Nuclear DBF-2-related Kinases Are Essential Regulators of Cytokinesis in Bloodstream Stage Trypanosoma brucei

Nuclear DBF-2-related (NDR) kinases are essential regulators of cell cycle progression, growth, and development in many organisms and are activated by the binding of an Mps One Binder (MOB) protein partner, autophosphorylation, and phosphorylation by an upstream STE20 family kinase. In the protozoan parasite, Trypanosoma brucei, the causative agent of human African trypanosomiasis, the NDR kinase, PK50, is expressed in proliferative life cycle stages and was shown to complement a yeast NDR kinase mutant cell line. However, the function of PK50 and a second NDR kinase, PK53, in T. brucei has not been determined to date, although trypanosome MOB1 is known to be essential for cytokinesis, suggesting the NDR kinases may also be involved in this process. Here, we show that specific depletion of PK50 or PK53 from bloodstream stage trypanosomes resulted in the rapid accumulation of cells with two nuclei and two kinetoplasts, indicating that cytokinesis was specifically inhibited. This led to a deregulation of the cell cycle and cell death and provides genetic validation of these kinases as potential novel drug targets for human African trypanosomiasis. Recombinant active PK50 and PK53 were produced and biochemically characterized. Both enzymes autophosphorylated, were able to trans-phosphorylate generic kinase substrates in vitro, and were active in the absence of phosphorylation by an upstream kinase. Additionally, both enzymes were active in the absence of MOB1 binding, which was also demonstrated to likely be a feature of the kinases in vivo. Biochemical characterization of recombinant PK50 and PK53 has revealed key kinetic differences between them, and the identification of in vitro peptide substrates in this study paves the way for high throughput inhibitor screening of these kinases. Nuclear DBF-2-related (NDR) kinases are essential regulators of cell cycle progression, growth, and development in many organisms and are activated by the binding of an Mps One Binder (MOB) protein partner, autophosphorylation, and phosphorylation by an upstream STE20 family kinase. In the protozoan parasite, Trypanosoma brucei, the causative agent of human African trypanosomiasis, the NDR kinase, PK50, is expressed in proliferative life cycle stages and was shown to complement a yeast NDR kinase mutant cell line. However, the function of PK50 and a second NDR kinase, PK53, in T. brucei has not been determined to date, although trypanosome MOB1 is known to be essential for cytokinesis, suggesting the NDR kinases may also be involved in this process. Here, we show that specific depletion of PK50 or PK53 from bloodstream stage trypanosomes resulted in the rapid accumulation of cells with two nuclei and two kinetoplasts, indicating that cytokinesis was specifically inhibited. This led to a deregulation of the cell cycle and cell death and provides genetic validation of these kinases as potential novel drug targets for human African trypanosomiasis. Recombinant active PK50 and PK53 were produced and biochemically characterized. Both enzymes autophosphorylated, were able to trans-phosphorylate generic kinase substrates in vitro, and were active in the absence of phosphorylation by an upstream kinase. Additionally, both enzymes were active in the absence of MOB1 binding, which was also demonstrated to likely be a feature of the kinases in vivo. Biochemical characterization of recombinant PK50 and PK53 has revealed key kinetic differences between them, and the identification of in vitro peptide substrates in this study paves the way for high throughput inhibitor screening of these kinases. IntroductionNuclear DBF2-related (NDR) 3The abbreviations used are: NDRnuclear DBF2-relatedGSTglutathione S-transferaseMBPmyelin basic proteinMOBMps One BinderRACErapid amplification of cDNA endsMBPmyelin basic proteinRNAiRNA interferenceDAPI4,6-diamidino-2-phenylindoleCHAPS3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acidMOPS4-morpholinepropanesulfonic acid. kinases are important regulators of a variety of cellular processes, including centrosome duplication, mitotic exit, cytokinesis, morphogenesis, and cell growth and development. The founding member of this family, DBF2, is part of the mitotic exit network in Saccharomyces cerevisiae (1Mah A.S. Jang J. Deshaies R.J. Proc. Natl. Acad. Sci. U.S.A. 2001; 98: 7325-7330Crossref PubMed Scopus (160) Google Scholar), although its counterpart in fission yeast, Sid2, is a component of the septation initiation network (2Hou M.C. Guertin D.A. McCollum D. Mol. Cell. Biol. 2004; 24: 3262-3276Crossref PubMed Scopus (55) Google Scholar). Both kinases were recently shown to phosphorylate CDC14 phosphatase, promoting its retention in the cytoplasm and allowing it to inactivate mitotic cyclin/cyclin-dependent kinase and promote cell cycle progression (3Chen C.T. Feoktistova A. Chen J.S. Shim Y.S. Clifford D.M. Gould K.L. McCollum D. Curr. Biol. 2008; 18: 1594-1599Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar, 4Mohl D.A. Huddleston M.J. Collingwood T.S. Annan R.S. Deshaies R.J. J. Cell Biol. 2009; 184: 527-539Crossref PubMed Scopus (81) Google Scholar). Other NDR kinases in these yeasts (CBK1 in S. cerevisiae and Orb6 in Schizosaccharomyces pombe) regulate cellular morphogenesis (5Weiss E.L. Kurischko C. Zhang C. Shokat K. Drubin D.G. Luca F.C. J. Cell Biol. 2002; 158: 885-900Crossref PubMed Scopus (163) Google Scholar, 6Hou M.C. Wiley D.J. Verde F. McCollum D. J. Cell Sci. 2003; 116: 125-135Crossref PubMed Scopus (69) Google Scholar). In contrast, the fungal pathogen, Candida albicans, expresses only a single DBF2 orthologue, which is essential for viability and appears to have evolved multiple functions, being required for mitotic spindle organization, actomyosin ring contraction, and hyphal growth (7González-Novo A. Labrador L. Pablo-Hernando M.E. Correa-Bordes J. Sánchez M. Jiménez J. Vázquez de Aldana C.R. Mol. Microbiol. 