Encyclopedia of Signaling Molecules

2018 Edition
| Editors: Sangdun Choi

MK-STYX

Reference work entry
DOI: https://doi.org/10.1007/978-3-319-67199-4_205

Synonyms

Historical Background

MK-STYX was first identified bioinformatically due to its homology with the dual specificity phosphatases (DUSPs), a family of enzymes known for their ability to dephosphorylate not only tyrosine residues but also serine and threonine residues (Wishart and Dixon 1998). Specifically, MK-STYX resembles a MAP kinase phosphatase (MKP), which contains a rhodenase or  Cdc25 homology (CH2) domain in its N-terminus, while containing a DUSP domain in its C-terminus. The name of the protein reflects this similarity, as MK-STYX was coined from the phrase MAP Kinase Phosphatase-like Serine Threonine Tyrosine interaction domain (Wishart and Dixon 1998). The Ser/Thr/Tyr interaction (STYX) domain was so named based on the observation that, though this domain shares significant homology to a DUSP domain, it lacks a critical cysteine residue necessary for catalysis; the conservative substitution of a serine in place of this residue renders this enzyme completely inactive. Mechanistically, this is explained though examination of the enzymatic reaction necessary for DUSPs to carry out the dephosphorylation reaction. After binding a phosphorylated substrate, a classical DUSP will utilize a conserved cysteine residue to nucleophilically attack the phosphate group which is to be removed from its substrate (Fig. 1a). This cysteine contains a thiolate intermediate, kept in its ionic state by an environment promoting an extremely low pKa within the pocket of the phosphatase. The substitution of a cysteine to a serine, however, abolishes this thiolate intermediate, and even the very low pKa of the phosphatase pocket is not sufficient to render the hydroxyl group on this serine as a potent nucleophile (Fig. 1b). Thus, the serine mutation does not affect the binding of the substrate, but rather the initiaton of the first step of catalysis. This Cys → Ser substitution has been exploited by researchers in the field of phosphatase biology to create what are known as “substrate-trapping mutants” (Flint et al. 1997). These mutants are utilized as a tool in which phosphatases bind their substrates in a more stable fashion, allowing for easier identification and characterization of binding partners and downstream effector molecules. Thus, MK-STYX is an endogenous “substrate-trapping mutant,” although whether this molecule uses its STYX domain as a tool to bind phosphorylated residues on other molecules has yet to be determined.
MK-STYX, Fig. 1

Catalytic mechanism of DUSPs and MK-STYX. All dual specificity phosphatases contain a consensus motif, DXnCX5R, which defines their phosphatase domain. The cysteine is critical for the initial nucleophilic attack of the phosphoryl group to be removed from the phosphorylated substrate (a, step 1). The phosphorylated residue is released from the enzyme, and the aspartate and arginine are involved in recycling the enzyme back to its active state (a, steps 2 and 3). In MK-STYX, the initial catalytic cysteine is substituted with a serine group, which is not a potent nucleophile (b, step 1). Due to the loss of this cysteine, the enzyme cannot instigate dephosphorylation of the substrate (b, steps 1 and 2). Instead, the phosphatase pocket creates a stable intermediate, with the inactive phosphatase “trapping” its substrate through docking in this binding pocket (b, step 3)

MK-STYX is part of a family of two known proteins in humans with a STYX domain. The other protein, simply named STYX, is also catalytically inactive, due to a Cys → Gly mutation within its active site (Wishart and Dixon 1998). Reversion of the glycine back to a cysteine is sufficient to restore catalytic activity, demonstrating the preservation of the phosphatase domain and binding pocket in this protein (Wishart et al. 1995). Due to this structural preservation, it has been speculated that the STYX domain could bind to phosphorylated proteins, analogous to an SH2 domain binding phosphotyrosine residues. This would be a particularly interesting possibility, as DUSPs are able dephosphorylate both tyrosine residues, as well as serine/threonine residues due to a particularly shallow pocket which accommodates the active site. Indeed, a recent study demonstrated that the mutation of two residues within the active site, the serine → cysteine along with the −1 position, is sufficient to promote phosphatase activity of MK-STYX (Hinton et al. 2010). These data suggest that while the enzyme is catalytically inactive, it retains a phosphatase binding pocket that could be important in its cellular functions.

Another interesting possibility of MK-STYX function is to regulate the localization or enzymatic activity of an active phosphatase, presumably a DUSP. Surprisingly, there are numerous phosphatases present in the human genome which are rendered catalytically inactive due to mutations at various points within their active sites (Tonks 2006). This includes the D2 domains of many receptor protein tyrosine phosphatases (R-PTPs), which are implicated in controlling enzymatic activity. Interestingly, a family of lipid phosphatases, the myotubularins, consists of 14 different members, 6 of which are catalytically inactive (Begley and Dixon 2005). Importantly, these catalytically inactive myotubularins are critical for modulating enzymatic activity and/or cellular sublocalization of their active counterparts, creating an additional regulatory layer into lipid phosphatase biology (Begley et al. 2006; Robinson et al. 2008). While a phosphatase interactor for STYX and/or MK-STYX has yet to be found, the possibility that these proteins could be similar to a regulatory module for active phosphatases is an attractive model with clear precedence within the cell.

