InteractiveFly: GeneBrief

puffyeye: Biological Overview | References

The essential and highly conserved role of Myc in organismal growth and development is dependent on the control of Myc protein abundance. It is now well established that Myc levels are in part regulated by ubiquitin-dependent proteasomal degradation. Using a genetic screen for modifiers of Drosophila Myc (dMyc)-induced growth, this study identified and characterized a ubiquitin-specific protease (USP), Puffyeye (Puf), as a novel regulator of dMyc levels and function in vivo. puf genetically and physically interacts with dMyc and the ubiquitin ligase archipelago (ago) to modulate a dMyc-dependent cell growth phenotype, and varying Puf levels in both the eye and wing phenocopies the effects of altered dMyc abundance. Puf containing point mutations within its USP enzymatic domain failed to alter dMyc levels and displayed no detectable phenotype, indicating the importance of deubiquitylating activity for Puf function. dMyc induces Ago, indicating that dMyc triggers a negative-feedback pathway that is modulated by Puf. In addition to its effects on dMyc, Puf regulates both Ago and its cell cycle substrate Cyclin E. Therefore, Puf influences cell growth by controlling the stability of key regulatory proteins (Li, 2013).

The mammalian Myc gene family, comprising Myc, Mycn and Mycl, is known to be crucial for growth and development. Myc proteins control multiple cellular processes, including cell growth, proliferation, metabolism and apoptosis, and deregulation of Myc plays an important role in oncogenesis (Dang, 2012). Non-mammalian Myc has been most intensively studied in Drosophila melanogaster where the absolute requirement for Drosophila Myc (dMyc) (dm -- FlyBase) function during development has been demonstrated by the fact that dMyc-null mutants die at an early larval stage (Li, 2013).

Myc transcript and protein abundance are subject to regulation at multiple levels ranging from transcriptional control by numerous mitogenic signaling pathways to extensive post-translational modifications. Of particular interest, given the relatively short half-life of Myc proteins, is the post-translational modification of Myc by the ubiquitin system (Müller, 2009; Thomas, 2011). Protein ubiquitylation is a fundamental and versatile post-translational modification that controls multiple cellular events by marking proteins as substrates for either degradation or non-degradative processing. In mammals, distinct ubiquitin E3 ligase complexes, including Skp2 and Fbw7, have been reported to influence Myc protein stability and activity (Li, 2013).

The Drosophila ortholog of Fbw7, Archipelago (Ago) is the only ligase identified thus far as involved in proteasome-mediated ubiquitin-dependent turnover of dMyc proteins (Moberg, 2004). Ago mutant alleles were first identified in a genetic screen for regulators of tissue growth in the eye, where it was initially shown to bind and regulate Cyclin E (CycE) levels (Moberg, 2001). Later work demonstrated that Ago also physically interacts with dMyc, and controls dMyc stability and biological function (Moberg, 2004). Unlike c-Myc, which was shown to have a single Myc BoxI phosphodegron associated with Fbw7 binding, several domains containing putative Ago-interacting motifs were shown in dMyc to mediate Casein kinase 1 (CK1)α-, CK1ε- and GSK3β-dependent protein degradation. Although their link to Ago function has not been precisely established, it is clear that GSK3β plays a key role in Ago-mediated dMyc ubiquitylation and degradation (Galletti, 2009; Moberg, 2004; Parisi, 2011; Li, 2013 and references therein).

Protein ubiquitylation is a reversible process in which removal of ubiquitin chains is mediated by deubiquitylating enzymes (DUBs), and the role of DUBs in controlling various cellular processes has attracted considerable interest (Clague, 2012; Reyes-Turcu, 2009). DUBs are classified into five subfamilies based on their deubiquitylating domain. Ubiquitin-specific proteases (USPs), which constitute the largest DUB subfamily, share a structurally conserved USP domain of ~350 to 450 amino acids. The USP domain is the catalytic core that mediates the cleavage of ubiquitin conjugates, whereas domains required for protein-protein interaction and substrate specificity are located within N and/or C termini of the USP protein (Komander, 2009; Ventii, 2008). Although several ubiquitin E3 ligases have been implicated in modulating c-Myc stability, only one deubiquitylating enzyme, USP28, has been demonstrated to catalyze the deubiquitylation of Myc in mammals (Popov, 2007a). Thus far, no deubiquitylating enzyme has been identified that modulates dMyc function or antagonizes Ago-mediated dMyc degradation. Of the 41 predicted Drosophila DUBs, 21 are predicted to have a mammalian USP ortholog (Tsou, 2012). Interestingly, Drosophila does not encode an USP28 ortholog, suggesting that a distinct USP may be responsible for reversing dMyc ubiquitylation in Drosophila. This study reports the identification and characterization of Puffyeye (Puf), a Drosophila USP that antagonizes Ago function and interacts genetically and physically with dMyc. Evidence is presented that Puf regulates dMyc activity at the level of cell and organ growth (Li, 2013).