2009; 72: 1364-1378Crossref PubMed Scopus (16) Google Scholar). In Drosophila, Tricornered/NDR1 (Trc/NDR1) regulates dendritic tiling and branching (8Emoto K. He Y. Ye B. Grueber W.B. Adler P.N. Jan L.Y. Jan Y.N. Cell. 2004; 119: 245-256Abstract Full Text Full Text PDF PubMed Scopus (184) Google Scholar) as well as morphogenesis of epidermal outgrowths (9Geng W. He B. Wang M. Adler P.N. Genetics. 2000; 156: 1817-1828PubMed Google Scholar). A second NDR kinase, Warts/Lats (Wts/Lts), in addition to a role in dendrite maintenance (10Emoto K. Parrish J.Z. Jan L.Y. Jan Y.N. Nature. 2006; 443: 210-213Crossref PubMed Scopus (165) Google Scholar), acts as a tumor suppressor, coordinating cell proliferation and apoptosis (11Huang J. Wu S. Barrera J. Matthews K. Pan D. Cell. 2005; 122: 421-434Abstract Full Text Full Text PDF PubMed Scopus (1309) Google Scholar), and phosphorylates the transcriptional co-activator, Yorkie, which stimulates transcription of cyclin E and the apoptosis inhibitor, Diap1. NDR phosphorylation of Yorkie promotes its binding to 14-3-3 proteins in the cytoplasm and hence inhibits its nuclear localization and function (12Oh H. Irvine K.D. Development. 2008; 135: 1081-1088Crossref PubMed Scopus (315) Google Scholar). Human cells express four NDR kinases, NDR1/2 and LATS1/2. NDR1/2 regulate centrosome duplication (13Hergovich A. Lamla S. Nigg E.A. Hemmings B.A. Mol. Cell. 2007; 25: 625-634Abstract Full Text Full Text PDF PubMed Scopus (91) Google Scholar), and LATS1/2 phosphorylate the Yorkie homologue, Yes-associated protein (YAP) (14Zhao B. Wei X. Li W. Udan R.S. Yang Q. Kim J. Xie J. Ikenoue T. Yu J. Li L. Zheng P. Ye K. Chinnaiyan A. Halder G. Lai Z.C. Guan K.L. Genes Dev. 2007; 21: 2747-2761Crossref PubMed Scopus (2031) Google Scholar), regulate organ size (15Dong J. Feldmann G. Huang J. Wu S. Zhang N. Comerford S.A. Gayyed M.F. Anders R.A. Maitra A. Pan D. Cell. 2007; 130: 1120-1133Abstract Full Text Full Text PDF PubMed Scopus (1721) Google Scholar), and have tumor suppressor functions, regulating progression through mitosis (16Hisaoka M. Tanaka A. Hashimoto H. Lab. Invest. 2002; 82: 1427-1435Crossref PubMed Scopus (81) Google Scholar).NDR kinases are members of the AGC group of protein kinases and are known to be activated by the binding of a Mps One Binder (MOB) protein partner and also via phosphorylation of the kinase domain activation loop and a C-terminal hydrophobic motif. In most AGC kinases, an upstream kinase phosphorylates both sites. However, NDR kinases autophosphorylate on the activation loop residue (Ser-281 in human NDR1) (17Stegert M.R. Tamaskovic R. Bichsel S.J. Hergovich A. Hemmings B.A. J. Biol. Chem. 2004; 279: 23806-23812Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar). They also possess a basic insert sequence just before the activation loop, which negatively regulates activity. MOB protein binding at the N terminus of the kinase relieves this inhibition, stimulates autophosphorylation, and activates the kinase (18Bichsel S.J. Tamaskovic R. Stegert M.R. Hemmings B.A. J. Biol. Chem. 2004; 279: 35228-35235Abstract Full Text Full Text PDF PubMed Scopus (107) Google Scholar). It has also been suggested that in S. pombe, MOB protein binding reduces an inhibitory self-association of the Sid2 NDR kinase (2Hou M.C. Guertin D.A. McCollum D. Mol. Cell. Biol. 2004; 24: 3262-3276Crossref PubMed Scopus (55) Google Scholar). STE20-like kinases further activate NDR kinases by phosphorylating the hydrophobic motif (Thr-444 in human NDR1) and also indirectly by phosphorylating MOB, increasing its binding affinity for the NDR kinase (18Bichsel S.J. Tamaskovic R. Stegert M.R. Hemmings B.A. J. Biol. Chem. 2004; 279: 35228-35235Abstract Full Text Full Text PDF PubMed Scopus (107) Google Scholar, 19Hirabayashi S. Nakagawa K. Sumita K. Hidaka S. Kawai T. Ikeda M. Kawata A. Ohno K. Hata Y. Oncogene. 2008; 27: 4281-4292Crossref PubMed Scopus (49) Google Scholar, 20Praskova M. Xia F. Avruch J. Curr. Biol. 2008; 18: 311-321Abstract Full Text Full Text PDF PubMed Scopus (299) Google Scholar). Scaffolding proteins are also known to enhance the NDR/MOB interaction (21Hergovich A. Stegert M.R. Schmitz D. Hemmings B.A. Nat. Rev. Mol. Cell Biol. 2006; 7: 253-264Crossref PubMed Scopus (253) Google Scholar, 22Chiba S. Ikeda M. Katsunuma K. Ohashi K. Mizuno K. Curr. Biol. 2009; 19: 675-681Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar), and MOB binding has been shown to target the NDR kinase to the plasma membrane, bringing it into close proximity with its STE20 kinase activator (23Hergovich A. Bichsel S.J. Hemmings B.A. Mol. Cell. Biol. 2005; 25: 8259-8272Crossref PubMed Scopus (90) Google Scholar, 24Hergovich A. Schmitz D. Hemmings B.A. Biochem. Biophys. Res. Commun. 