The Aurora-A (AurA) kinase is a member of the evolutionarily conserved Ipl family of kinases (reviewed in Marumoto et al
). AurA is most abundant at the centrosome in G2 to M phase2
, and in studies performed in mammals and model organisms including Drosophila
, AurA has been shown to be activated at mitotic entry, performing critical functions in regulating entry into and passage through mitosis1
. Activation of AurA at mitosis is supported by a complex set of interactions between the protein and a number of partners, including Ajuba4
. These interactions typically occur at the centrosome, with the partner proteins supporting AurA localization and stability, and inducing allosteric changes that contribute to AurA activation. Because of the complex nature of the interactions, the exact mechanism timing the abrupt AurA activation at the mitotic boundary has not been well resolved.
In the past several years, AurA has attracted increasing attention because it has been found to be overexpressed and hyperactivated in a high percentage of tumours arising in breast, colon, ovary and other tissues10
. Abnormally high AurA activity is oncogenic in various cell line models and is associated with defective cytokinesis and aneuploidy15
. AurA is now being actively exploited as a target for development of new anti-cancer agents (reviewed in Andrews19
) on the basis of this known role as a mitotic regulator. Interestingly, the overexpressed AurA associated with cancer cells has a number of activities that are not specific to function in a mitotic compartment. For example, AurA directly phosphorylates and regulates the activity of the RalA GTPase, an EGFR/Ras effector important in many cancers20
, with this activity observed in interphase cells. AurA is not typically mutationally activated in cancer, making the mechanism of its activation in interphase cells somewhat elusive.
Intriguingly, a number of studies have emerged in recent years to challenge the idea that AurA is solely a mitotic kinase even in normal cells. Serum induces AurA activation at the basal body of the cell cilium in non-cycling G0/G1-phase mammalian cells, causing AurA-dependent ciliary resorption21
, and hence indirectly impacting the functionality of the cilia-dependent and cancer-relevant Hedgehog signalling cascades22
. AurA also has been reported to regulate microtubule dynamics in interphase cells23
, and has been shown to be abundantly expressed in some human adult non-cycling tissues such as kidney24
. All these studies strongly imply a non-mitotic activity for AurA, and indeed, a distant ortholog of AurA in the green algae Chlamydomonas
regulates both resorption and excision of the flagella (a structure analogous to the mammalian cilium) in response to altered ionic conditions or cues for mating25
, again suggesting important non-mitotic roles for AurA, although across a great evolutionary distance. However, to date, little effort has been made to investigate the non-mitotic activation of AurA in either normal cells or tumours, and mechanistic insight into factors governing such activation is essentially absent.
Our previous work demonstrating that AurA activation occurs before ciliary resorption in interphase cells21
was based on studies in Chlamydomonas
, which implicated an AurA ortholog, CALK, as important for resorption of flagella in that organism25
. Stimuli that lead to loss of flagella in Chlamydomonas
include mating response to pheromone and transient ionic shock25
. Recent studies in Chlamydomonas
have also suggested increasing intraflagellar calcium concentrations during the mating response26
, shortly before the activation of the CALK kinase. Independently, a study of the Chlamydomonas
flagellar excision process emphasized the importance of rapid spatiotemporal patterning of calcium ion distribution as a critical mediating signal27
. Interestingly, a recent study of oocyte maturation in Xenopus
indicated that inhibition of Ca2+
signalling led to eventual failure to accumulate and activate AurA28
, although no direct connection was investigated. Cumulatively, these studies led us to hypothesize that AurA might be a direct target of cellular calcium signalling in mammals.
Our work presented here demonstrates that elevated cytoplasmic calcium signals are transmitted rapidly and transiently through calmodulin (CaM) to activate AurA. This activation involves direct CaM binding to two sites in the unstructured regions of AurA, and induced by multiple inducers of calcium release from intracellular stores in the endoplasmic reticulum (ER). These results provide the first clear mechanism for AurA activation in non-mitotic cells and potential insight into the timing of activation of AurA at mitosis.
O.V.P. performed the substantive majority of the experiments described, based on pilot studies performed by E.N.P. R.L.D. performed the structural prediction analysis. E.A.G. provided overall guidance on study design and execution, and wrote the paper.