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The Drosophila clock protein Timeless is a member of the Arm/HEAT family

2002; Elsevier BV; Volume: 12; Issue: 18 Linguagem: Inglês

10.1016/s0960-9822(02)01130-2

1879-0445

Nicolas Vodovar, Jonathan D. Clayton, Rodolfo Costa, Mark Odell, Charalambos P. Kyriacou,

Light effects on plants

In higher eukaryotes, the circadian clock is generated by a system of negative and positive auto-regulatory feedback loops [1.Young M.W Kay S.A Time zones: a comparative genetics of circadian clocks.Nat. Rev. Genet. 2001; 2: 702-715Crossref PubMed Scopus (922) Google Scholar]. In Drosophila, the negative limb of this mechanism involves the Timeless (Tim) and Period (Per) proteins, which must physically associate in order to be translocated to the nucleus. Once inside, the Per–Tim heterodimer negatively regulates the expression of the per and tim genes. Per is a founder member of the PAS (Per-Arnt-Sim) protein family [2.Huang Z.J Edery I Rosbash M PAS is a dimerization domain common to Drosophila PERIOD and several transcription factors.Nature. 1993; 364: 259-262Crossref PubMed Scopus (414) Google Scholar] and contains two PAS domains that are involved in protein dimerization. The Drosophila genome encodes two different tim genes, the clock relevant dtim, and a second, ancestral gene, dtim2 or timeout, which is the true orthologue of mammalian Tim[3.Gotter A.L Manganaro T Weaver D.R Kolakowski L.F Possidente B Sriram S MacLaughlin D.T Reppert S.M A time-less function for mouse Timeless.Nat. Neurosci. 2000; 3: 755-756Crossref PubMed Scopus (147) Google Scholar, 4.Benna C Scannapieco P Piccin A Sandrelli F Zordan M Rosato E Kyriacou C.P Valle G Costa R A second timeless gene in Drosophila shares greater sequence similarity with mammalian tim.Curr. Biol. 2000; 10: R512-R513Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar]. mTim does not appear to play a significant role in the murine clock [5.Reppert S.M Weaver D.R Comparing clockworks: Mouse versus fly.J. Biol. Rhythms. 2000; 15: 357-364Crossref PubMed Scopus (74) Google Scholar]. No protein domains have yet been identified in Tim or Tim2, so in order to gain some insight into the possible biochemical and conformational nature of Tim proteins, we have applied structural predictions that suggest them to be members of the Arm/HEAT family. A PSI-BLAST reiterative analysis of the ancestral Drosophila Tim2 protein revealed similarities between the first 516 residues of dTim2, and two amino-terminal regions of dTim (23%, BLOSUM 80, regions 24–284 and 508–790; Figure 1). Further rounds of PSI-BLAST failed to reveal any significant similarities to other protein classes. Protein structure prediction using PSA (bmerc-www.bu.edu/psa/) suggested that this region in both proteins was entirely α-helical. We therefore used 3D-PSSM [6.Kelley L.A MacCallum R.M Sternberg M.J.E Enhanced genome annotation using structural profiles in the program 3D-PSSM.J. Mol. Biol. 2000; 299: 499-520Crossref PubMed Scopus (1120) Google Scholar] to compare further the amino-terminal dTim2 region to the SCOP database [7.Murzin A.G Brenner S.E Hubbard T Chothia C SCOP: a structural classification of proteins database for the investigation of sequences and structures.J. Mol. Biol. 1995; 247: 536-540Crossref PubMed Scopus (5584) Google Scholar]. The search returned five highly significant values (E < 0.05; note that in 3D-PSSM, E < 0.5 is considered significant), representing members of the Armadillo (Arm) repeat superfamily from the fold of alpha–alpha superhelical proteins. Three were from the Arm (yeast and murine importin-α, murine β-catenin) and two from the HEAT (human importin-β and the PRb65/A subunit of protein phosphatase 2A) families (Figure 1). Similar searches using hTIM and mTIM also returned significant similarities (E < 0.06) with