2006; 345: 50-58Crossref PubMed Scopus (137) Google Scholar). Recently, phosphorylation of MOB1 by CDK1 in S. cerevisiae was reported to inhibit DBF2 kinase activity without interfering with the MOB1/DBF2 interaction (25König C. Maekawa H. Schiebel E. J. Cell Biol. 2010; 188: 351-368Crossref PubMed Scopus (60) Google Scholar). Regulation of NDR kinase activity is therefore complex and not yet fully understood.The protozoan parasite, Trypanosoma brucei, causes devastating diseases of humans (human African trypanosomiasis) and animals (N′gana) in sub-Saharan Africa. It has a digenetic life cycle, split between mammalian and tsetse fly hosts. In the mammalian bloodstream, the surface coat of the parasite undergoes antigenic variation, and therefore a vaccine against the parasite has so far proved elusive (26Kennedy P.G. Ann. Neurol. 2008; 64: 116-126Crossref PubMed Scopus (163) Google Scholar). Chemotherapies are available to treat the disease but can cause undesirable and even dangerous side effects, and only two drugs, melarsoprol and difluoromethylornithine (DFMO or eflornithine), both parenterally administered, are effective against late stage disease, characterized by the parasite crossing the blood-brain barrier (27Barrett M.P. Boykin D.W. Brun R. Tidwell R.R. Br. J. Pharmacol. 2007; 152: 1155-1171Crossref PubMed Scopus (259) Google Scholar). The recently approved nifurtimox-eflornithine combination therapy (28Priotto G. Kasparian S. Mutombo W. Ngouama D. Ghorashian S. Arnold U. Ghabri S. Baudin E. Buard V. Kazadi-Kyanza S. Ilunga M. Mutangala W. Pohlig G. Schmid C. Karunakara U. Torreele E. Kande V. Lancet. 2009; 374: 56-64Abstract Full Text Full Text PDF PubMed Scopus (383) Google Scholar) offers improved safety over DFMO monotherapy, but new drugs are still urgently needed.The trypanosome cell cycle displays several unusual features compared with mammalian cells, and it is likely that novel signal transduction pathways regulate these cellular processes. Elucidation of these pathways could identify novel drug targets. In particular, cytokinesis occurs via a distinctive physical mechanism (29Hammarton T.C. Mol. Biochem. Parasitol. 2007; 153: 1-8Crossref PubMed Scopus (114) Google Scholar, 30Hammarton T.C. Monnerat S. Mottram J.C. Curr. Opin. Microbiol. 2007; 10: 520-527Crossref PubMed Scopus (52) Google Scholar). Whereas mammalian cells divide using a contractile actomyosin ring, in the parasite, a furrow ingresses unidirectionally from the anterior to the posterior along the helical axis of the cell to divide the microtubule cytoskeleton and cell contents. Parasite cell division is highly precise; a number of single copy organelles must be replicated, segregated, and positioned accurately so that the furrow can pass between them to generate two daughter cells with equivalent complements of organelles. Any perturbation of cytokinesis results in disastrous consequences for the parasite (30Hammarton T.C. Monnerat S. Mottram J.C. Curr. Opin. Microbiol. 2007; 10: 520-527Crossref PubMed Scopus (52) Google Scholar), suggesting that inhibiting a parasite cytokinesis regulator might have therapeutic potential for human African trypanosomiasis.T. brucei expresses two MOB1 proteins (MOB1A and MOB1B) and two putative NDR kinases, PK50 and PK53 (31García-Salcedo J.A. Nolan D.P. Gijón P. Gómez-Rodriguez J. Pays E. Mol. Microbiol. 2002; 45: 307-319Crossref PubMed Scopus (23) Google Scholar, 32Hammarton T.C. Lillico S.G. Welburn S.C. Mottram J.C. Mol. Microbiol. 2005; 56: 104-116Crossref PubMed Scopus (52) Google Scholar). The MOB1 proteins play essential roles during furrow ingression in bloodstream stage trypanosomes, because their depletion leads to an accumulation of post-mitotic cells with partially ingressed furrows, resulting in deregulation of the cell cycle (32Hammarton T.C. Lillico S.G. Welburn S.C. Mottram J.C. Mol. Microbiol. 2005; 56: 104-116Crossref PubMed Scopus (52) Google Scholar). Previous data also suggested that MOB1A and PK50 can interact in procyclic (insect) form T. brucei (32Hammarton T.C. Lillico S.G. Welburn S.C. Mottram J.C. Mol. Microbiol. 2005; 56: 104-116Crossref PubMed Scopus (52) Google Scholar). The functions of the NDR kinases in trypanosomes have not been studied previously, although it was reported that PK50 is expressed in dividing parasite life cycle stages and is able to complement an Orb6 fission yeast mutant (31García-Salcedo J.A. Nolan D.P. Gijón P. Gómez-Rodriguez J. Pays E. Mol. Microbiol. 2002; 45: 307-319Crossref PubMed Scopus (23) Google Scholar). We therefore sought to determine whether the trypanosome NDR kinases were, like the MOB1 proteins, also key cell cycle regulators, to investigate the interactions between the NDR kinases and MOB1 proteins and to evaluate the potential of the NDR kinases as novel drug targets for human African trypanosomiasis.DISCUSSIONThis study demonstrates that the two trypanosome NDR kinases are essential enzymes in bloodstream stage T. brucei, thus providing genetic validation for these enzymes as putative novel drug targets. Depletion of either kinase disrupted cytokinesis, leading to cell cycle deregulation and cell death. The early increase in 2N2K cells, observed at 5 h post-induction of PK50 or PK53 RNAi, argues that depletion of either of these kinases has a specific effect on cytokinesis, because a growth arrest and large numbers of abnormal cells in the population were only observed after the appearance of increased numbers of 2N2K cells. The phenotypes observed following depletion of the two kinases were not identical, however. Following PK50 depletion, most of the abnormal cells had multiple nuclei and kinetoplasts, and, like the