Cilia and flagella have been described as cellular “antennas”, sensing a multiplicity of extracellular stimuli to induce an intracellular response (Singla and Reiter, 2006
). In addition to undergoing regulated resorption induced by extracellular cues, for over four decades cilia have been known to be dynamically resorbed and resynthesized throughout the cell cycle. Taken in sum, our data suggest a model () in which the serum growth factor-induced activation of a HEF1-AurA complex allows AurA to phosphorylate and activate HDAC6, which destabilizes the ciliary axoneme by deacetylating tubulin. Unexpectedly, activation of AurA is a central component of this cascade even during the G1 resorption wave, indicating a non-mitotic activity for AurA in animals.
An important finding of this work is the novel connection between AurA and HDAC6. HDAC6 tightly interacts with α and β tubulins through its HDAC domain, which may restrict its enzymatic activity, based on reports that taxol treatment causes HDAC6 to accumulate on microtubules, and is accompanied by increased tubulin acetylation (Zhang et al., 2003
). Localized phosphorylation by AurA may increase the turnover of HDAC6 at microtubules, thus increasing the active pool of HDAC6 at cilia. Interestingly, studies in Chlamydomonas
indicate that an important element of flagellar resorption is destabilization of the microtubule-based axoneme, suggesting this signaling cascade may be evolutionarily conserved (Pan and Snell, 2005
; Pan et al., 2004
). Further supporting the idea of conservation, the C. elegans
gene MEC-12 encodes an α-tubulin variant that is specifically required only in mechanosensing neurons, which depend on intact cilia: MEC-12 is the only α-tubulin in this species with a conserved site for acetylation (Fukushige et al., 1999
). Interestingly, HDAC6 has been reported to associate with protein phosphatase 1 (PP1) (Brush et al., 2004
), which binds microtubules (Liao et al., 1998
), and dephosphorylates and inactivates AurA kinase. Such feedback may limit AurA activation at cilia.
A number of growth stimuli induce HEF1 expression and phosphorylation, influencing its protein interactions. These include PDGF, which is here shown to partially induce ciliary disassembly (Natarajan et al., 2006
). Intriguingly, recent studies of p130Cas, a protein structurally similar to HEF1, indicate that p130Cas acts as a stretch sensor; HEF1 contains all sequence motifs necessary for similar function (Kostic and Sheetz, 2006
). As one major function of cilium is to sense fluid flow, and overly persistent flow has been reported to induce ciliary disassembly (Iomini et al., 2004
), stretch sensation may be an important action of HEF1. Our data suggest that HEF1 both activates AurA and stabilizes the protein from degradation; it will be interesting to determine if the HEF1 scaffolding activity also contributes to AurA interaction with its effector HDAC6. Our data also indicate that AurA activity influences IFT88 localization during disassembly, and suggest integrity of the IFT system is important for the disassembly process in animals, as in Chlamydomonas
(Pan and Snell, 2005
Our establishment of a HEF1-AurA-HDAC6 cascade at cilia also informs the understanding of the mitotic activities of these proteins. Dynamic changes in microtubule acetylation and deacetylation characterize the stages of mitosis, and HDAC inhibitors that inhibit family members with microtubule deacetylase activity induce mitotic arrest (Blagosklonny et al., 2002
). The identification here of HDAC6 as an AurA target suggests that HEF1-AurA regulation of tubulin deacetylation at mitosis through HDAC6 might offer a mechanism to fine-tune the mechanical properties of the mitotic spindle. This signaling cascade may also influence reestablishment of focal adhesions at and following cytokinesis, given the growing appreciation of the role of microtubules in guiding the formation of these structures (Ezratty et al., 2005
; Strickland et al., 2005
). Further, one intriguing possibility is that the common use of an AurA-HEF1-HDAC6 switch at the basal body of quiescent cells and the centrosome of G2/M cells may serve as part of a checkpoint mechanism coordinating responsiveness to extracellular cues at different points in cell cycle. In this context, our observation that inhibition of AurA causes appearance of mitotically arrested cells possessing both spindles and cilia (results not shown) may reflect triggering of such a centrosomally based checkpoint.
These results also have implications for the understanding and treatment of cancer. Tumor cells commonly do not have cilia, and both HEF1 and AurA are often upregulated in cancer. The roles for these proteins at the centrosome and focal adhesions described earlier already offer two mechanisms by which these proteins may promote tumor initiation and progression. The current study indicates a third mechanism, in which elevation of HEF1 or AurA in tumors may destabilize cilia, thus conditioning cellular response to external cues and impacting multiple signaling pathways. Further, AurA is regarded as a promising chemotherapeutic target, with agents inhibiting this protein currently in clinical trials (Andrews, 2005
). TSA and other broad-spectrum agents targeting HDACs are used in the clinic (Vanhaecke et al., 2004
), with more focused agents such as tubacin in preclinical development (Hideshima et al., 2005
). Our data suggest that AurA- or HDAC-targeted drugs may have previously unappreciated in vivo effects involving cilia, that may contribute to the observed efficacy and/or side effects of these agents.
PKD is one of the best-described cilia-related diseases (Wilson, 2001
), with mutation of the cilia-localized polycystin proteins 1 and 2 (PKD1 and PKD2) responsible for the significant majority of PKD patients. p130Cas interacts directly with complexes containing PKD1 and PKD2, and also with nephrocystins, cilia-associated proteins that are mutated in a second renal cystic syndrome, nephronophthisis (Benzing et al., 2001
). Although an association of HEF1 with these proteins has never been assessed, HEF1 is abundant in the kidney and conserves many protein interaction sequences with p130Cas. It is also tantalizing to consider that closer connections exist between dysplastic disorders leading to cysts and cancer than have previously been appreciated. One of the surprising results of a recent large study to analyze the cancer genome was the identification of the PKHD1 protein, a ciliary protein which is mutant in autosomal recessive PKD, as commonly mutated in colorectal cancer (Sjoblom et al., 2006
). Overall, deregulated AurA/HEF1/HDAC6 signaling may have broad implications for studies of human development and disease.