these same Arm/HEAT containing proteins. We also examined with 3D-PSSM the mouse and fly clock proteins, Per, Clock, Bmal1 (and Cycle), CKlε (and Dbt), and GSK-3 (and Sgg), but no Arm/HEAT domains were detected. dTim has two amino-terminal isoforms with methionine initiation at +1 and +24, respectively [8.Rosato E Trevisan A Sandrelli F Zordan M Kyriacou C.P Costa R Conceptual translation of timeless reveals alternative initiating methionines in Drosophila.NAR. 1997; 25: 455-457Crossref PubMed Scopus (54) Google Scholar], so we analysed the structural motifs common to the amino termini of both variants (Figure 1). This region aligned with human importin-β using 3D-PSSM (E = 0.08). Because the similarity with Arm/HEAT repeats was slightly less striking than for dTim2, we analysed dTim by SAM-T99 [9.Karplus K Barrett C Cline M Diekhans M Grate L Hughey R Predicting protein structure using only sequence information.Proteins. 1999; 37: 121-125Crossref PubMed Google Scholar] based on Hidden Markov Modelling and by hydrophobic cluster analysis, HCA [10.Callebaut I Labesse G Durand P Poupon A Canard L Chomilier J Henrissat B Mornon J.P Deciphering protein sequence information though hydrohobic cluster analysis. Current status and perspectives.Cell. Mol. Life Sci. 1997; 53: 621-645Crossref PubMed Scopus (432) Google Scholar]. Both of these methods confirmed the existence of an Arm superfamily homologous region in the amino-terminal region (data not shown). The final borders of the Arm region in dTim were defined by 3D-PSSM reiteration to lie between residues 24–422. To characterise any additional structural elements present in the dTim proteins, we analysed the regions downstream of residues 500. HCA analysis revealed a region consistent with a second Arm superfamily domain located between residues 570–990 in dTim. Examination of this region by 3D-PSSM suggested that the domain borders were slightly larger, apparently extending between residues 582–1052 with homologies to Arm superfamily members (yeast importin-α and human importin-β E < 0.5; murine β-catenin, E < 0.7). In dTim2 the presence of additional Arm/HEAT features was not as apparent as for dTim by 3D-PSSM (E > 1) although the presence of Arm superfamily motifs downstream of residue 511 was suggested by REP analysis of residues 512–936 (www.embl–heidelberg.de/∼andrade/papers/rep/search.html). Because of the similarity between residues 261–516 of dTim2 and 508–790 of dTim detected by PSI-BLAST analysis, we speculate that the amino-terminal Arm/HEAT domain of dTim2 may be divided in dTim by residues 284–582 which are largely specific to dTim (see Figure 1). This means that the first interaction domain in dTim that was identified by deletion analysis and associates with the first Per PAS repeat [11.Saez L Young M.W Regulation of nuclear entry of the Drosophila clock proteins period and timeless.Neuron. 1996; 17: 911-920Abstract Full Text Full Text PDF PubMed Scopus (269) Google Scholar] (see Figure 1) falls largely, if not entirely, between the two dTim Arm/HEAT regions. Arm/HEAT motifs are tandem repeats of ∼50 residues with highly degenerate sequences [12.Andrade M.A Petosa C O'Donoghue S.I Muller C.W Bork P Comparison of ARM and HEAT protein repeats.J. Mol. Biol. 2001; 309: 1-18Crossref PubMed Scopus (395) Google Scholar]. The crystal structures of two Arm family members, importin-α and β-catenin, have defined the ARM repeat as having three helices [13.Conti E Uy M Leighton L Blobel G Kuriyan J Crystallographic analysis of the recognition of a nuclear localization signal by the nuclear import factor karyopherin-α.Cell. 1998; 94: 193-204Abstract Full Text Full Text PDF PubMed Scopus (655) Google Scholar, 14.Huber A.H Nelson W.J Weis W.I Three-dimensional structure of the Armadillo repeat region of β-catenin.Cell. 1997; 90: 871-882Abstract Full Text Full Text PDF PubMed Scopus (567) Google Scholar], whereas the HEAT repeat, characterized from the structures of importins β1 and β2 and protein phosphatase 2A, consists of only two helices [15.Cingolani G Petosa C Weis K Muller C.W Structure of importin-β bound to the IBB domain of importin-α.Nature. 