majority of the 2N2K cells, lacked cleavage furrows. Very few zoids (0N1K cells), cells commonly formed following aberrant cytokinesis, or 2N1K cells, which can form following defects in basal body or kinetoplast replication or segregation, or as a result of inaccurate furrowing, were present following PK50 RNAi. Together, these data indicate that depletion of PK50 prevents bloodstream stage cells from initiating cytokinesis. In comparison, following PK53 depletion, partially furrowed 2N2K cells accumulated. Additionally, at later time points, 2N1K cells and zoids accumulated in equal numbers, suggesting that some 2N2K cells did manage to divide, but with a loss of accuracy of furrow positioning. However, the accumulation of cells with multiple nuclei and kinetoplasts partitioned into multiple cell bodies with multiple partially ingressed cleavage furrows (Fig. 3C) indicates that completion of furrow ingression was blocked in other dividing cells.The different phenotypes observed for PK50 and PK53 could indicate that these kinases act sequentially in a cytokinesis signaling pathway, most likely with PK50 above PK53 in the cascade. However, neither kinase was able to use the other as a substrate in vitro (data not shown). Alternatively, because both kinases were apparently depleted by similar proportions following RNAi induction (Fig. 1C), the different phenotypes observed may reflect different threshold levels for their function at different stages of cytokinesis. Both kinases could conceivably be required for the initiation and ingression of furrowing, but the amount of residual PK53 protein was sufficient for initiation of cytokinesis, whereas that of PK50 was not.Immunofluorescence was performed for PK50 and PK53 across the cell cycle. However, despite the RNAi phenotypes obtained, no specific localization to the cytokinesis furrow was observed. One of the problems inherent in performing immunofluorescence for any kinase is that, without a phosphospecific antibody, it is not possible to distinguish the active protein kinase pool from inactive forms. Hence, the total kinase pool may mask a specific localization of the active pool. It is also possible that these kinases do not localize to the cytokinesis furrow themselves but instead phosphorylate a substrate, which as a result then translocates to the furrow to bring about cytokinesis. The more restricted localization of PK53 during abscission and early stages in the cell cycle is intriguing but not easy to explain without further study. However, this finding may suggest additional roles for this kinase that have not been revealed by our analysis to date of the RNAi cell lines.PK50 and PK53 join a growing list of T. brucei signaling proteins with direct roles in cytokinesis. A chromosomal passenger complex has been reported to regulate the mitosis to cytokinesis transition (51Li Z. Lee J.H. Chu F. Burlingame A.L. Günzl A. Wang C.C. Plos ONE. 2008; 3: e2354Crossref PubMed Scopus (65) Google Scholar, 52Li Z. Umeyama T. Wang C.C. Plos ONE. 2008; 3: e3814Crossref PubMed Scopus (43) Google Scholar, 53Li Z. Umeyama T. Wang C.C. Plos Pathogens. 2009; 5: e1000575Crossref PubMed Scopus (53) Google Scholar) in bloodstream and procyclic trypanosomes. TbRACK1 is required for furrow ingression in procyclic T. brucei, although its depletion in bloodstream trypanosomes appears to prevent cytokinesis initiation (54Rothberg K.G. Burdette D.L. Pfannstiel J. Jetton N. Singh R. Ruben L. J. Biol. Chem. 2006; 281: 9781-9790Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar), and TbRACK1 is thought to facilitate continued translation during cytokinesis (55Regmi S. Rothberg K.G. Hubbard J.G. Ruben L. Mol. Microbiol. 2008; 70: 724-745Crossref PubMed Scopus (19) Google Scholar). The Polo-like kinase, PLK1, is essential for furrow ingression in the bloodstream form (56Hammarton T.C. Kramer S. Tetley L. Boshart M. Mottram J.C. Mol. Microbiol. 2007; 65: 1229-1248Crossref PubMed Scopus (82) Google Scholar), as is the F-box protein, CFB2 (57Benz C. Clayton C.E. Mol. Biochem. Parasitol. 2007; 156: 217-224Crossref PubMed Scopus (22) Google Scholar), and MOB1 (32Hammarton T.C. Lillico S.G. Welburn S.C. Mottram J.C. Mol. Microbiol. 2005; 56: 104-116Crossref PubMed Scopus (52) Google Scholar). MOB1 also plays roles in accuracy of furrow ingression in procyclic trypanosomes. RNAi of each of these regulators gives slightly different phenotypes, and given the incomplete nature and varied kinetics of protein knockdown, as well as the relatively few protein/protein interactions that have been determined between these regulators, it is difficult to interpret the temporal order of the signaling events that control trypanosome cytokinesis at present.This study provides the first biochemical analysis of the trypanosome NDR kinases. Despite MOB1 proteins being known activators of NDR kinases in a wide range of organisms, the recombinant trypanosome NDR kinases were active in the absence of MOB1 activation or phosphorylation by upstream kinases. For PK50, this property was not unduly influenced by the affinity tag, but for PK53, the C-terminal His6 tag led to autophosphorylation being favored over MBP phosphorylation (supplemental Fig. S3), although the reason for this is not clear at present. In mammalian cells, protein phosphatase 2A has been shown to remove the activatory phosphate groups (in the activation loop and C-terminal hydrophobic motif) to inhibit NDR1 (21Hergovich A. Stegert M.R. Schmitz D. Hemmings