tricornered (trc) encodes the Drosophila Ndr protein kinase (Nuclear Dbf2 related; Geng, 2000). The Ndr kinases are members of a subfamily of serine/threonine kinases that includes Sax1 (Caenorhabditis elegans), Cbk1 (Saccharomyces cerevisiae), Dbf2 (S. cerevisiae), Warts/Lats (Drosophila), Orb6 (Schizosaccharomyces pombe) and Cot-1 (Neurospora), which regulate cell growth, cell division and cell morphology. In S. cerevisiae Cbk1 and Dbf2/Dbf20 play central roles in the RAM (regulation of AceII activity and cellular morphogenesis) and MEN (mitotic exit network) pathways. Mutants of Cbk1 or other RAM pathway genes including its binding partners Mob2 and Tao3 (Pag1) fail to activate the AceII transcription factor in daughter cells and result in rounder than normal cells due to a defect in axial growth of the bud. Little is known about the in vivo function of the two human Ndr genes but extensive study of their biochemical characteristics has been carried out for nearly 10 years (He, 2005b and references therein).
The function of trc and its partner furry (fry) is required for the development of epidermal hairs, sensory bristles, arista laterals, and dendrite arborization (da) sensory neuron dendrites. The morphogenesis of these cell extensions involves the regulated activation of both the actin and microtubule cytoskeletons. Mutations in trc and fry result in split and multipled hairs and laterals, split and deformed bristles and dendrites with extra branching and tiling defects (He, 2005b and references therein).
The single warts/large tumor suppressor (wts/lats) gene is the Drosophila kinase most closely related to trc (45% identical and 65% similar over 418 amino acids). Once again there are two wts homologues in mammals. There is no wts ortholog in yeast. wts was first identified in Drosophila as a tumor suppressor. Homozygous wts/lats mutant cells display defects in morphogenesis (such as deformed bristles and altered cuticle morphology) and extensive overgrowths (He, 2005b and references therein).
Ndr, like many kinases, is regulated by phosphorylation. The phosphorylation of the activation segment site Ser-281 and the hydrophobic motif site Thr-444 of Ndr increase Ndr kinase activity in vitro. Ser-281 phosphorylation is thought to be due to autophosphorylation, whereas Thr-444 is targeted by an as yet unidentified upstream kinase. These sites are also important regulatory sites for Trc function in the Drosophila epidermis and nervous system. The mutation of these sites in trc to alanine results in dominant negative proteins (He, 2005b and references therein).
Several Ndr family kinases have been shown to function with members of the Furry protein family, which consists of large conserved proteins that lack informative motifs. The first member of this family to be characterized was the Drosophila fry gene. Both genetic and biochemical experiments have shown that in flies trc and fry function in a common process, are present together in a complex and that Fry is required for Trc kinase activity. Mutations in both result in similar phenotypes in both the epidermis and sensory neurons. In addition, the subcellular localization/accumulation of Trc and Fry is interdependent in pupal wing cells. The subcellular localization of Cbk1 and Tao3 in S. cerevisiae and Orb6 and Mor2 in S. pombe has also been found to be interdependent, although the relationships differ in these systems (Tao3 and Mor2 are the Fry homologues in these systems) (He, 2005b and references therein).
The Trc and Furry family proteins appear to be conserved both in terms of sequence and function in a wide range of eukaryotes. This suggests that homologues of other members of the RAM pathway in S. cerevisiae will also play similar roles in higher eukaryotes. The Mob2 protein of yeast has been shown to bind to Cbk1 and be essential for Cbk1 kinase activity (Weiss, 2002). In vivo Mob2 is required along with Cbk1 for both mother/daughter separation after cytokinesis and the maintenance of polarized cell growth (Weiss, 2002). Furthermore, Mob2 and Cbk1 show interdependent localization (Nelson, 2003). A similar situation exists for the related Dbf2 kinase, which is a component of the mitotic exit network (MEN). Dbf2 binds to Mob1 (which is related to Mob2) and this complex is essential for activity (Mah, 2001). Similarly, S. pombe Mob2 interacts physically with the Orb6 protein kinase and is required for Orb6 function in the coordination of cell polarity with the cell cycle (Hou, 2003). Multicellular organisms possess multiple mob genes. Recently, it was shown that a basic sequence within the insert in the catalytic domain of Ndr has an autoinhibitory function and that Human Mob1 may stimulate Ndr activity by releasing the autoinhibitory effect of this sequence (Bichsel, 2004; Devroe, 2004; He, 2005b and references therein).
