Genetic and biochemical studies in yeast, flies, and toads have identified some of the estimated 150–200 components of the centrosome and mitotic spindle, but few such proteins have been identified in mammalian cells (for review see Kalt and Schliwa, 1993
). Here we describe a novel mammalian kinase, IAK1, whose expression is tightly regulated, temporally and spatially, in the cell cycle. IAK1 has a distinct expression pattern, being localized to the separated, duplicated centrosomes and mitotic spindle of dividing cells. In cells recovering from nocodazole treatment and taxol-treated cells, IAK1 is associated with each MTOC, suggesting that it may play a role in microtubule formation and/or stabilization. Because of its inherent protein kinase activity and the timing of its expression and association with the centrosome and spindle, IAK1 is clearly a candidate regulator of phosphorylation changes on these structures during mitosis. The localization of IAK1 that we describe here is strikingly similar to another serine/threonine kinase, the Polo-like kinase (Plk1) (Golsteyn et al., 1995
). Interestingly, Plk1 was detected in the centrosomes of interphase cells and remained on those structures during prophase and mitosis (Golsteyn et al., 1995
). Plk1 and IAK1 may interact to regulate each other's function, but we have been unable to demonstrate physical association of these kinases in immunoprecipitation and Western blot analyses (Chase, D., G. Gopalan, P.J. Donovan, and D. Ferris, unpublished observations). Nevertheless, Plk1 and IAK1 may be members of a common signal transduction cascade. One striking difference between Plk1 and IAK1 is that Plk1, unlike IAK1, is detected in the cytoplasm of interphase cells. Our Western blot data demonstrates that IAK1 protein may be present in the cell in S-phase before its immunocytochemical detection in the duplicated centrosomes at the G2
phase of the cell cycle. This suggests either that IAK1 may be present on the centrosome in S-phase, but that the antigenic epitope recognized by the COOH-terminal antiserum is masked, or that IAK1 may be present in the centrosome or cytoplasm at levels undetectable with our current reagents. Analysis of IAK1 protein levels in subcellular fractions of S-phase cells should address this question.
In cells treated with nocodazole, IAK1 remains associated with the centriole, suggesting that IAK1 association with this structure is independent of the presence of polymerized microtubules. IAK1 localization in MTOCs and spindles of cells recovering from nocodazole is consistent with it playing some role in microtubule polymerization or stabilization. A similar conclusion can be reached from the localization of IAK1 to MTOCs in taxol-treated cells. One potential role of IAK1 might be to stabilize microtubules, which could lead to centrosome separation and microtubule-mediated chromosome movements at mitosis. Alternatively, IAK1 might act on microtubule motor proteins that have been implicated in the process of centrosome separation and chromosome segregation (for review see Walczak and Mitchison 1996
). Indeed, Glover and colleagues have suggested that the Drosophila
aurora kinase might act on a kinesin-related protein (Glover et al., 1995
). In this regard, it is apparent that the localization pattern of IAK1 is very similar to that described for the kinesin-related protein Eg5 in vertebrate cell lines (Houliston et al., 1994
). Whether IAK1 interacts with and phosphorylates Eg5 (or other kinesin-related proteins) is being examined. The ability of IAK1 to disrupt mitotic spindle architecture and to affect the segregation of chromosome mass in ts ipl1
mutants strongly suggests that it can interact with a component of the yeast Ipl1 pathway or a closely related pathway. Thus, our current efforts to identify such a protein may facilitate our further understanding of the mode of action of IAK1.
What is the function of IAK1? The recent localization of the Ipl1p protein kinase to the yeast mitotic spindle (Chan, C., unpublished results) demonstrates that IAK1 is similar to Ipl1p not only in sequence but also in subcellular localization. Our data show that IAK1 cannot rescue the ipl1-4
mutant phenotype and therefore may not perform identical functions as Ipl1p. Nevertheless, the data presented here also demonstrate that IAK1 exacerbates ipl1
mutant phenotypes since it causes inviability of ipl1-4
mutant but not wild-type cells (Fig. ). These data suggest that IAK1 proteins act as dominant-negative proteins whose effect is only seen when Ipl1p function is impaired. Similarly, Nurse and colleagues showed that a kinase-dead Xenopus
CDK2 gene disrupted cell cycle progression in a ts cdc2
fission yeast mutant but not in wild-type cells (Paris et al., 1994
). This effect is most likely brought about by the ability of the expressed proteins to interfere with the activity of the endogenous kinase by competing for interacting proteins. Expression of other kinases (as well as several other genes or mutations) in ts ipl1
mutants does not cause lethality of the ts ipl1
mutant at the permissive temperature (Chan, C., unpublished observations). However, expression of the Drosophila
aurora kinase, like IAK1, caused lethality in ipl1-4
mutants but not in wild-type cells at the permissive temperature (Chan, C., unpublished observations). This strongly suggests that the observed phenotype is specific and that IAK1 is not simply causing nonspecific lethality. Some of these cytological defects are also observed in conditional ipl1
mutant cells incubated at their restrictive temperatures and are also reminiscent of the effect of the aurora
mutation in Drosophila.
These observations strongly suggest that IAK1 interferes with the function of Ipl1p (or a parallel pathway), thus leading to lethality in cells that already have compromised Ipl1p function (due to the ipl1-4
mutation). IAK1 could interact with Ipl1p itself, an upstream regulator of Ipl1p, a downstream effector (substrate) of Ipl1p, or a protein in a parallel pathway. Disruption of IAK1 activity in mammalian cells will probably address its role in centrosome and mitotic spindle function in mammalian cells.
The inability of IAK1 to rescue the ipl-4 mutation suggests that IAK1 may not perform the identical functions as Ipl1p. It is possible that an, as yet unidentified, relative of IAK1, fulfills the Ipl1p function or that there is no single functional homologue of Ipl1p in mammalian cells. Conceivably the function(s) carried out by Ipl1p in yeast could be carried out in mammalian cells by multiple kinases which have evolved more complex or distinct forms of regulation. Interestingly, in addition to IAK1 we have identified another mammalian kinase, related to Ipl1 and aurora, which is not expressed in NIH 3T3 cells or most adult tissues, but is expressed in germ cells (Gopalan, G., J. Centanni, and P.J. Donovan, manuscript in preparation). This kinase, which shares 52% identity at the amino acid level to IAK1, may play a similar role to IAK1 in meiotic germ cells or may function during postmeiotic germ cell differentiation. Another cell cycle–regulated kinase of unknown function, STK1, has also recently been identified and shows homology to IAK1 (see Fig. B). The mouse genome therefore contains at least three members of this subfamily and, based on chromosome localization studies, may also contain another related gene (our own unpublished observations). A Xenopus cDNA sequence (Eg2) present in the GenBank database (accession number Z17207)also shows a high degree of homology to IAK1, STK1, Ipl1, and aurora (Fig. B). As well as these other genes, we have identified two related genes each in human and worm (Gopalan, G., J. Schumacher, A. Golden, and P.J. Donovan, unpublished observations). These data suggest that aurora, IAK1, STK1, Eg2, and Ipl1 may be the first members of an emerging subfamily of the serine/threonine kinase superfamily that could play important roles in centrosome and mitotic spindle function during mitosis. Analysis of these related proteins in a variety of species will probably allow a more complete understanding of the function and mode of action of this protein family.