MK-STYX and STYX Domains Throughout Evolution

MK-STYX seems to be a relatively recent molecular addition in the evolution of animals, as definitive homologues of the protein can only be found within the phylum Chordata, which includes deuterosomes such as the sea cucumber as well as zebrafish, mice, and humans. It is not conserved, however, in insects, C. elegans, or yeast. It is interesting to note, however, that “STYX-domain” containing proteins do exist in these organisms (Wishart and Dixon 1998). While they are probably not direct homologues of MK-STYX, it is notable that catalytically inactive phosphatases have been utilized in even early life forms, though little is known about the functionality of these genes.

MK-STYX Regulation

As MK-STYX is relatively uncharacterized, it comes as no surprise that little is known about its regulation at a transcriptional or posttranscriptional level. There are multiple studies, however, that have demonstrated that MK-STYX transcript levels increase in the context of the Ewing’s Sarcoma fusion product, EWS-FLI (Guillon et al. 2009; Siligan et al. 2005). EWS-FLI is a transcription factor with aberrant-binding capacities which is known to be sufficient in causing Ewing’s Sarcoma. While MK-STYX transcript levels have been shown to increase to this aberrant fusion protein, presumably through transcriptional upregulation, little is known of the physiological significance of this observation, as MK-STYX has also been hypothesized to function as a tumor suppressor (see RNAi phenotypes, below).

MK-STYX has also been identified as being transcriptionally upregulated by the induction of the p21 protein (Chang et al. 2000), though this would have to be an indirect form of regulation, as p21 itself is not a direct transcriptional regulator. It is interesting, however, in light of the potential tumor suppressive functions of MK-STYX uncovered by MacKeigan et al., that p21 could upregulate MK-STYX to halt cell cycle progression or promote an apoptotic phenotype. The broad applicability p21-mediated control of MK-STYX expression will have to be confirmed and explored in much more detail before a clear understanding of these implications is uncovered.

RNAi-Mediated Phenotypes of MK-STYX Knockdown

Interestingly, MK-STYX has been identified as having numerous phenotypes in multiple RNAi screens designed to study very different cellular processes. The first study aimed to identify novel kinases and phosphatases involved in cellular survival or apoptotic potential (MacKeigan et al. 2005). The authors used siRNA sequences to all known and putative human kinases and phosphatases and transfected them into cancer cells. RNAi-mediated loss of MK-STYX promoted the most highly chemoresistant phenotype of all enzymes assayed. Importantly, this chemoresistance was shown in response to multiple drugs with different mechanisms of action, implicating a general cellular mechanism of chemoresistance. As chemoresistance is a highly significant clinical problem for patients with advanced and recurrent cancers, studies on the significance of this gene in treatment response and/or prediction of response rate could be an important future direction in oncology research.

A second RNAi-screening paper identified MK-STYX as potentially tumor suppressive in the context of breast cancer. This paper demonstrated that the RNAi-mediated loss of MK-STYX promoted a highly aberrant migratory phenotype in MCF-10A cells, a nontransformed mammary epithelial cell line (Simpson et al. 2008). This seemed to be coupled with a striking loss in cell polarity, which is a typical feature of cancer cells.

An additional independent study identified loss of MK-STYX within a set of genes whose downregulation is associated with breast cancer metastasis to the brain (Bos et al. 2009). In the study, two cell lines were passaged in vivo to create a daughter cell line which was highly metastatic to the brain. Importantly, both cell models had statistically significant downregulation of MK-STYX in the metastatic cell lines relative to the parental lines. This data, coupled with the RNAi-screening data, suggests that MK-STYX could be a potent tumor and/or metastasis suppressor in breast cancers.

While no molecular mechanism was worked out for either of these phenotypes, it is interesting to note that the loss of MK-STYX seems to promote both resistance to therapy, as well as a prometastatic phenotype. These data suggest that the loss of MK-STYX could be an important event in the later stages of cancer progression and could be a valuable therapeutic target if these observations validate in follow-up studies.

MK-STYX and Stress Granule Formation

Currently, the only known protein identified as an interaction partner of MK-STYX is the RNA-binding protein G3BP1. A recent study has shown that MK-STYX can bind to endogenous G3BP, a protein intimately involved in the formation and maintenance of stress granules within the cell (Hinton et al. 2010). Importantly, MK-STYX overexpression alleviated stress granule formation within cells, implicating a functional role to this interaction. Interestingly, the authors noted that the interaction between MK-STYX and G3BP is abrogated when MK-STYX is reverted to an active enzyme through the mutation of two key residues within its active site, suggesting that the MK-STYX-G3BP1 interaction axis is mediated through its STYX domain, potentially through a substrate-trapping mechanism. It will be interesting in the future to understand whether G3BP is phosphorylated and whether this phosphorylation is what mediates the interaction of these two proteins.

