Tousled-like kinase in a microbial eukaryote regulates spindle assembly and S-phase progression by interacting with Aurora kinase and chromatin assembly factors

Ziyin Li 0 Stphane Gourguechon 0 Ching C. Wang ) 0 0 Department of Pharmaceutical Chemistry, University of California , San Francisco, CA 94158-2280 , USA Summary The Tousled-like kinases are an evolutionarily conserved family of proteins implicated in DNA repair, DNA replication and mitosis in metazoans and plants. Their absence from the yeasts and other eukaryotic 'microbes' suggests a specific role for them in the development of multicellular organisms. In this study, two closely related ce Tousled-like kinase homologs, TLK1 and TLK2, were n identified in Trypanosoma brucei, a unicellular protozoan e ic parasite. Only TLK1 plays an essential role in cell growth, S and a deficiency in TLK1 led to an enrichment of S-phase lle cells, defective spindle formation and aberrant C chromosome segregation. Although both TLK proteins fo localize to the nucleus, only TLK1 also concentrates in the l spindle poles during mitosis. Both TLK proteins are a n phosphorylated by the Aurora kinase (AUK1), and both ru can autophosphorylate and phosphorylate histone H3 and o J - Introduction The Tousled-like kinases (TLKs) are a family of highly conserved serine/threonine protein kinases in both plants and animals. Tousled kinase is essential for proper flower and leaf development in Arabidopsis (Roe et al., 1993), whereas its homologs regulate DNA repair, DNA replication and mitosis in human and Drosophila (Sillj et al., 1999; Groth et al., 2003; Carrera et al., 2003). Mammalian TLK activity peaks during S phase, when it phosphorylates the anti-silencing function protein 1 (Asf1), a histone H3/H4 chaperone and a chromatin assembly factor involved in DNA repair and chromatin assembly (Sillj et al., 1999; Sillj and Nigg, 2001). In Caenorhabditis elegans, the Tousled-like kinase TLK-1 acts as a substrate activator of the Aurora B kinase AIR-2, a chromosomal passenger controlling chromosome segregation and cytokinesis (Han et al., 2005). TLK-1 is phosphorylated by AIR-2 and the phosphorylated TLK-1 in turn increases the kinase activity of AIR-2 towards histone H3. No TLK homolog is present in the unicellular eukaryote Saccharomyces cerevisiae (Sillj et al., 1999) or any other microbial eukaryotes examined thus far. These led to the hypothesis that TLKs are specific for multicellular organisms and probably function in some fundamental aspects of development common to both plants and animals (Sillj et al., 1999). the chromatin assembly factors Asf1A and Asf1B in vitro, but only TLK1 is autophosphorylated and capable of oligomerizing and interacting with AUK1, Asf1A and Asf1B in vivo. These discrepancies between the two TLK proteins can be attributed to minor differences between their N- and C-terminal sequences. In summary, TLK1 cooperates with Aurora kinase to regulate spindle assembly and chromosome segregation, and it performs a role in DNA replication probably by regulating histone modification in trypanosomes. Supplementary material available online at http://jcs.biologists.org/cgi/content/full/120/21/3883/DC1 Two close homologs of TLK were, however, identified in Trypanosoma brucei, a unicellular parasite that causes sleeping sickness in humans and nagana in cattle. This organism is deeply branched in the phylogenetic tree and classified to be more ancient than the yeasts (Baldauf, 2003; Bapteste and Gribaldo, 2003). It is transmitted between the mammalian host and the insect vector in three multiplying forms, the bloodstream and two insect (procyclic and epimastigote) forms, displaying distinctive cellular morphology and biological features. The trypanosome cell cycle has the usual sequential G1, S, G2 and M phases (McKean, 2003) but differs from other eukaryotes by having a unique mitochondrial DNA complex, the kinetoplast, whose division is mediated by basal body segregation (Woodward and Gull, 1990). A delicate coordination between the progression of the nuclear cycle and basal body/kinetoplast cycle is thus important for cytokinesis (Ploubidou et al., 1999). In the procyclic form, initiation of cytokinesis is dependent primarily on the basal body/kinetoplast cycle (Ploubidou et al., 1999; Li and Wang, 2003; Hammarton et al., 2003). Inhibition of mitosis in the procyclic form does not totally block cytokinesis and results in the formation of anucleate cells with a single kinetoplast. In the bloodstream form, however, inhibition of mitosis prevents cytokinesis but does not affect additional rounds of organelle replication (Hammarton et al., 2003; Tu and Wang, 2004; Li and Wang, 2006). An Aurora kinase homolog, AUK1, is essential for spindle formation, chromosome segregation and cytokinesis in both bloodstream and procyclic forms of T. brucei (Tu et al., 2006; Li and Wang, 2006), suggesting that, like the Aurora kinase Ark1 in Schizosaccharomyces pombe, AUK1 has the functions of both the Aurora A and Aurora B kinases possessed by the higher eukaryotes (Petersen et al., 2001). AUK1 displays a subcellular localization pattern typical of a chromosomal passenger that is, being concentrated in the nucleus during prophase but translocated to the spindle midzone in late anaphase (Tu et al., 2006; Li and Wang, 2006). This localization is mediated by the formation of a chromosomal passenger complex between Aurora B kinase and several other proteins such as Survivin, Borealin/Dasra and INCENP, the substrate activator of Aurora B kinase, in metazoa (Ducat and Zheng, 2004). However, homologs of these proteins are absent from the trypanosome genome (Z.L. and C.C.W., unpublished). Furthermore, homologs of the majority of the kinetochore components, which are all conserved among yeasts, plants and mammals and some of which are substrates of Aurora B kinase, are also all absent from the genome of T. brucei (Berriman et al., 2005). These ce raise the interesting question of how the Aurora kinase AUK1 n from T. brucei is regulated in the absence of its regulatory e ic proteins. S As T. brucei possesses two homologs of TLK, which acts as lle a substrate activator of Aurora B kinase in C. elegans and C phosphorylates the chromatin assembly factor Asf1 in metazoa fo and plants, we analyzed their potential involvement with l AUK1 and the Asf1 homologs in regulating cell cycle a n progression in T. brucei. We observed that, despite the 89% ru sequence identity between TLK1 and TLK2, only TLK1 Jo cooperates with AUK1 to regulate spindle assembly and chromosome segregation and maintains the S-phase progression by regulating Asf1A and Asf1B. This is the first time, to our knowledge, that a functioning TLK has been identified in a unicellular microorganism. Results Identification of TLK homologs in T. brucei Two close homologs of TLK were identified in T. brucei (Tb927.4.5180 and Tb927.8.7220) and designated TLK1 and TLK2, respectively. They reside on different chromosomes and are 89% identical in their amino acid sequences. They also showed a 29% identity to the Arabidopsis Tousled (supplementary ma (...truncated)