1999; 399: 221-229Crossref PubMed Scopus (448) Google Scholar, 16.Chook X Blobel Y Structure of the nuclear transport complex karyopherin-β2-Ran–GppNHp.Nature. 1999; 399: 230-237Crossref PubMed Scopus (291) Google Scholar, 17.Groves M.R Hanlon N Turowski P Hemmings B.A Bartford D The structure of the protein phosphatase 2A PR65/A subunit reveals the conformation of its 15 tandemly repeated HEAT motifs.Cell. 1999; 96: 99-110Abstract Full Text Full Text PDF PubMed Scopus (353) Google Scholar]. Nevertheless, these two motifs bear a striking resemblance to each other at the conformational level. As dTim is required for the nuclear import of dPer [18.Vosshall L.B Price J.L Sehgal A Saez L Young M.W Block in nuclear localisation of Period protein by a second clock mutation, timeless.Science. 1994; 263: 1606-1609Crossref PubMed Scopus (288) Google Scholar] the structural similarity with importins takes on an immediate biological relevance. Furthermore, dPer is retained in the cytoplasm via its cytoplasmic localisation domain (CLD), which associates with dTim residues 747–946 within the second putative Arm domain (Figure 1 and [11.Saez L Young M.W Regulation of nuclear entry of the Drosophila clock proteins period and timeless.Neuron. 1996; 17: 911-920Abstract Full Text Full Text PDF PubMed Scopus (269) Google Scholar]). Consequently a scenario may be envisaged in which the second Arm domain of dTim 'mimics' the plasma membrane component (such as β-catenin), to attract the CLD of dPer. Finally, within the fly circadian mechanism, Doubletime (casein kinase 1ε) and Shaggy (glycogen synthase kinase-3) are key players in regulating the stabilities of both Per and Tim, respectively [19.Kloss B Price J.L Saez L Blau J Rothenfluh A Wesley C.S Young M.W The Drosophila clock gene double-time encodes a protein closely related to human casein kinase I epsilon.Cell. 1998; 92: 97-107Abstract Full Text Full Text PDF Scopus (594) Google Scholar, 20.Martinek S Inonog S Manoukian A.S Young M.W A role for the segment polarity gene shaggy/GSK-3 in the Drosophila circadian clock.Cell. 2001; 105: 769-779Abstract Full Text Full Text PDF PubMed Scopus (453) Google Scholar]. These two kinases also carry out similar roles in Wnt signalling, where they regulate the stability of β-Catenin [21.van Noort M Meeldijk J van der Zee R Destree O Clevers H Wnt signaling controls the phosphorylation status of β-catenin.J. Biol. Chem. 2002; 277: 17901-17905Crossref PubMed Scopus (420) Google Scholar, 22.Amit S Hatzubai A Birman Y Andersen J.S Ben-Shushan E Mann M Ben-Neriah Y Alkalay I Axin-mediated CKI phosphorylation of β-catenin at Ser 45: a molecular switch for the Wnt pathway.Genes & Dev. 2002; 16: 1066-1076Crossref PubMed Scopus (589) Google Scholar] which contains Arm repeats. Our finding that Tim may contain domains of Arm/HEAT repeats thus provides the focal point for implicating the Wingless/Wnt signalling module in the circadian mechanism, and suggests that other members of this pathway should be investigated for possible roles in the generation of biological oscillations. Clearly, unequivocal evidence for Arm/HEAT domains within Tim proteins must await a three-dimensional experimental analysis. Nevertheless, our results raise interesting questions concerning the evolutionary origins of this signalling module, and how some components came to be shared among the developmental and circadian pathways. This work was supported by HFSP and BBSRC grants to CPK, a Socrates studentship to NV and a MURST-British Council grant to CPK and RC.