Summary and Future Directions

MK-STYX, while relatively uncharacterized, is suggested to have numerous independent and interesting cellular phenotypes. Many studies remain to be done to elucidate how a single gene could have such pleiotropic effects; subcellular localization, regulation, and turnover, as well as identification of interaction partners will be critical for these analyses. Although many facts remain to be uncovered about this gene, the few studies that have been done on this protein suggest that it could play a very interesting role in the etiology of diseases, such as cancer. As such, studies in the future should take note of this interesting gene, and efforts should be made to uncover its cellular function and what role it plays in the etiology of disease.

References

  1. Begley MJ, Dixon JE. The structure and regulation of myotubularin phosphatases. Curr Opin Struct Biol. 2005;15:614–20.PubMedCrossRefGoogle Scholar
  2. Begley MJ, Taylor GS, Brock MA, Ghosh P, Woods VL, Dixon JE. Molecular basis for substrate recognition by MTMR2, a myotubularin family phosphoinositide phosphatase. Proc Natl Acad Sci U S A. 2006;103:927–32.PubMedPubMedCentralCrossRefGoogle Scholar
  3. Bos PD, Zhang XH, Nadal C, Shu W, Gomis RR, Nguyen DX, Minn AJ, van de Vijver MJ, Gerald WL, Foekens JA, Massague J. Genes that mediate breast cancer metastasis to the brain. Nature. 2009;459:1005–9.PubMedPubMedCentralCrossRefGoogle Scholar
  4. Chang BD, Watanabe K, Broude EV, Fang J, Poole JC, Kalinichenko TV, Roninson IB. Effects of p21Waf1/Cip1/Sdi1 on cellular gene expression: implications for carcinogenesis, senescence, and age-related diseases. Proc Natl Acad Sci U S A. 2000;97:4291–6.PubMedPubMedCentralCrossRefGoogle Scholar
  5. Flint AJ, Tiganis T, Barford D, Tonks NK. Development of “substrate-trapping” mutants to identify physiological substrates of protein tyrosine phosphatases. Proc Natl Acad Sci U S A. 1997;94(5):1680.PubMedPubMedCentralCrossRefGoogle Scholar
  6. Guillon N, Tirode F, Boeva V, Zynovyev A, Barillot E, Delattre O. The oncogenic EWS-FLI1 protein binds in vivo GGAA microsatellite sequences with potential transcriptional activation function. PLoS One. 2009;4:e4932.PubMedPubMedCentralCrossRefGoogle Scholar
  7. Hinton SD, Myers MP, Roggero VR, Allison LA, Tonks NK. The pseudophosphatase MK-STYX interacts with G3BP and decreases stress granule formation. Biochem J. 2010;427:349–57.PubMedPubMedCentralCrossRefGoogle Scholar
  8. MacKeigan JP, Murphy LO, Blenis J. Sensitized RNAi screen of human kinases and phosphatases identifies new regulators of apoptosis and chemoresistance. Nat Cell Biol. 2005;7:591–600.PubMedCrossRefGoogle Scholar
  9. Robinson FL, Niesman IR, Beiswenger KK, Dixon JE. Loss of the inactive myotubularin-related phosphatase Mtmr13 leads to a charcot-marie-tooth 4B2-like peripheral neuropathy in mice. Proc Natl Acad Sci U S A. 2008;105:4916–21.PubMedPubMedCentralCrossRefGoogle Scholar
  10. Siligan C, Ban J, Bachmaier R, Spahn L, Kreppel M, Schaefer KL, Poremba C, Aryee DN, Kovar H. EWS-FLI1 target genes recovered from Ewing’s sarcoma chromatin. Oncogene. 2005;24:2512–24.PubMedCrossRefGoogle Scholar
  11. Simpson KJ, Selfors LM, Bui J, Reynolds A, Leake D, Khvorova A, Brugge JS. Identification of genes that regulate epithelial cell migration using an siRNA screening approach. Nat Cell Biol. 2008;10:1027–38.PubMedCrossRefGoogle Scholar
  12. Tonks NK. Protein tyrosine phosphatases: from genes, to function, to disease. Nat Rev Mol Cell Biol. 2006;7:833–46.PubMedCrossRefGoogle Scholar
  13. Wishart MJ, Dixon JE. Gathering STYX: phosphatase-like form predicts functions for unique protein-interaction domains. Trends Biochem Sci. 1998;23:301–6.PubMedCrossRefGoogle Scholar
  14. Wishart MJ, Denu JM, Williams JA, Dixon JE. A single mutation converts a novel phosphotyrosine binding domain into a dual-specificity phosphatase. J Biol Chem. 1995;270:26782–5.PubMedCrossRefGoogle Scholar

Copyright information

© Springer International Publishing AG 2018

Authors and Affiliations

  1. 1.Van Andel Institute Graduate SchoolVan Andel Research InstituteGrand RapidsUSA
  2. 2.Center for Cancer Genomics and Quantitative BiologyVan Andel Research InstituteGrand RapidsUSA