There are 4 Drosophila genes related to the yeast mob genes. Evidence for a two-hybrid interaction between CG13852 (Dmob1/mats) and Trc was described in a genome scale experiment; however; no evidence for such interactions were seen between Trc and any of the other Drosophila Mobs (Giot, 2003). Nor was there any indication in that paper that any of the Drosophila Mobs function with the related Warts/Lats kinase. Evidence is provided that Mob1 (Mats), interacts with both trc and wts and that at least one additional member of the Dmob gene family CG11711 (Dmob2: FlyBase confusingly terms this gene Mob1) can interact with trc and wts. While this paper was in revision, Lai (2005) reported that CG13852 interacted with and activated Wts. They named CG13852 mats, and the He (2005b) study follows their lead and uses that name (He, 2005b).
The S. cerevisiae Cbk1 and Mob2 proteins are known to interact physically as do the S. pombe homologues (Orb6 and Mob2; Weiss, 2002; Hou, 2003). In addition the related Dbf2 and Mob1 proteins of S. cerevisiae also interact physically (Komarnitsky, 1998). Thus, it was expected that Trc and at least one of the Drosophila Mobs would interact physically. Indeed, Trc and Dmob1 (CG13582) were identified as interacting proteins in a genome scale two-hybrid screen (Giot, 2003). None of the other Dmobs were reported as being able to interact with Trc, and no Dmob was reported as interacting with Wts in that study (Giot, 2003), although a recent article (Lai, 2005) demonstrated an interaction between Wts and Mats (He, 2005b).
A yeast two-hybrid screen of a Drosophila cDNA library was performed using full-length trc cDNA as 'bait'. Most of the clear positive clones recovered contained fusions of segments of Dmob2 (FlyBase terms the Dmob2 gene Mob1) fused to the GAL4 activation domain. No clones were recovered of any of the other Dmobs. Perhaps they were not present in the library screened. To determine whether Mats and Trc interact in the yeast two-hybrid system, a cDNA clone for mats was obtained from the BDGP collection and it was subcloned into pGADT7. This plasmid was used and it was confirmed that Trc and Mats interact in the yeast-two-hybrid system. Thus, Trc appears to be able to interact with at least two different Mob family proteins in Drosophila. Similarly it was tested and confirmed that Wts is able to interact with both Mats and Dmob2 in the two hybrid system. Thus, no evidence was seen of specificity in the Drosophila NDR/Mob family interactions (He, 2005b).
To determine what portion of the Trc protein interacts with Dmobs, a set of plasmids was generated that contained trc C-terminal truncations, and they were assayed for an interaction with Dmob2 and Mats using the two-hybrid system. Similar but not identical results were obtained with these two mob family members. All of the Trc deleted proteins interacted strongly with Mats and the larger Trc proteins interacted strongly with Dmob2. However, Trc proteins that contained only amino acids 1-60 or 1-119 interact. The data for the binding of human Ndr1 to hMob1 indicates that important residues are found in the amino terminal region of Ndr1 (Bichsel, 2004). In hNdr, Tyr-31, Arg-41, Thr-74, and Arg-78 were found to be absolutely required for interaction, whereas the Lys-24, Arg-44, and Leu-79 mutants displayed reduced interaction (Bichsel, 2004). The corresponding residues in Trc are Tyr35, Arg45, Thr78, Arg81, Lys28, Arg48, and Leu82. Hence the data argue that for Mats binding to Trc requires only a subset of the residues needed in human Ndr1, whereas Dmob2 binding is enhanced by additional residues. The significance of these differences is not clear, but each of these examples is consistent with the amino terminal region of the NDR kinase family members being essential for the interaction with Mob family members (He, 2005b).