B.A. Nat. Rev. Mol. Cell Biol. 2006; 7: 253-264Crossref PubMed Scopus (253) Google Scholar, 58Millward T.A. Hess D. Hemmings B.A. J. Biol. Chem. 1999; 274: 33847-33850Abstract Full Text Full Text PDF PubMed Scopus (88) Google Scholar). It will be interesting to discover whether the activities of the trypanosome kinases are regulated in a similar manner in vivo.Because of GST:MOB1A/B being unable to bind to recombinant NDR kinases, we were unable to determine whether MOB1 binding might have increased the activity of the kinases still further. It is possible that the GST tag on the MOB1 proteins prevented them from interacting with the NDR kinases, or that GST:MOB1 was not folded correctly, possibilities that we have not been able to rule out at present. However, we were unable to co-immunoprecipitate the NDR kinases and MOB1A/B proteins in vivo either, at least under the conditions used in this study, suggesting the intriguing possibility that the trypanosome NDR kinases are regulated by different mechanisms in the trypanosome compared with other eukaryotes. The reason for the discrepancy between the interaction data generated here and previous data indicating an interaction between PK50 and MOB1Aty in procyclic trypanosomes is unclear and not possible to determine, given that supplies of the original rabbit anti-PK50 antibody have been exhausted. Although technical issues with the original experiment cannot be ruled out, it is also possible that the rabbit anti-PK50 antibody (31García-Salcedo J.A. Nolan D.P. Gijón P. Gómez-Rodriguez J. Pays E. Mol. Microbiol. 2002; 45: 307-319Crossref PubMed Scopus (23) Google Scholar) used previously recognized an ∼50-kDa protein in trypanosome cell lysates that was not PK50, since a PK50 RNAi cell line was not available at the time to confirm its in vivo specificity. Because of the more comprehensive analysis of the NDR kinase/MOB1 interactions, we believe that the data set presented here is the more robust of the two analyses.Given the data presented here, we favor a hypothesis whereby the NDR kinases function largely independently of the MOB1 proteins. However, we cannot completely rule out that interactions between subpopulations of the MOB1 proteins and NDR kinases do occur, perhaps very transiently. A specific phosphorylation event or a scaffold protein could be required for an NDR/MOB1 interaction to occur. Phosphorylation of mammalian MOB1 at Thr-74 by the STE20 kinase, MST2, is essential for its interaction with, and activation of, NDR1 (19Hirabayashi S. Nakagawa K. Sumita K. Hidaka S. Kawai T. Ikeda M. Kawata A. Ohno K. Hata Y. Oncogene. 2008; 27: 4281-4292Crossref PubMed Scopus (49) Google Scholar), and in Drosophila, phosphorylation of MOBKL1A/B at Thr-12 and Thr-35 by MST1/2 kinases increases their affinity for LATS1 (20Praskova M. Xia F. Avruch J. Curr. Biol. 2008; 18: 311-321Abstract Full Text Full Text PDF PubMed Scopus (299) Google Scholar). Scaffolding proteins containing multiple HEAT/Armadillo-like repeats can promote the activation of NDR kinases in a variety of organisms (21Hergovich A. Stegert M.R. Schmitz D. Hemmings B.A. Nat. Rev. Mol. Cell Biol. 2006; 7: 253-264Crossref PubMed Scopus (253) Google Scholar). In Drosophila, Trc/NDR1 and MOB2 were shown to interact with the Furry scaffolding protein, leading to activation of NDR1 (22Chiba S. Ikeda M. Katsunuma K. Ohashi K. Mizuno K. Curr. Biol. 2009; 19: 675-681Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar, 59He Y. Fang X. Emoto K. Jan Y.N. Adler P.N. Mol. Biol. Cell. 2005; 16: 689-700Crossref PubMed Scopus (54) Google Scholar). In this system, the MOB2/Furry interaction was dependent on phosphorylation of MOB2. A recent phosphoproteomic study of bloodstream T. brucei (60Nett I.R. Martin D.M. Miranda-Saavedra D. Lamont D. Barber J.D. Mehlert A. Ferguson M.A. Mol. Cell. Proteomics. 2009; 8: 1527-1538Abstract Full Text Full Text PDF PubMed Scopus (133) Google Scholar) revealed that Ser-23 of MOB1A (Ser-40 in MOB1B) was phosphorylated in vivo, but further investigation will be required to determine the significance of this modification.PK50 and PK53 have been shown to be phosphorylated in vivo on Ser-434 and Ser-298, respectively (60Nett I.R. Martin D.M. Miranda-Saavedra D. Lamont D. Barber J.D. Mehlert A. Ferguson M.A. Mol. Cell. Proteomics. 2009; 8: 1527-1538Abstract Full Text Full Text PDF PubMed Scopus (133) Google Scholar). Ser-298 of PK53 is the activation loop serine, whereas Ser-434 of PK50 corresponds to the most C-terminal serine residue and is not conserved in other NDR kinases. Hence, this could indicate a trypanosome-specific phosphoregulatory event. Phosphorylation of the activation loop serine (Ser-251) or the hydrophobic motif threonine (Thr-408) of PK50 was not detected in the study by Nett et al. (60Nett I.R. Martin D.M. Miranda-Saavedra D. Lamont D. Barber J.D. Mehlert A. Ferguson M.A. Mol. Cell. Proteomics. 