inactivated in a mats cell, then these latter observations suggest that trc/fry/mats could function antagonistically and upstream of mats/wts. In this situation a lack of trc function would result in increased wts function (and increased cell size and delayed hair formation). A lack of wts function would result in the reduced cross section and advanced hair morphogenesis. In a mats mutant a reduction is expected in both trc and wts function. This would result in a wts-like phenotype because a lack of trc inhibition of wts would be of no consequence in cells that already lack wts activity. However, this model does not explain the multiple hair cell interactions. Given the difficulties in any single model it is suggested that the interactions are context dependent and/or the two modules function entirely in parallel (He, 2005b).

The tumor phenotype of mats

The 'tumors' produced by mats clones were characterized by altered cell shape and proliferation. The altered cell shape could be seen in the cuticle of clone cells. The altered proliferation of clone cells was associated with them outcompeting neighboring cells so that, in wings in which recombination was induced at a high level using vg-Gal4 and UAS-flp, most cells in the wing were mutant and the wing was grossly larger than normal. These observations are very similar to those seen in wts/lats mutant clones. It is unclear how the altered cell shape and proliferation are related. Excess proliferation of a clone of cells that is surrounded by slower growing normal cells is expected to lead to compression of the faster growing clone cells and their immediate neighbors. This could be responsible for the decreased cross sectional area of mats and wts cells and their bulged apical surface. However, if compression is responsible for the change in cell shape, it would be expected that this change would smoothly spread into the surrounding wild-type cells and would be more severe in the center of clones than near their periphery. This does not appear to be the case although this issue deserves further study. The cells in entirely mutant discs also appeared to have a bulged shape, which further argues against excess growth-mediated compression being responsible for the cell shape changes (He, 2005b).

The tumors produced by mats clones and their ability to outcompete neighbors suggests the possibility that mats mutant cells grow and/or divide more rapidly than normal. Thus, it was surprising when it was found that discs in mats homozygous and hemizygous mutants are smaller than normal. This could be due to the mutant larvae being impaired in feeding, digestion, or absorption of nutrients. This could lead the disk cells to be effectively starved reducing their growth. Alternatively it could be due to the mutation resulting in a defect in the secretion of a growth factor. It remains possible, however, that the overgrowth requires the direct contact of mats mutant and wild-type cells (He, 2005b).

GENE STRUCTURE

cDNA clone length - 1268

Bases in 5' UTR - 418

Exons - 3

Bases in 3' UTR - 190

PROTEIN STRUCTURE

Amino Acids - 219

Structural Domains

BLAST searches identified four Drosophila genes that shared similarity to the yeast mob1 and mob2 genes: CG13852 (mats or Dmob1), CG11711 (Dmob2: FlyBase terms this gene Mob1), CG4946 (Dmob3), and CG3403 (Dmob4). The BLAST analysis and additional clustering analyses did not allow identification of one or two of the fly genes as being clear orthologues of either yeast mob1 or yeast mob2. CG13852 and CG4946 are the two most closely related to both yeast genes, and CG3403 is the most distantly related. Gene chip analysis of pupal wing RNA showed all four of the fly mob genes are expressed in the pupal wing before, during, and after hair morphogenesis (He, 2005b).

date revised: 21 December 2005

Home page: The Interactive Fly © 1995, 1996 Thomas B. Brody, Ph.D.

The Interactive Fly resides on the
Society for Developmental Biology's Web server.