- Research article
- Open Access
SPOC: A widely distributed domain associated with cancer, apoptosis and transcription
BMC Bioinformatics volume 5, Article number: 91 (2004)
The Split ends (Spen) family are large proteins characterised by N-terminal RNA recognition motifs (RRMs) and a conserved SPOC (Spen paralog and ortholog C-terminal) domain. The aim of this study is to characterize the family at the sequence level.
We describe undetected members of the Spen family in other lineages (Plasmodium and Plants) and localise SPOC in a new domain context, in a family that is common to all eukaryotes using profile-based sequence searches and structural prediction methods.
The widely distributed DIO (Death inducer-obliterator) family is related to cancer and apoptosis and offers new clues about SPOC domain functionality.
The aim of this study was to characterize the Spen family at the sequence level. The Spen family of proteins participates in various biological processes. It is involved in neuronal cell fate, survival and axonal guidance [1–3], cell cycle regulation , and repression of head identity in the embryonic trunk . More recently it has been shown that the split ends gene participates in Wingless signalling in the eye, wing and leg imaginal discs  in the fly. The human Spen protein SHARP (SMRT/HDAC1-associated repressor protein) has been identified as a component of transcriptional repression complexes in both nuclear receptor and Notch/RBP-Jkappa signalling pathways [7–9]. Therefore, the Spen family of proteins appears to regulate transcription in several signalling pathways. In addition, the human Spen protein RBM15 (RNA-binding motif protein 15) is involved in the recurrent t(1;22) translocation whose product is the RBM15-MKL1 fusion protein. This aberrant protein is related to the megakaryoblastic leukemia 1 (MKL1) [10, 11].
On the other hand, DIO and its homologue PHF3 (PHD finger protein 3) are human proteins that contain a PHD (Plant Homeodomain) finger and a TFIIS (Transcription Factor S-II) domain: both domains are usually associated with transcription [12, 13]. The DIO-1 protein regulates the early stages of cell death in mouse and humans [14, 15]. Experimental evidence shows that the PHF3 protein is ubiquitously expressed in normal tissues, including brain. However, its expression is dramatically reduced or lost in glioblastoma, the most frequent tumour reported in human brain [12, 13]. Although this family has been shown to be involved in apoptosis and cancer, the underlying molecular mechanisms are unclear.
Sequence profiles of the C-terminal conserved region of DIO family found the SPOC domain of Spen family at E-values of 0.083. Reciprocally, the profile of the SPOC domain of Spen detected the DIO family with an E-value of 0.05. We localised new members of the Spen and DIO families in different eukaryote lineages (Figures 1 and 2). Statistically significant E-values connected all the SPOC domain-containing families. None of these HMMer profile searches retrieved any new unrelated sequences and, as stated above, reciprocal searches produced convergent results.
For the DIO family of sequences, secondary structure predictions were performed for the SPOC. These predictions showed good agreement with the crystal structure of the SPOC domain of SHARP  (Figure 1).
To investigate whether fold recognition analysis generated consistent results, we submitted the SPOC domain of DIO-1 (swissprot-id: DAT1_HUMAN, residues 1093 to 1199) as a query to an independent fold assignment system (see methods). The template 1OW1 (the SPOC domain of SHARP protein) was found with a Z-score of -12.2 (estimated error rate <1%) despite its low sequence homology (16%).
Considering the E-values of the HMMer searches, the reliability of secondary structure predictions, and the fold assignment results, we are confident that the SPOC domain is present in the DIO family of proteins.
To highlight the degree of fold-conservation, we generated a structural model (Fig. 3B) of the SPOC domain of DIO. In the sequence alignment showed in figure 1, the C-terminal region of SPOC is missing. This region includes: two small helices (named E and F), which do not form part of the core and are not well conserved within the SHARP family, and the β sheet 7, which is part of the β-barrel core. The high sequence divergence in these region, made impossible to extend the alignment for automatic methods. However, for modelling purposes, the alignment was carefully extended to the C-terminal region and a beta sheet was detected in DIO, while the two helices were missing. Therefore, SPOC domain of DIO adopts a similar fold than the SPOC domain of SHARP and the seven strands β-barrel core is maintained (Figure 3).