Because the protein kinase domain of Trc extends from residue 90-393, these results indicate that the kinase domain does not have to be intact for Trc to interact with Dmob2 or Mats. Mutations in the conserved regulatory phosphorylation sites, S292A+T453A did not interfere with the interaction. Thus it appears clear that the kinase activity of Trc is not important for its ability to bind to Dmob2. These results were similar to those seen (Komarnitsky, 1998) between kinase inactive Dbf2 and Mob1 (He, 2005b).
In S. cerevisiae Mob1 a number of sites have been identified as being important for the binding of Mob1 and Cbk1 (Luca, 1998). To test whether these sites are functionally conserved within the Dmob family, the sequence of the four Drosophila Mobs were aligned with yeast, human, and frog to identify the Dmob2 amino acids that correspond to the important sites in yeast Mob1 for interaction with Dbf2. Similar mutants in Dmob2 (RE70633) were generated to confirm the conservation of the Mob-Ndr interaction. Most of these mutations in Dmob2 disrupt the binding to Trc, consistent with the conclusion that the mechanism of interaction has been conserved. Most of the residues noted above are also conserved in Mats, Dmob3, and Dmob4, consistent with all Mob family members interacting with Ndr family members in the same manner (He, 2005b).
To assess whether these proteins were capable of associating in vivo in Drosophila cells, immunoprecipitation experiments were carried out in Drosophila S2 cells expressing both Trc and Dmob2. Trc was found in anti-Dmob2-8x HA, consistent with these two proteins interacting in vivo. As an additional test of these proteins interacting in vivo, the subcellular localization of Trc and Dmob2 protein was examined in wing cells. Trc distribution was examined with an anti-FLAG monoclonal antibody (Sigma) using UAS-trcWT and UAS-trcDN transgenes driven by ptc-GAL4. The proteins encoded by these transgenes carry an amino terminal FLAG epitope. It was found that the FLAG staining pattern of overexpressed Trc was the same as the endogenous Trc detected by anti-Trc antibody staining (He, 2005a). Dmob2 was localized in a similar way using a CFP tag because an anti-Dmob2 antibody was not available. Confocal microscopy demonstrated that before hair formation Trc is cytoplasmic and concentrated at the cell periphery. During hair outgrowth Trc accumulates in the hair, as is the case for the endogenous Trc (He, 2005a). Both before and after hair initiation Dmob-2 is localized similarly to Trc. The subcellular localization of these proteins is similar in flies that carry a single transgene or both UAS-trc and UAS-Dmob2 transgenes. It is concluded that Trc and Mob proteins can interact in vivo in Drosophila cells (He, 2005b).
Ten deficiencies from the 68C region were tested and the enhancing region was further mapped to a small interval (68C11-13) that contained CG11711 (Dmob2). Deficiencies from this region were able to similarly enhance the phenotypes that resulted from the directed expression of other dominant negative Trc proteins. The genome project annotation of Dmob2 suggests it is a complicated gene that encodes at least four variant mRNAs from exons that span >40 kb. These mRNAs encode four proteins with a common c-terminal segment but with different amino terminal regions. There are P insertions in a large intron of Dmob2, but these do not inactivate the gene to produce a mutant phenotype. Attempts to use imprecise excision to produce a deletion that would ensure that no Dmob2 protein could be made were not successful, because only small deletions were obtained that would eliminate one isoform. These did not produce a mutant phenotype. As an alternative approach transgenic flies were generated that carried UAS constructs that encoded either a tagged full-length Dmob2 (GH07469) protein or partial proteins aa 1-157 (Dmob2-N) and aa 148-354 (Dmob2-C) that might act as dominant negative proteins. The directed expression of the wild-type Dmob2 and Dmob2-N proteins by ap-GAL4 did not cause any notable visible phenotype. The interpretation of these results is limited by the fact that only one of 4 CG11711 isoforms was expressed. In contrast, overexpression of the common Dmob2-C protein segment resulted in a weak trc-like multiple hair cell phenotype and it also enhanced the dominant negative trc wing hair phenotype in a dose-sensitive way, consistent with Dmob2-C being a dominant negative and the normal function of Dmob2 being to activate Trc. In addition, overexpression of Dmob2-C caused an extra vein phenotype. This phenotype was enhanced by increasing the number of UAS-mob2c transgenes and it was also enhanced by heterozygosity for a deletion for CG11711. Thus, Dmob2-C acts as a dominant negative for this phenotype. A similar, but weaker vein phenotype was also seen when trcDN was overexpressed (He, 2005b).