Although a great deal has been learned recently concerning ubiquitin ligases that interact with Myc proteins (Müller, 2009), to date only one DUB has been reported that targets Myc (Popov, 2007b). This study has employed a genetic screen based on the rough eye phenotype induced by dMyc overexpression in the eye (GMM) in Drosophila. This screen led to the identification of a USP-type DUB, which was named Puffyeye (Puf; CG9754), as a novel regulator of dMyc function in vivo. Reduced puf expression suppresses, whereas puf overexpression augments, the GMM phenotype. This phenotype is largely an effect of cell overgrowth (Secombe, 2007), yet overgrowth in the eye due to cyclin D/Cdk4 was not influenced by altered Puf abundance. Moreover, knockdown of four other USPs had no effect on the GMM phenotype. This suggests that puf possesses specificity for dMyc-induced growth in the eye. Indeed, puf itself induced a dose-dependent rough eye phenotype, displaying augmented ommatidial size that can be modulated by altering dMyc levels. In the wing disc, dMyc and Puf also were found to collaborate in cell growth. It was also found that Puf is essential for normal development, consistent with a crucial role for Puf in cell growth (Li, 2013).

dMyc levels markedly increase in cells in which puf is overexpressed, whereas dMyc levels are decreased in Puf hypomorphic mutants. These changes in dMyc are predominantly post-translational. This is consistent with the finding that Puf overexpression results in a dramatic increase in dMyc protein stability. Importantly, all of the biological effects of Puf, as well as its effects on dMyc abundance and turnover, are abolished by point mutations in the highly conserved Puf USP catalytic domain. It is surmised that Puf stabilizes Myc through its function as a deubiquitylating enzyme that antagonizes the activity of the Ago ubiquitin ligase, previously shown to target Myc for ubiquitylation and degradation (Moberg, 2004). Importantly, increased Puf exacerbates, and decreased Puf suppresses, the effect of Ago heterozygotes in enhancing the GMM phenotype. The notion that Puf and Ago act as antagonists receives further support from the findings that Puf protein physically associates with both dMyc and Ago in vivo. Interactions between DUBs and their antagonistic E3 ligases, as well as their substrates have been reported previously (Popov, 2007b; Sowa, 2009). The ability of both the Puf short and long isoforms to modify the dMyc-mediated eye phenotype, and stabilize dMyc and Ago proteins in an ubiquitylation domain-dependent manner suggests that domain(s) required for Puf to interact with dMyc or Ago are located in a region N-terminal to the core catalytic domain (Li, 2013).

It was also found that Puf stabilizes CycE, another known Ago substrate, suggesting that Puf antagonizes Ago function in regulating other targets that are crucial for cell cycle control. Indeed, flies homozygous for puf and ago double mutations do not survive, raising the possibility that, in addition to regulating common substrates, they each possess unique targets, as shown for other ubiquitin ligases and DUBs. Notch would be another potential candidate for Puf activity (Moberg, 2004); however, no significant effect of Puf on Notch levels was found in wing discs. In mammalian cells, the ubiquitin-specific protease USP28 was demonstrated to regulate the turnover of c-Myc by binding and antagonizing the activity of Fbw7α, the vertebrate ortholog of Ago (Popov, 2007b). However, Puf and USP28 are not homologs: they appear to be two very distinct USPs in terms of their overall size and amino acid sequence similarity in both their core enzymatic domains and the protein sequence as a whole. The closest mammalian homolog of Puf is USP34 (3546aa). Puf and USP34 are highly homologous in their core catalytic domains (67% identity; 80% similarity) with the catalytic triad conserved, whereas the overall similarity between the two proteins is ~52% (~37% identity) (Li, 2013).

Previous studies have shown that multiple signaling pathways regulate Ago and Fbw7 expression and activity (Nicholson, 2011). This study found that Ago levels are increased upon dMyc, as well as upon Puf overexpression. Although the mechanisms by which dMyc and Puf regulate Ago expression are unclear, dMyc-dependent Ago expression may provide a mechanism for dMyc autoregulation, whereas Puf may stabilize Ago by deubiquitylating it. Indeed, Fbw7 has been shown to be regulated through ubiquitylation (Min, 2012). A similar type of dynamic relationship has been reported for the ubiquitin ligase Mdm2 and deubiquitylase HAUSP/USP7 in regulating the stability and function of the tumor suppressor p53 (Brooks, 2011). Taken together, these data suggest that Ago and Puf represent a regulatory node that controls degradation of Myc and CycE, and very likely other growth control factors. Further studies will be required to identify additional substrates of Puf and to understand the physiological importance of Puf-mediated regulation of protein degradation in Drosophila (Li, 2013).