Reported as a protein-protein interaction domain, the structure of the SPOC domain of SHARP contains a basic cluster essential for interaction with SMRT (silencing mediator for retinoid and thyroid receptors) the co-repressor . The Arg 3552 of SHARP is localised in this basic cluster. Interestingly, this arginine is fully conserved in the SPOC alignment (Figure 1 and 3). The full conservation of Arg 3552 may be important, especially when equivalent substitutions in the protein-protein interaction interface (e.g. Lysine) might have little effect on the binding capability of this cluster. Therefore, one explanation for this fully conserved arginine could be its specific post-translational modification. Arginine methylation is a common post-translational modification in transcription regulation proteins that are catalysed by type I and II protein arginine methyltransferases . Arginine methylation modulates transcriptional activity and it has recently been related with a wide range of cellular processes .
For instance, proteins like the coactivator of nuclear receptors CBP (CREB-binding protein) follow this schema. In this protein there is a methylation site at Arg 600. This residue is essential for stabilising the structure of the domain implicated in CREB recruitment. There is a critical interaction between Arg 600 and Tyr 640 . Disruption of this interaction by arginine methylation could lead to conformational changes . Analogously, a similar interaction is observed on the surface of SPOC between the conserved residues Arg 3552 and Tyr 3602 (Figure 3) .
As arginine is one of the aminoacids most frequently found at the active sites of enzymes , alternative functional hypothesis for this conserved arginine is that it might form part of an active site of unknown function in the SPOC domain, or contribute to a specific catalytic function in other proteins, analogous to the "Arginine Finger" in Ras-GAP proteins .
The SPOC domain is present in different domain architectures among all the eukaryote lineages (Figures 1 and 2). This study shows that, with the exponential growth of the sequence databases, sequence analysis sheds new light on biological function, even when structure is already available. The fact that this domain has been identified in cancer and apoptosis related proteins emphasises its importance in transcriptional regulation. Additional experimental approaches using different members of the SPOC domain-containing families are required to confirm these hypotheses.
For the sequence analysis we related distant protein families via intermediate searches  using global hidden Markov model profiles (using hmmsearch of HMMer http://hmmer.wustl.edu/) . To improve the profile quality we followed two approaches: first, BLAST searches against unfinished genomes , and secondly, additional searches against EST (expressed sequence tags) databases . This sequence enrichment improved the quality of the profile that was used to perform the searches against the non-redundant protein databases. We used NAIL to view and analyse the HMMer results . The alignment was produced with HMMer  and T-Coffee software  using default parameters and was slightly refined manually. It is viewed with the Belvu program .
Structural predictions and modeling
Secondary structure predictions were performed using PHD . Fold recognition analyses were performed using the FFAS  server http://ffas.ljcrf.edu/. The model was based on the published crystal structure from the SPOC domain of SHARP protein  and obtained using swiss-model . The model was evaluated using PSQS  and WHATIF  tools. Illustrations were generated with MOLMOL .
LSP and AR carried out the sequence and structural analysis of the domain.
KVW and CMA provided with the initial input of the research.
LSP, AR, KVW, CMA and AV authored the manuscript.
RNA Recognition motifs
Spen paralog and ortholog C-terminal
SMRT/HDAC1-associated repressor protein
silencing mediator for retinoid and thyroid receptors
RNA-binding motif protein 15
megakaryoblastic leukemia 1
PHD finger protein 3
Transcription Factor S-II
GTPase Activating Protein
hidden Markov model
expressed sequence tags
BRM and KIS domain.
Chen F, Rebay I: split ends, a new component of the Drosophila EGF receptor pathway, regulates development of midline glial cells. Curr Biol 2000, 10: 943–946. 10.1016/S0960-9822(00)00625-4
Kuang B, Wu SC, Shin Y, Luo L, Kolodziej P: split ends encodes large nuclear proteins that regulate neuronal cell fate and axon extension in the Drosophila embryo. Development 2000, 127: 1517–1529.
Rebay I, Chen F, Hsiao F, Kolodziej PA, Kuang BH, Laverty T, Suh C, Voas M, Williams A, Rubin GM: A genetic screen for novel components of the Ras/Mitogen-activated protein kinase signaling pathway that interact with the yan gene of Drosophila identifies split ends, a new RNA recognition motif-containing protein. Genetics 2000, 154: 695–712.