Tissue growth and organ size are determined by coordinated cell proliferation and apoptosis in development. Recent studies have demonstrated that Hippo (Hpo) signaling plays a crucial role in coordinating these processes by restricting cell proliferation and promoting apoptosis. Mob as tumor suppressor protein, Mats, functions as a key component of the Hpo signaling pathway. Mats associates with Hpo in a protein complex and is a target of the Hpo serine/threonine protein kinase. Mats phosphorylation by Hpo increases its affinity with Warts (Wts)/large tumor suppressor (Lats) serine/threonine protein kinase and ability to upregulate Wts catalytic activity to target downstream molecules such as Yorkie (Yki). Consistently, epistatic analysis suggests that mats acts downstream of hpo. Coexpression analysis indicated that Mats can indeed potentiate Hpo-mediated growth inhibition in vivo. These results support a model in which Mats is activated by Hpo through phosphorylation for growth inhibition, and this regulatory mechanism is conserved from flies to mammals (Wei, 2007).
Two protein kinases Hippo [Hpo and Warts (Wts)/large tumor suppressor (Lats)], and a scaffold protein Salvador (Sav)/Shar-pei, are key components of this pathway. Moreover, two FERM-domain proteins, Merlin (Mer) and Expanded (Ex), function upstream of Hpo, and Mob as tumor suppressor (Mats), associates with Wts to stimulate the catalytic activity of the Wts protein kinase. Recently, both putative receptor and ligand that function further upstream of, or in parallel with, Hpo signaling have been identified (Hariharan, 2006). A major signal output of this growth inhibitory pathway is to inactivate a transcription coactivator Yorkie (Yki) via phosphorylation by Wts kinase. In addition to Cyclin E and Drosophila inhibitor of apoptosis 1 (diap1), the bantam microRNA is also found to be a target of the Hpo pathway. Most components in this emerging signaling pathway are conserved from yeast to flies and humans, suggesting that this pathway plays a fundamental role in cellular regulation (Wei, 2007).
The function of Mob proteins has been better studied in yeast, Drosophila and mammalian cells, which revealed a conserved property of Mob proteins as a binding partner as well as a coactivator of protein kinases of the Ndr (nuclear Dbf2-related) family (Hergovich, 2006b). As stated above, Drosophila Mats/dMob1 is required for mediating Hpo signaling by regulating Wts kinase activity in growth inhibition and tumor suppression. All four Drosophila mob genes dMob1-4 genetically interact with trc (tricornered) (He, 2005a), the fly Ndr homolog important for maintaining integrity of epidermal outgrowths and regulating dentritic tiling and branching (Emoto, 2004; He, 2005b). In the budding yeast Saccharomyces cerevisiae, Mob1 binds to and activates Dbf2/Dbf20 protein kinases for controlling mitotic exit and cytokinesis (Komarnitsky, 1998; Lee, 2001; Mah, 2001). Similarly, Mob1 is required for the activation of Sid2, an Ndr family kinase in the fission yeast Schizosaccharomyces pombe essential for cytokinesis (Hou, 2000; Hou, 2004). In human, hLats1 preferentially interacts with hMob1/hMats, but not hMob2 protein, and appeared to be required for promoting mitotic exit (Bothos, 2005), as well as cytokinesis (Yang, 2004). Importantly, the function of Mob proteins has been highly conserved in evolution. For instance, the human Mob1A/Mats1 protein has been shown to act as a kinase activator and can rescue the lethality and tumor phenotypes ofDrosophila mats mutants (Lai, 2005; Wei, 2007 and references therein).