Search PubMed for articles about Drosophila Puffyeye

Brooks, C. L. and Gu, W. (2011). p53 regulation by ubiquitin. FEBS Lett 585: 2803-2809. PubMed ID: 21624367

Clague, M. J., Coulson, J. M. and Urbe, S. (2012). Cellular functions of the DUBs. J Cell Sci 125: 277-286. PubMed ID: 22357969

Dang, C. V. (2012). MYC on the path to cancer. Cell 149: 22-35. PubMed ID: 22464321

Galletti, M., Riccardo, S., Parisi, F., Lora, C., Saqcena, M. K., Rivas, L., Wong, B., Serra, A., Serras, F., Grifoni, D., Pelicci, P., Jiang, J. and Bellosta, P. (2009). Identification of domains responsible for ubiquitin-dependent degradation of dMyc by glycogen synthase kinase 3beta and casein kinase 1 kinases. Mol Cell Biol 29: 3424-3434. PubMed ID: 19364825

Komander, D., Clague, M. J. and Urbe, S. (2009). Breaking the chains: structure and function of the deubiquitinases. Nat Rev Mol Cell Biol 10: 550-563. PubMed ID: 19626045

Li, L., Anderson, S., Secombe, J. and Eisenman, R. N. (2013). The Drosophila ubiquitin-specific protease Puffyeye regulates dMyc-mediated growth. Development 140: 4776-4787. PubMed ID: 24173801

Min, S. H., Lau, A. W., Lee, T. H., Inuzuka, H., Wei, S., Huang, P., Shaik, S., Lee, D. Y., Finn, G., Balastik, M., Chen, C. H., Luo, M., Tron, A. E., Decaprio, J. A., Zhou, X. Z., Wei, W. and Lu, K. P. (2012). Negative regulation of the stability and tumor suppressor function of Fbw7 by the Pin1 prolyl isomerase. Mol Cell 46: 771-783. PubMed ID: 22608923

Moberg, K. H., Bell, D. W., Wahrer, D. C., Haber, D. A. and Hariharan, I. K. (2001). Archipelago regulates Cyclin E levels in Drosophila and is mutated in human cancer cell lines. Nature 413: 311-316. PubMed ID: 11565033

Moberg, K. H., Mukherjee, A., Veraksa, A., Artavanis-Tsakonas, S. and Hariharan, I. K. (2004). The Drosophila F box protein archipelago regulates dMyc protein levels in vivo. Curr Biol 14: 965-974. PubMed ID: 15182669

Müller, J. and Eilers, M. (2008). Ubiquitination of Myc: proteasomal degradation and beyond. Ernst Schering Found Symp Proc: 99-113. PubMed ID: 19198066

Nicholson, S. C., Nicolay, B. N., Frolov, M. V. and Moberg, K. H. (2011). Notch-dependent expression of the archipelago ubiquitin ligase subunit in the Drosophila eye. Development 138: 251-260. PubMed ID: 21148181

Parisi, F., Riccardo, S., Daniel, M., Saqcena, M., Kundu, N., Pession, A., Grifoni, D., Stocker, H., Tabak, E. and Bellosta, P. (2011). Drosophila insulin and target of rapamycin (TOR) pathways regulate GSK3 beta activity to control Myc stability and determine Myc expression in vivo. BMC Biol 9: 65. PubMed ID: 21951762

Popov, N., Herold, S., Llamazares, M., Schulein, C. and Eilers, M. (2007a). Fbw7 and Usp28 regulate myc protein stability in response to DNA damage. Cell Cycle 6: 2327-2331. PubMed ID: 17873522

Popov, N., Wanzel, M., Madiredjo, M., Zhang, D., Beijersbergen, R., Bernards, R., Moll, R., Elledge, S. J. and Eilers, M. (2007b). The ubiquitin-specific protease USP28 is required for MYC stability. Nat Cell Biol 9: 765-774. PubMed ID: 17558397

Reyes-Turcu, F. E., Ventii, K. H. and Wilkinson, K. D. (2009). Regulation and cellular roles of ubiquitin-specific deubiquitinating enzymes. Annu Rev Biochem 78: 363-397. PubMed ID: 19489724

Secombe, J., Li, L., Carlos, L. and Eisenman, R. N. (2007). The Trithorax group protein Lid is a trimethyl histone H3K4 demethylase required for dMyc-induced cell growth. Genes Dev 21: 537-551. PubMed ID: 17311883

Sowa, M. E., Bennett, E. J., Gygi, S. P. and Harper, J. W. (2009). Defining the human deubiquitinating enzyme interaction landscape. Cell 138: 389-403. PubMed ID: 19615732

Thomas, L. R. and Tansey, W. P. (2011). Proteolytic control of the oncoprotein transcription factor Myc. In Advances in Cancer Research, Vol. 110 (ed. K. George), pp. 77-106. Academic Press.

Tsou, W. L., Sheedlo, M. J., Morrow, M. E., Blount, J. R., McGregor, K. M., Das, C. and Todi, S. V. (2012). Systematic analysis of the physiological importance of deubiquitinating enzymes. PLoS One 7: e43112. PubMed ID: 22937016

Ventii, K. H. and Wilkinson, K. D. (2008). Protein partners of deubiquitinating enzymes. Biochem J 414: 161-175. PubMed ID: 18687060

date revised: 25 November 2013

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