2009; 8: 1527-1538Abstract Full Text Full Text PDF PubMed Scopus (133) Google Scholar. No other in vivo phosphosites were detected for PK53, and the C-terminal hydrophobic motif is not conserved in this protein, again suggesting trypanosome-specific regulation of this kinase may occur. In this study, recombinant GST:PK50 was shown to be phosphorylated on Ser-434, mimicking the in vivo situation, and Ser-251 and Thr-408 were identified as putative phosphorylation sites. Additional phosphorylation sites were identified in the recombinant protein at Tyr-25, Ser-36, Ser-216/Thr-217/Ser-218, Thr-394, and Ser-402. GST:PK53 was found to be phosphorylated on Ser-298, mimicking the in vivo data, and additional phosphorylation sites were detected at Ser-2/Thr-3, Thr-17/Ser-20/Ser-23, Ser-31, Ser-33, Thr-48, Ser-50/Thr-53, Ser-114/Ser-118, Ser-218, and Tyr-458. The in vivo significance of these phosphorylations is not clear at present. However, although the presence of these phosphosites suggests that PK50 and PK53 are able to autophosphorylate on these residues in vitro, this does not rule out that these modifications are performed by an upstream kinase in vivo. NDR2 kinase is known to autophosphorylate its hydrophobic motif threonine in vitro, although this event is primarily performed by the STE20 kinase MST3 in vivo (17Stegert M.R. Tamaskovic R. Bichsel S.J. Hergovich A. Hemmings B.A. J. Biol. Chem. 2004; 279: 23806-23812Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar, 61Stegert M.R. Hergovich A. Tamaskovic R. Bichsel S.J. Hemmings B.A. Mol. Cell. Biol. 2005; 25: 11019-11029Crossref PubMed Scopus (126) Google Scholar). Additionally, although it is likely that some of these residues, e.g. Ser-251 (activation loop) and Thr-408 (hydrophobic motif) in PK50, are phosphorylated in vivo, similar to NDR kinases in other organisms, it is not yet clear whether the other phosphosites identified represent bone fide in vivo modifications of the NDR kinases.NDR kinases are known to phosphorylate substrates with the consensus sequence RXXS (3Chen C.T. Feoktistova A. Chen J.S. Shim Y.S. Clifford D.M. Gould K.L. McCollum D. Curr. Biol. 2008; 18: 1594-1599Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar, 50Mah A.S. Elia A.E. Devgan G. Ptacek J. Schutkowski M. Snyder M. Yaffe M.B. Deshaies R.J. BMC Biochem. 2005; 6: 22Crossref PubMed Scopus (85) Google Scholar), although relatively few in vivo substrates have been identified for these kinases. Our substrate screen showed our kinases to favor peptides with the RXXS consensus sequence. Both kinases phosphorylated the peptide KKLNRTLSFAEPG, but GST:PK50 was unable to phosphorylate the GST:PK53 substrate RARTLSFAEPG, whereas the GST:PK50-preferred substrate, KKLRRTLSVA, was relatively poorly phosphorylated by GST:PK53. The in vivo substrate specificities of these enzymes are still unknown, but it is clear from the lethal RNAi phenotypes obtained upon depletion of either kinase that they are not redundant enzymes in bloodstream stage T. brucei. Biochemical characterization also revealed differences between these kinases. Although GST:PK50 and GST:PK53 were shown to have similar cation preferences (for both kinases, the in vitro activity dropped dramatically in presence of Mg2+ and was restored by Mn2+), they differ in their catalytic pocket as demonstrated by their different affinities for ATP and the different potencies of staurosporine. These findings are consistent with the presence of a 36-amino acid insertion in the catalytic pocket of PK53 compared with PK50.PK50 and PK53 are about as similar to each other at the amino acid level (∼30% identity and ∼45% similarity, rising to ∼35% identity and ∼50% similarity over the kinase domain) as they are to human NDR/LATS kinases. It has been reported that for kinases sharing >60% identity over their catalytic domain, there is a high likelihood that these enzymes will be inhibited by the same group of low molecular weight compounds (62Vieth M. Higgs R.E. Robertson D.H. Shapiro M. Gragg E.A. Hemmerle H. Biochim. Biophys. Acta. 2004; 1697: 243-257Crossref PubMed Scopus (202) Google Scholar) and suggested that there may therefore be a higher probability of being able to identify selective small molecule inhibitors for kinases sharing <60% identity (43Naula C. Parsons M. Mottram J.C. Biochim. Biophys. Acta. 2005; 1754: 151-159Crossref PubMed Scopus (184) Google Scholar). The difference in affinity of GST:PK50 and GST:PK53 for the generic kinase inhibitor staurosporine provides preliminary evidence that selective inhibition of these kinases is possible. Indeed, using recombinant GST:PK50 and GST:PK53, high throughput screening for small molecule inhibitors has now been performed. 4R. Grimaldi, J. Ma, L. Cleghorn, C. Stockdale, C. Benz, A. Woodland, P. Wyatt, T. C. Hammarton, and J. Frearson, manuscript in preparation. Inhibitors based on distinct scaffolds with sub-micromolar IC50 values were identified for each recombinant kinase that were selective against the majority of human kinases tested. Work is ongoing to determine the trypanosome activity and mode of action of these inhibitors. IntroductionNuclear DBF2-related (NDR) 3The abbreviations used are: NDRnuclear DBF2-relatedGSTglutathione S-transferaseMBPmyelin basic proteinMOBMps One BinderRACErapid amplification of cDNA endsMBPmyelin basic proteinRNAiRNA interferenceDAPI4,6-diamidino-2-phenylindoleCHAPS3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acidMOPS4-morpholinepropanesulfonic acid. kinases are important regulators of a variety of cellular processes, including centrosome duplication, mitotic exit, cytokinesis, morphogenesis, and cell growth and development. The founding member of this family, DBF2, is part of the mitotic exit network in Saccharomyces cerevisiae (1Mah A.S. Jang J. Deshaies R.J. Proc. Natl. Acad. Sci. U.S.A. 2001; 98: 7325-7330Crossref PubMed Scopus (160) Google Scholar), although its counterpart in fission yeast, Sid2, is a component of the septation initiation network (2Hou M.C. Guertin D.A. McCollum D. Mol. Cell. Biol. 2004; 