Structural analysis of a human Mob1 protein, Mob1A/Mats1, revealed several important features of Mob family proteins (Stavridi, 2003). One is that several highly conserved residues are responsible for generating an atypical Cys2-His2 zinc-binding site, which is predicted to contribute to the stability of the Mob protein. Another striking feature is that there is a flat surface rich in acidic residues on one side of the protein. This property provides the structural basis for a Mob protein to interact with its partner, such as Ndr family kinases through electrostatic forces. Indeed, a 65-amino-acid region rich in basic residues exists in the N-terminal side of the kinase domain of Ndr family kinases, and alterations in the basic residues can prevent the kinases from binding to Mob proteins (Bichsel, 2004; Bothos, 2005; Hergovich, 2006b). Finally, hMob1A adopts a globular structure involving residues throughout the polypeptide. Mob proteins are small and usually do not carry any other structural motifs other than the Mob domain (Wei, 2007).
Although previous studies suggest that Ndr family kinases can be activated by upstream regulators such as Cdc15, Hpo and Mst kinases via phosphorylation in yeast, flies or human cells, very little is known about how Mob is regulated. Studies carried out in yeast and mammalian cells suggested that Mob proteins may be regulated through phosphorylation. For instance, yeast Mob1 was shown to be essential for the phosphorylation of Dbf2 by an upstream protein kinase Cdc15 and Mob1 itself was also phosphorylated by Cdc15 (Mah, 2001). However, the functional significance of this modification has not been elucidated. Work on human Mob1A/Mats1 also suggested that phosphorylation might provide a mechanism for regulating hMob1A activity (Bichsel, 2004). This study has tested a hypothesis that Mats is directly activated by Hpo kinase to regulate Wts kinase activity for growth inhibition and tumor suppression. Using the Drosophila system, it was found that Mats can be complexed with Hpo and is a target of the Hpo protein kinase. Similarly, human Mats1 is also a target protein of mammalian Mst kinases. Mats phosphorylation by Hpo increases its affinity with Wts protein kinase and ability to increase Wts activity to target Yki. Moreover, epistatic analysis suggested that mats acts downstream of hpo. Genetic analysis indicated that Mats functions together with Hpo for mediating growth inhibition of developing organs. Therefore, the Mob as tumor suppressor protein, Mats, functions as a critical component of the Hpo signaling pathway. The results support a model in which Mats is activated by Hpo through phosphorylation for growth inhibition, and this regulatory mechanism is conserved from flies to mammals (Wei, 2007).
Recent studies have defined an emerging growth inhibitory pathway mediated by Fat, Mer/Ex, Hpo/Sav and Wts/Mats proteins in tissue growth and organ size control in Drosophila. Previous work has shown that Mats functions as a coactivator of the Wts protein kinase (Lai, 2005). This study has focused on addressing how Mats is activated to regulate Wts kinase activity. Fenetic analysis suggests that Mats acts downstream of Hpo and is a critical component of the Hpo signaling pathway. Moreover, evidence is provided that Hpo-mediated phosphorylation increases Mats's activity as a coactivator of the Wts protein kinase, and this regulatory mechanism is conserved from flies to humans. Therefore, Hpo-mediated phosphorylation of Mats significantly contributes to Wts activation. In a simple model, Hpo needs to directly phosphorylate Wts as well as Mats in order for Wts kinase to be fully activated. Although both Wts and Mats are activated by Hpo-mediated phosphorylation, further investigations are needed to address how Hpo phosphorylation and Mats binding are coordinated for Wts activation (Wei, 2007).
This report provides evidence that Mats is a target of Hpo/Mst protein kinases and Hpo/Mst-mediated phosphorylation positively regulates Mats protein's coactivator activity for Wts protein kinase. Importantly, it was found that Mats exists as a phosphoprotein in living cells, indicating that Mats phosphorylation occurs under physiological conditions. In addition to Hpo/Mst, Wts kinase has also been shown to target Mats for phosphorylation (Lai, 2005), although the physiological effect of this modification has not been elucidated. In S. cerevisiae, the founding member of the Mob superfamily Mob1 was found to be a phosphoprotein and a substrate for the Mps1 kinase. Mob1 is also phosphorylated by an upstream regulator Cdc15 kinase (Mah, 2001). However, the role of Cdc15 in Mob1 phosphorylation has not been revealed even though Mob1 is known to be required for Cdc15-mediated activation of its binding partner Dbf2 kinase. In mammalian cells, protein phosphatase 2A inhibition by OA treatment caused phosphorylation of a Mob family protein (Moreno, 2001). Moreover, OA-induced modification on hMob1 was shown to be critical for its binding to its partner Ndr kinase (Bichsel, 2004). Thus, phosphorylation appears to be a common mechanism for Mob regulation (Wei, 2007).
