The novel human protein RB6K is shown to be a mitotic KLP that has a distinct function in the final stages of the cell cycle. Its expression is highly regulated to peak during M phase, with low intracellular mRNA and protein concentrations during G1
and S phase (Fig. and ). This is highly similar to the expression kinetics of cyclin B, a protein of which the expression has been extensively studied in relation to regulation of the cell cycle (23
). The observed fluctuations of the mRNA levels are at least in part based on cell cycle-dependent promoter regulation (Fig. and ). Similar to the cell cycle-regulated promoters of cdc25C, cyclin A, cdc2
, and plk
), the RB6K promoter contains a CDE-CHR element (Fig. B) that is known to induce repression of transcription during G1
). In contrast to typical cell cycle-regulatory proteins of which the intracellular concentrations are regulated by ubiquitin-mediated proteolysis (12
), RB6K protein shows a rather gradual decline that continues in early G1
(Fig. B). Indeed, no consensus sequences for ubiquitination were found, and no changes in molecular weight indicative of ubiquitination were observed (Fig. B and data not shown). The importance of correctly tuned de novo synthesis and proteolysis of RB6K is illustrated by the finding that constitutive overexpression of RB6K in interphase cells leads to cell death, probably caused by a bundling of interphase microtubuli (Fig. C, panels 3 and 4). This microtubule binding was not observed for the endogenous RB6K in interphase cells. Therefore, incorrectly regulated expression of RB6K might directly interfere with interphase microtubule function. The observed tight expression regulation of RB6K during the cell cycle represents a means by which the cell can adapt its transport capacity to the specific requirements of the various stages of the cell cycle.
Human RB6K showed 91% homology and 86% identity to mouse RB6K (11
) and was therefore considered to be its human equivalent. Murine RB6K was initially isolated and characterized as a protein tightly binding the Golgi-localized GTPase Rab6. Overexpression in HeLa cells of GFP-tagged murine RB6K caused dispersion of the Golgi apparatus, suggesting a role for murine RB6K in Golgi dynamics (6
). Like murine RB6K, the human RB6K is able to bind Rab6 in a two-hybrid assay (A. Echard and B. Goud, unpublished results) and is localized to the Golgi apparatus in interphase cells (Fig. A). Our study, however, also covered M phase, during which we found RB6K to be expressed at levels considerably higher than that in interphase cells. Concomitantly, RB6K no longer localizes exclusively to the Golgi but appears in the prophase nucleus. This localization is in good agreement with a PSORT-II prediction (60.9% nuclear versus 17.4% cytoplasmic). For MKLP-1, it was suggested that sequestration in the nucleus prevents undesirable binding to the interphase microtubuli and only after dispersal of the nuclear envelope after prophase the KLP is released and allowed to interact with the then-formed mitotic spindle (21
). For RB6K, an explanation should concern both the function of RB6K in interphase Golgi and the function in M phase. The relatively small RB6K pool on the interphase Golgi is involved in retrograde transport and is regulated by the effector GTPase Rab6. However, Fig. B shows that localization of most of the M phase-induced RB6K is independent of Rab6, which seemingly contradicts a function related to the mitotic Golgi apparatus (28
). Rather, taking into consideration the fact that several proteins that are critically involved in cytokinesis initially localize to the prophase nucleus (29
), it can be hypothesized that localization to the nucleus of the RB6K pool that is synthesized at the onset of M phase is instrumental in binding cargo and/or modulation of RB6K function by regulatory proteins. This hypothesis will require an unbiased search for proteins that interact with RB6K in various stages of the cell cycle.
Concomitant with the onset of cytokinesis during anaphase, RB6K concentrates in the equatorial zone of the cell. Consistent with this localization, effects of interference with RB6K function by injection of specific antibodies are restricted to cytokinesis. Phylogenetic analysis of the motor domain sequences has been used to classify most of the KLPs into 8 to 10 subfamilies that, in addition to having related motor domain sequences, usually have a related domain structure and show similarity with regard to motility behavior and cellular functions (2
; website of Greene and Henikoff). Detailed information on the function of members of the MKLP1 family has been derived from mutant analysis in the case of the Drosophila melanogaster
) and the C. elegans
) or from antibody microinjection studies of the MKLP-1 (CHO1 antigen) protein in mammalian cells (21
). When antibodies raised against the CHO1 protein are injected in mammalian cells before onset of anaphase, cells are arrested in metaphase, showing partially impaired congression of chromosomes and a disorganized spindle, indicating that the antibodies interfere in a stage preceding anaphase A. Injection after onset of anaphase has little effect on completion of cell division (21
). In contrast, RB6K antibody microinjection does not affect mitosis before anaphase B, indicating that its function temporally follows that of MKLP-1 rather than being redundant with it. As RB6K, like the other MKLP-1 family members, has the ability to cross-link antiparallel microtubules, our microinjection results might indicate interference with dynamics of midzone microtubules in anaphase B. It should be noted, however, that RB6K is only distantly related to MKLP-1, PAV-KLP, and ZEN-4, as sequence homology is confined to the motor domain.
Presently, the combined databases do not contain KLPs from other species that could be considered to be functional equivalents of human and mouse RB6K by showing even distant homology to the C-terminal 350 residues. Still, the phenotype resulting from microinjection of anti-RB6K antibodies is comparable to that of zen-4, polo
, and pav
), suggesting that RB6K may fulfill a separate but comparable role in cytokinesis. In mammalian cells, Polo-like kinase has been shown to colocalize and interact with MKLP-1 in vivo and to phosphorylate MKLP-1 in vitro (16
). ZEN-4 and PAV-KLP, show 50 and 58% overall homology to MKLP-1, including the C terminus that is supposed to be involved in cargo binding. In both cases, it was speculated that, in addition to a role in spindle function, the KLPs may also contribute to cytokinesis by transporting Polo-like kinase and Polo, respectively (1
). Indeed, several studies, both in Drosophila
and in cultured cells (5
), have established that the spindle midzone provides stimuli for cytokinesis. Although the underlying molecular mechanisms are only partially understood, relocation of several proteins along microtubuli seems to be involved (5
). Based on these observations, we can speculate that RB6K is possibly involved in transport of one of the many essential components of the cleavage furrow that are currently emerging (4
The failure of cytokinesis that we observed after anti-RB6K antibody microinjection is compatible with both a function for RB6K on the spindle midzone and a function more related to the cleavage furrow, since both processes are intimately linked, as evidenced by Giansanti et al. (8
). These authors showed the cooperative interaction between the contractile ring and the spindle midzone, and if either of these stuctures is perturbed, the assembly of the other is disrupted. A more detailed understanding of RB6K function therefore requires knowledge of RB6K interacting proteins. At present, our data provide evidence for a tight cell cycle-regulated expression of RB6K and show that it is an essential, nonredundant component of the cell cycle that is required for successful completion of cytokinesis.