24: 3262-3276Crossref PubMed Scopus (55) Google Scholar). Both kinases were recently shown to phosphorylate CDC14 phosphatase, promoting its retention in the cytoplasm and allowing it to inactivate mitotic cyclin/cyclin-dependent kinase and promote cell cycle progression (3Chen C.T. Feoktistova A. Chen J.S. Shim Y.S. Clifford D.M. Gould K.L. McCollum D. Curr. Biol. 2008; 18: 1594-1599Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar, 4Mohl D.A. Huddleston M.J. Collingwood T.S. Annan R.S. Deshaies R.J. J. Cell Biol. 2009; 184: 527-539Crossref PubMed Scopus (81) Google Scholar). Other NDR kinases in these yeasts (CBK1 in S. cerevisiae and Orb6 in Schizosaccharomyces pombe) regulate cellular morphogenesis (5Weiss E.L. Kurischko C. Zhang C. Shokat K. Drubin D.G. Luca F.C. J. Cell Biol. 2002; 158: 885-900Crossref PubMed Scopus (163) Google Scholar, 6Hou M.C. Wiley D.J. Verde F. McCollum D. J. Cell Sci. 2003; 116: 125-135Crossref PubMed Scopus (69) Google Scholar). In contrast, the fungal pathogen, Candida albicans, expresses only a single DBF2 orthologue, which is essential for viability and appears to have evolved multiple functions, being required for mitotic spindle organization, actomyosin ring contraction, and hyphal growth (7González-Novo A. Labrador L. Pablo-Hernando M.E. Correa-Bordes J. Sánchez M. Jiménez J. Vázquez de Aldana C.R. Mol. Microbiol. 2009; 72: 1364-1378Crossref PubMed Scopus (16) Google Scholar). In Drosophila, Tricornered/NDR1 (Trc/NDR1) regulates dendritic tiling and branching (8Emoto K. He Y. Ye B. Grueber W.B. Adler P.N. Jan L.Y. Jan Y.N. Cell. 2004; 119: 245-256Abstract Full Text Full Text PDF PubMed Scopus (184) Google Scholar) as well as morphogenesis of epidermal outgrowths (9Geng W. He B. Wang M. Adler P.N. Genetics. 2000; 156: 1817-1828PubMed Google Scholar). A second NDR kinase, Warts/Lats (Wts/Lts), in addition to a role in dendrite maintenance (10Emoto K. Parrish J.Z. Jan L.Y. Jan Y.N. Nature. 2006; 443: 210-213Crossref PubMed Scopus (165) Google Scholar), acts as a tumor suppressor, coordinating cell proliferation and apoptosis (11Huang J. Wu S. Barrera J. Matthews K. Pan D. Cell. 2005; 122: 421-434Abstract Full Text Full Text PDF PubMed Scopus (1309) Google Scholar), and phosphorylates the transcriptional co-activator, Yorkie, which stimulates transcription of cyclin E and the apoptosis inhibitor, Diap1. NDR phosphorylation of Yorkie promotes its binding to 14-3-3 proteins in the cytoplasm and hence inhibits its nuclear localization and function (12Oh H. Irvine K.D. Development. 2008; 135: 1081-1088Crossref PubMed Scopus (315) Google Scholar). Human cells express four NDR kinases, NDR1/2 and LATS1/2. NDR1/2 regulate centrosome duplication (13Hergovich A. Lamla S. Nigg E.A. Hemmings B.A. Mol. Cell. 2007; 25: 625-634Abstract Full Text Full Text PDF PubMed Scopus (91) Google Scholar), and LATS1/2 phosphorylate the Yorkie homologue, Yes-associated protein (YAP) (14Zhao B. Wei X. Li W. Udan R.S. Yang Q. Kim J. Xie J. Ikenoue T. Yu J. Li L. Zheng P. Ye K. Chinnaiyan A. Halder G. Lai Z.C. Guan K.L. Genes Dev. 2007; 21: 2747-2761Crossref PubMed Scopus (2031) Google Scholar), regulate organ size (15Dong J. Feldmann G. Huang J. Wu S. Zhang N. Comerford S.A. Gayyed M.F. Anders R.A. Maitra A. Pan D. Cell. 2007; 130: 1120-1133Abstract Full Text Full Text PDF PubMed Scopus (1721) Google Scholar), and have tumor suppressor functions, regulating progression through mitosis (16Hisaoka M. Tanaka A. Hashimoto H. Lab. Invest. 2002; 82: 1427-1435Crossref PubMed Scopus (81) Google Scholar).NDR kinases are members of the AGC group of protein kinases and are known to be activated by the binding of a Mps One Binder (MOB) protein partner and also via phosphorylation of the kinase domain activation loop and a C-terminal hydrophobic motif. In most AGC kinases, an upstream kinase phosphorylates both sites. However, NDR kinases autophosphorylate on the activation loop residue (Ser-281 in human NDR1) (17Stegert M.R. Tamaskovic R. Bichsel S.J. Hergovich A. Hemmings B.A. J. Biol. Chem. 2004; 279: 23806-23812Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar). They also possess a basic insert sequence just before the activation loop, which negatively regulates activity. MOB protein binding at the N terminus of the kinase relieves this inhibition, stimulates autophosphorylation, and activates the kinase (18Bichsel S.J. Tamaskovic R. Stegert M.R. Hemmings B.A. J. Biol. Chem. 2004; 279: 35228-35235Abstract Full Text Full Text PDF PubMed Scopus (107) Google Scholar). It has also been suggested that in S. pombe, MOB protein binding reduces an inhibitory self-association of the Sid2 NDR kinase (2Hou M.C. Guertin D.A. McCollum D. Mol. Cell. Biol. 2004; 24: 3262-3276Crossref PubMed Scopus (55) Google Scholar). STE20-like kinases further activate NDR kinases by phosphorylating the hydrophobic motif (Thr-444 in human NDR1) and also indirectly by phosphorylating MOB, increasing its binding affinity for the NDR kinase (18Bichsel S.J. Tamaskovic R. Stegert M.R. Hemmings B.A. J. Biol. Chem. 2004; 279: 35228-35235Abstract Full Text Full Text PDF PubMed Scopus (107) Google Scholar, 19Hirabayashi S. Nakagawa K. Sumita K. Hidaka S. Kawai T. Ikeda M. Kawata A. Ohno K. Hata Y. Oncogene. 