In the present study, we concluded a preliminary functional analysis of all the structural homologues of cyclin and CRK identified in T. brucei [1, 2]. From the previous investigations, CycE1/CRK1 is known to regulate the G1/S passage [24–26, 28], CycE1/CRK2 controls the posterior extension in the procyclic form during G1/S transition [28, 39], whereas CycE1/CRK3 and CycB2/CRK3 are involved in regulating the passage from the G2 phase to the mitotic phase [24, 26–28]. The new experimental data from the current study covered the rest of the homologues that have not yet been analyzed. They added one more protein kinase CRK9 that plays an essential role in promoting the growth of procyclic form but apparently not the bloodstream form of trypanosome. For the rest of the newly as well as the previously identified homologues of cyclins and CRKs, their knockdowns showed little apparent effect on trypanosome cell growth. Among those CRK's whose knockdowns in the present study did not register any phenotype, their sequences indicate that they either lack or have mutated certain residues in the glycine-rich box or the conserved glutamate residue immediately downstream of the gatekeeper that are known to be a part of the kinase active site (see Additional File 3). They could be thus either pseudogenes or their protein products may not possess any kinase activity. If they still play any role at all in cell cycle regulation, they might perform either an auxiliary or redundant function.
In other eukaryotic organisms, certain cyclin/Cdk pairs are dispensable, because they are known to play an auxiliary role. For example, cyclin D/Cdk4 in mammalian cells promotes exit from G0 into G1 by countering the activity of anti-proliferating agent such as Rb and p21 [40–42]. These auxiliary cyclin/Cdk pairs are generally required for stress responses and adaptive conditions [6, 43–45]. Thus, CycE2, E3, E4, B1, B3 and 10-11, CRK4, 6-8,10-12, found without an apparent function in cell growth in the previous and present studies, could be the auxiliary proteins dispensable for cell cycle regulation in T. brucei. Given that the latest bioinformatics analysis of the T. brucei kinome appears complete, this work may have completed the identification of all the primary cell cycle regulators in trypanosome.
Further analysis of CRK9 indicated that the procyclic form is enriched in the G2/M phase upon the depletion of CRK9. The protein is localized to the nucleus and binds to CycB2 in vitro. These indications suggest that CRK9 may form complex with CycB2 and play an essential role in regulating the G2/M passage in procyclic-form T. brucei.
The arrested mitosis in procyclic cells depleted of CRK9, however, does not lead to appreciable formation of anucleated cells as observed previously from depleting the mitotic kinase CRK3 . This is attributed to the fact that CRK9 plays also another role in regulating cytokinesis by controlling kinetoplast replication/segregation in the procyclic trypanosome. Thus, in addition to a mitotic arrest, CRK9 ablation leads also to a failure in kinetoplast replication/segregation, which causes a block of basal body segregation and cytokinetic initiation in the procyclic form. This blockade is apparently also responsible for a drastic morphological change of cells to a round shape, which has not yet been observed previously when cytokinesis was inhibited in the procyclic form of T. brucei by other means [32, 33, 35, 46]. Thus, CRK9 may be involved in cytokinetic regulation with a mechanism completely different from that of the phosphatases PP1 or PP2A, targets of okadeic acid inhibition, (Das et al., 1994), TbAUK1 [32, 46] and TbPLK . These enzymes are known to control cytokinesis, but their depletion does not cause a morphological change of the procyclic cells. Okadaic acid, the inhibitor of PP1 and PP2A, is known to arrest the cytokinesis of procyclic cells resulting in a single kinetoplast, a single basal body, a single flagellum but multiple nuclei in each cell. Thus, cytokinesis is blocked at a very early phase prior to kinetoplast/basal body replication by this inhibitor. A depletion of TbAUK1, however, results in cytokinesis-arrested procyclic cells with two widely segregated kinetoplast/basal body pairs, indicating that TbAUK1 acts downstream from CRK9. A TbPLK depletion leads to cytokinesis-arrested cells with multiple nuclei, kinetoplasts, basal bodies, flagella, and is known to play a role downstream from that of TbAUK1 in regulating cytokinesis [33, 46]. Thus in a series of regulators of cytokinetic initiation, their order of actions is most likely; the phospatases→CRK9→TbAUK1→TbPLK. Furthermore, an active extension of microtubule corset toward the posterior end is known to take place only during the G1/S transition in the procyclic form [16, 28, 39, 47]. It was postulated that this extension is coupled with the segregation between the two kinetoplast/basal body pairs. Assuming that the microtubule extension might continue while the kinetoplast segregation is inhibited by a depletion of CRK9, a distortion of cellular morphology could result from such a lack of coordination. As for the question on how a nuclear protein CRK9 plays a role in controlling basal body/kinetoplast replication/segregation, we don't have a ready answer yet. But we could hypothesize that CRK9 is a part of a signaling network coordinating basal body replication/segregation with nuclear duplication. It may phosphorylate a yet un-identified protein(s) that promotes basal body replication/segregation in procyclic cells. A pursuit of the mechanism of CRK9 regulation of cytokinesis will be thus a major interest in our future investigations.
CRK9 thus distinguishes itself from the other CRKs in T. brucei by playing essential roles in controlling both mitosis and cytokinesis in the procyclic trypanosome. There is only one other protein kinase in T. brucei known to play essential roles in both mitosis and cytokinesis. It is an orthologue of Aurora B, TbAUK1, which does not belong to the CRK family [32, 46]. Considerable knowledge has been accumulated on this protein kinase in recent years. It is a component of the unique chromosomal passenger complex that is confined in the nucleus during mitosis, but migrates out of the nucleus and trans-localize to the potential cleavage furrow on the dorsal side of the cell in telophase to initiate cytokinesis . CRK9 could have a similar pattern of trans-localization like that of TbAUK1 toward the end of mitosis, i.e., to move out of the nucleus and become associated with the organelles involved with cytokinetic initiation. Only a detailed further analysis of CRK9 localization in a synchronized cell population would shed some light on this interesting possibility.
A depletion of TbAUK1 leads to an elimination of the spindle structure in the nucleus, mitotic arrest and cytokinetic block with two kinetoplast/basal body pairs in the procyclic cells, which are similar, albeit not identical, to that observed from CRK9 depletion. The multiple functions of TbAUK1 can be, however, observed in both procyclic and bloodstream forms of trypanosome [32, 46]. A depletion of TbAUK1 from the bloodstream form resulted in multiple kinetoplasts/basal bodies/flagella, reflecting a dissociation between organelle multiplication and cytokinetic initiation in bloodstream form. The lack of a role of CRK9 in controlling cytokinesis in the bloodstream form could be also attributed to such a dissociation, i.e., cytokinesis could still proceed while the kinetoplast replication/segregation has failed.
The absence of any effect on the mitosis in bloodstream form from knocking down CRK9 is a little more difficult to comprehend at the present time. But one did observe different phenotypes between the two forms when mitotic cyclin B2 or mitotic protein kinase CRK3 was knocked down in the previous studies [24, 26, 27]. Mitosis in the procyclic form was stopped with a nucleus approximately twice the size of that in the G1 phase. But the same knockdowns in bloodstream form resulted in an extremely large aggregate of apparently un-segregated nuclei, suggesting that, without a mitotic exit, the cells can still enter a new G1 phase repeatedly. A check point on mitosis could be thus absent from the bloodstream form though it may be still present and functioning in the procyclic form. Assuming that the role of CRK9 in regulating G2/M passage may depend on its interaction with some of the yet unidentified check point proteins, it could explain why CRK9 functions in one but not the other form.