Lane ME, Elend M, Heidmann D, Herr A, Marzodko S, Herzig A, Lehner CF: A screen for modifiers of cyclin E function in Drosophila melanogaster identifies Cdk2 mutations, revealing the insignificance of putative phosphorylation sites in Cdk2. Genetics 2000, 155: 233–244.
Wiellette EL, Harding KW, Mace KA, Ronshaugen MR, Wang FY, McGinnis W: spen encodes an RNP motif protein that interacts with Hox pathways to repress the development of head-like sclerites in the Drosophila trunk. Development 1999, 126: 5373–5385.
Lin HV, Doroquez DB, Cho S, Chen F, Rebay I, Cadigan KM: Splits ends is a tissue/promoter specific regulator of Wingless signaling. Development 2003, 130: 3125–3135. 10.1242/dev.00527
Shi Y, Downes M, Xie W, Kao HY, Ordentlich P, Tsai CC, Hon M, Evans RM: Sharp, an inducible cofactor that integrates nuclear receptor repression and activation. Genes Dev 2001, 15: 1140–1151. 10.1101/gad.871201
Oswald F, Kostezka U, Astrahantseff K, Bourteele S, Dillinger K, Zechner U, Ludwig L, Wilda M, Hameister H, Knochel W, Liptay S, Schmid RM: SHARP is a novel component of the Notch/RBP-Jkappa signalling pathway. EMBO J 2002, 21: 5417–5426. 10.1093/emboj/cdf549
Ariyoshi M, Schwabe JW: A conserved structural motif reveals the essential transcriptional repression function of Spen proteins and their role in developmental signaling. Genes Dev 2003, 17: 1909–1920. 10.1101/gad.266203
Mercher T, Coniat MB, Monni R, Mauchauffe M, Khac FN, Gressin L, Mugneret F, Leblanc T, Dastugue N, Berger R, Bernard OA: Involvement of a human gene related to the Drosophila spen gene in the recurrent t(1;22) translocation of acute megakaryocytic leukemia. Proc Natl Acad Sci USA 2001, 98: 5776–5779. 10.1073/pnas.101001498
Ma Z, Morris SW, Valentine V, Li M, Herbrick JA, Cui X, Bouman D, Li Y, Mehta PK, Nizetic D, Kaneko Y, Chan GC, Chan LC, Squire J, Scherer SW, Hitzler JK: Fusion of two novel genes, RBM15 and MKL1, in the t(1;22) (p13;q13) of acute megakaryoblastic leukemia. Nat Genet 2001, 28: 220–221. 10.1038/90054
Fischer U, Struss AK, Hemmer D, Michel A, Henn W, Steudel WI, Meese E: PHF3 expression is frequently reduced in glioma. Cytogenet Cell Genet 2001, 94: 131–136. 10.1159/000048804
Struss AK, Romeike BF, Munnia A, Nastainczyk W, Steudel WI, Konig J, Ohgaki H, Feiden W, Fischer U, Meese E: PHF3-specific antibody responses in over 60% of patients with glioblastoma multiforme. Oncogene 2001, 20: 4107–4114. 10.1038/sj.onc.1204552
Garcia-Domingo D, Ramirez D, Gonzalez de Buitrago G, Martinez-A C: Death inducer-obliterator 1 triggers apoptosis after nuclear translocation and caspase upregulation. Mol Cell Biol 2003, 23: 3216–3225. 10.1128/MCB.23.9.3216-3225.2003
Garcia-Domingo D, Leonardo E, Grandien A, Martinez P, Albar JP, Izpisua-Belmonte JC, Martinez-A C: DIO-1 is a gene involved in onset of apoptosis in vitro, whose misexpression disrupts limb development. Proc Natl Acad Sci USA 1999, 96: 7992–7997. 10.1073/pnas.96.14.7992
McBride AE, Silver PA: State of the arg: protein methylation at arginine comes of age. Cell 2001, 106: 5–8. 10.1016/S0092-8674(01)00423-8
Boisvert FM, Cote J, Boulanger MC, Richard S: A Proteomic Analysis of Arginine-methylated Protein Complexes. Mol Cell Proteomics 2003, 2: 1319–1330. 10.1074/mcp.M300088-MCP200
Xu W, Chen H, Du K, Asahara H, Tini M, Emerson BM, Montminy M, Evans RM: A transcriptional switch mediated by cofactor methylation. Science 2001, 294: 2507–2511. 10.1126/science.1065961