2008; 27: 4281-4292Crossref PubMed Scopus (49) Google Scholar, 20Praskova M. Xia F. Avruch J. Curr. Biol. 2008; 18: 311-321Abstract Full Text Full Text PDF PubMed Scopus (299) Google Scholar). Scaffolding proteins are also known to enhance the NDR/MOB interaction (21Hergovich A. Stegert M.R. Schmitz D. Hemmings B.A. Nat. Rev. Mol. Cell Biol. 2006; 7: 253-264Crossref PubMed Scopus (253) Google Scholar, 22Chiba S. Ikeda M. Katsunuma K. Ohashi K. Mizuno K. Curr. Biol. 2009; 19: 675-681Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar), and MOB binding has been shown to target the NDR kinase to the plasma membrane, bringing it into close proximity with its STE20 kinase activator (23Hergovich A. Bichsel S.J. Hemmings B.A. Mol. Cell. Biol. 2005; 25: 8259-8272Crossref PubMed Scopus (90) Google Scholar, 24Hergovich A. Schmitz D. Hemmings B.A. Biochem. Biophys. Res. Commun. 2006; 345: 50-58Crossref PubMed Scopus (137) Google Scholar). Recently, phosphorylation of MOB1 by CDK1 in S. cerevisiae was reported to inhibit DBF2 kinase activity without interfering with the MOB1/DBF2 interaction (25König C. Maekawa H. Schiebel E. J. Cell Biol. 2010; 188: 351-368Crossref PubMed Scopus (60) Google Scholar). Regulation of NDR kinase activity is therefore complex and not yet fully understood.The protozoan parasite, Trypanosoma brucei, causes devastating diseases of humans (human African trypanosomiasis) and animals (N′gana) in sub-Saharan Africa. It has a digenetic life cycle, split between mammalian and tsetse fly hosts. In the mammalian bloodstream, the surface coat of the parasite undergoes antigenic variation, and therefore a vaccine against the parasite has so far proved elusive (26Kennedy P.G. Ann. Neurol. 2008; 64: 116-126Crossref PubMed Scopus (163) Google Scholar). Chemotherapies are available to treat the disease but can cause undesirable and even dangerous side effects, and only two drugs, melarsoprol and difluoromethylornithine (DFMO or eflornithine), both parenterally administered, are effective against late stage disease, characterized by the parasite crossing the blood-brain barrier (27Barrett M.P. Boykin D.W. Brun R. Tidwell R.R. Br. J. Pharmacol. 2007; 152: 1155-1171Crossref PubMed Scopus (259) Google Scholar). The recently approved nifurtimox-eflornithine combination therapy (28Priotto G. Kasparian S. Mutombo W. Ngouama D. Ghorashian S. Arnold U. Ghabri S. Baudin E. Buard V. Kazadi-Kyanza S. Ilunga M. Mutangala W. Pohlig G. Schmid C. Karunakara U. Torreele E. Kande V. Lancet. 2009; 374: 56-64Abstract Full Text Full Text PDF PubMed Scopus (383) Google Scholar) offers improved safety over DFMO monotherapy, but new drugs are still urgently needed.The trypanosome cell cycle displays several unusual features compared with mammalian cells, and it is likely that novel signal transduction pathways regulate these cellular processes. Elucidation of these pathways could identify novel drug targets. In particular, cytokinesis occurs via a distinctive physical mechanism (29Hammarton T.C. Mol. Biochem. Parasitol. 2007; 153: 1-8Crossref PubMed Scopus (114) Google Scholar, 30Hammarton T.C. Monnerat S. Mottram J.C. Curr. Opin. Microbiol. 2007; 10: 520-527Crossref PubMed Scopus (52) Google Scholar). Whereas mammalian cells divide using a contractile actomyosin ring, in the parasite, a furrow ingresses unidirectionally from the anterior to the posterior along the helical axis of the cell to divide the microtubule cytoskeleton and cell contents. Parasite cell division is highly precise; a number of single copy organelles must be replicated, segregated, and positioned accurately so that the furrow can pass between them to generate two daughter cells with equivalent complements of organelles. Any perturbation of cytokinesis results in disastrous consequences for the parasite (30Hammarton T.C. Monnerat S. Mottram J.C. Curr. Opin. Microbiol. 2007; 10: 520-527Crossref PubMed Scopus (52) Google Scholar), suggesting that inhibiting a parasite cytokinesis regulator might have therapeutic potential for human African trypanosomiasis.T. brucei expresses two MOB1 proteins (MOB1A and MOB1B) and two putative NDR kinases, PK50 and PK53 (31García-Salcedo J.A. Nolan D.P. Gijón P. Gómez-Rodriguez J. Pays E. Mol. Microbiol. 2002; 45: 307-319Crossref PubMed Scopus (23) Google Scholar, 32Hammarton T.C. Lillico S.G. Welburn S.C. Mottram J.C. Mol. Microbiol. 2005; 56: 104-116Crossref PubMed Scopus (52) Google Scholar). The MOB1 proteins play essential roles during furrow ingression in bloodstream stage trypanosomes, because their depletion leads to an accumulation of post-mitotic cells with partially ingressed furrows, resulting in deregulation of the cell cycle (32Hammarton T.C. Lillico S.G. Welburn S.C. Mottram J.C. Mol. Microbiol. 2005; 56: 104-116Crossref PubMed Scopus (52) Google Scholar). Previous data also suggested that MOB1A and PK50 can interact in procyclic (insect) form T. brucei (32Hammarton T.C. Lillico S.G. Welburn S.C. Mottram J.C. Mol. Microbiol. 2005; 56: 104-116Crossref PubMed Scopus (52) Google Scholar). The functions of the NDR kinases in trypanosomes have not been studied previously, although it was reported that PK50 is expressed in dividing parasite life cycle stages and is able to complement an Orb6 fission yeast mutant (31García-Salcedo J.A. Nolan D.P. Gijón P. Gómez-Rodriguez J. Pays E. Mol. Microbiol. 2002; 45: 307-319Crossref PubMed Scopus (23) Google Scholar). We therefore sought to determine whether the trypanosome NDR kinases were, like the MOB1 proteins, also key cell cycle regulators, to investigate the interactions between the NDR kinases and MOB1 proteins and to evaluate the potential of the NDR kinases as novel drug targets for human African trypanosomiasis.