Consistent with the finding that Mats is activated by Hpo via phosphorylation for upregulating Wts kinase activity, epistatic analysis suggests that Mats is acting downstream of Hpo. This is the first case that Ste20 family protein kinase-mediated phosphorylation of Mob is critical for regulating the catalytic activity of Ndr family protein kinase such as Wts. At this point, it is not clear how Mob proteins function to activate Ndr family kinases. Based on the results from recent studies of human Mob1 and Ndr family kinases, a potential mechanism is that Ndr family kinase is rapidly recruited by hMob1 to the plasma membrane for activation (Hergovich, 2005; Hergovich, 2006a). It is speculate dthat Hpo phosphorylation might facilitate Mats to associate to the membrane through an unknown mechanism, which in turn recruits Wts to the membrane as evidenced by the observation that Hpo phosphorylated Mats has an increased affinity to Wts. Subsequently, Wts is activated by phosphorylations mediated by protein kinases such as Hpo. Mats as a target of Hpo kinase, is able to associate with Hpo in a protein complex. Since Hpo/Mst1 kinase was not present in the Mats/Wts protein complex (Lai, 2005), it appears that Mats simultaneously cannot associate with Hpo and Wts in the same protein complex (Wei, 2007).
In addition to the membrane recruitment model, the data also support an active and more direct role of Mats in upregulating Wts kinase. From in vitro kinase assays, it was found that Hpo-mediated phosphorylation increases the affinity between Mats and Wts, as well as the ability of Mats to activate Wts kinase activity in the absence of any membrane structures. The results support a model in which Mats binding likely causes a conformational change of Wts for Wts activation. In the case of human Ndr kinase, an autoinhibitory effect of hNdr can be released by hMob1 binding (Bichsel, 2004), which presumably induces a conformational change in hNdr for its activation. Finally, it was found that Mats increases the steady level of Wts protein, which contributes to the increase in Wts activity. Further investigation is needed to understand how Mats is able to stabilize and/or increase the production of Wts protein (Wei, 2007).
Previous work has shown that Mats negatively regulates tissue growth by binding to another tumor suppressor Wts and subsequently activating the catalytic activity of Wts kinase (Lai, 2005). Since loss of mats function leads to tissue overgrowth and tumor development, it suggests that Wts alone is not sufficient to inhibit tissue growth in the absence of Mats. Therefore, Mats is an indispensable component of the Hpo pathway, and Wts activation is dependent not only on Hpo-mediated phosphorylation, but also on Mats binding. Further studies are needed to understand how exactly Wts activation is coordinated by Hpo phosphorylation and Mats binding. This work has provide evidence that Mats activation can be mediated by Hpo phosphorylation (Wei, 2007).
The Hpo signaling pathway plays an important role in growth inhibition and tumor suppression in Drosophila, and this pathway appears to be also critical for tissue growth control and tumor suppression in mammals. For instance, mammalian NF2 tumor suppressor is a homolog of Drosophila Mer and Ex proteins, which are upstream regulators of the Hpo signaling pathway. Moreover, loss of Lats1 function in mouse causes soft tissue sarcomas and ovarian tumors. Recently, it was found that hMats1 can functionally replace fly Mats to suppress tumor development, and Mats1 is mutated in mammalian tumors (Lai, 2005). Thus, mechanisms for the control of Hpo signaling might be commonly used across species, and understanding such mechanisms should provide insights into tumor development in mammals. As shown in this report, one mechanism by which Hpo functions to control tissue growth is to target Mats for phosphorylation, and, consequently, Mats is activated to upregulate Wts kinase. Because mammalian Hpo orthologs, Mst kinases, regulates hMats1 in a similar manner, this mechanism is likely used in mammalian cells as well. Therefore, by understanding how Hpo/Mst kinases regulate Mats and Wts/Lats in normal as well as tumor cells, valuable insights will be gained into tissue growth inhibition and tumor suppression (Wei, 2007).
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