Wei Y, Horng JC, Vendel AC, Raleigh DP, Lumb KJ: Contribution to stability and folding of a buried polar residue at the CARM1 methylation site of the KIX domain of CBP. Biochemistry 2003, 42: 7044–7049. 10.1021/bi0343976
Bartlett GJ, Porter CT, Borkakoti N, Thornton JM: Analysis of catalytic residues in enzyme active sites. J Mol Biol 2002, 324: 105–121. 10.1016/S0022-2836(02)01036-7
Ahmadian MR, Stege P, Scheffzek K, Wittinghofer A: Confirmation of the arginine-finger hypothesis for the GAP-stimulated GTP-hydrolysis reaction of Ras. Nat Struct Biol 1997, 4: 686–689. 10.1038/nsb0997-686
Park J, Teichmann SA, Hubbard T, Chothia C: Intermediate sequences increase the detection of homology between sequences. J Mol Biol 1997, 273: 349–354. 10.1006/jmbi.1997.1288
Eddy SR: Profile hidden Markov models. Bioinformatics 1998, 14: 755–763. 10.1093/bioinformatics/14.9.755
Cummings L, Riley L, Black L, Souvorov A, Resenchuk S, Dondoshansky I, Tatusova T: Genomic BLAST: custom-defined virtual databases for complete and unfinished genomes. FEMS Microbiol Lett 2002, 216: 133–138. 10.1016/S0378-1097(02)00955-2
Boguski MS, Lowe TM, Tolstoshev CM: dbEST – database for "expressed sequence tags". Nat Genet 1993, 4: 332–333. 10.1038/ng0893-332
Sanchez-Pulido L, Yuan YP, Andrade MA, Bork P: NAIL-Network Analysis Interface for Linking HMMER results. Bioinformatics 2000, 16: 656–657. 10.1093/bioinformatics/16.7.656
Notredame C, Higgins DG, Heringa J: T-Coffee: A novel method for fast and accurate multiple sequence alignment. J Mol Biol 2000, 302: 205–217. 10.1006/jmbi.2000.4042
Rost B: PHD: predicting one-dimensional protein structure by profile-based neural networks. Methods Enzymol 1996, 266: 525–539. 10.1016/S0076-6879(96)66033-9
Rychlewski L, Jaroszewski L, Li W, Godzik A: Comparison of sequence profiles. Strategies for structural predictions using sequence information. Protein Sci 2000, 9: 232–241.
Schwede T, Kopp J, Guex N, Peitsch MC: SWISS-MODEL: an automated protein homology-modeling server. Nucleic Acids Res 2003, 31: 3381–3385. 10.1093/nar/gkg520
Koradi R, Billeter M, Wüthrich K: MOLMOL: a program for display and analysis of macromolecular structures. J Mol Graphics 1996, 14: 51–55. 10.1016/0263-7855(96)00009-4
Bateman A, Coin L, Durbin R, Finn RD, Hollich V, Griffiths-Jones S, Khanna A, Marshall M, Moxon S, Sonnhammer EL, Studholme DJ, Yeats C, Eddy SR: The Pfam protein families database. Nucleic Acids Res 2004, 32(Database issue):D138–141. 10.1093/nar/gkh121
Letunic I, Copley RR, Schmidt S, Ciccarelli FD, Doerks T, Schultz J, Ponting CP, Bork P: SMART 4.0: towards genomic data integration. Nucleic Acids Res 2004, 32(Database issue):D142–144. 10.1093/nar/gkh088
We are grateful to M. Tress (CNB-Spain) and R. Rycroft for their helpful comments on the manuscript. This study was financed in part by the VITH EU Framework Project QLGI-CT-2001-01536.
About this article
Cite this article
Sánchez-Pulido, L., Rojas, A.M., van Wely, K.H. et al. SPOC: A widely distributed domain associated with cancer, apoptosis and transcription. BMC Bioinformatics 5, 91 (2004). https://doi.org/10.1186/1471-2105-5-91
- Secondary Structure Prediction
- Hide Markov Model Profile
- Arginine Methylation
- Megakaryoblastic Leukemia
- Transcriptional Repression Complex