The results of this study for the first time establish HEF1 as a regulator of AurA and Nek2 activation, and of centrosome cohesion and amplification. Proteins initially defined as components of the cell attachment machinery, including APC 23
, have recently been found to also function in cell cycle controls, and elegant genetic studies in lower eukaryotic models for development such as C. elegans
(reviewed in 24,25
) have begun to elucidate a model in which dynamic interconnections between the centrosome and structures at the cell cortex controls the plane of mitotic spindle orientation, and cleavage furrow formation. It is likely that this mechanism will prove to be important for higher eukaryotes as well, given the need of many cells to limit cell division to specific planes (for example, to maintain barrier function). An economical view of cellular function would suggest that the re-use of proteins which govern cell attachment and cytoskeletal dynamics in interphase cells might not only be efficient, but might also provide a means to synchronize changes in cell contacts during the mitotic process. It is possible that the pools of HEF1 used for centrosome, mitotic spindle, and focal adhesions are completely distinct. However, it may be that migration of proteins such as HEF1 between these structures provides polarity and attachment cues that influence the entry to and exit from mitosis. Given the particular abundance of HEF1 in polarized epithelial and lymphoid cell populations, our work would define it as an excellent candidate for such a role.
Our data suggest a model in which in normal cells, HEF1 initially interacts with AurA in G2 prior to AurA activation, with the centrosome one important point of interaction. In this model, as mitosis approaches, focal adhesion disassembly releases more HEF1, and the increasing interaction of HEF1 with AurA promotes AurA activation. In turn, phosphorylation of HEF1 by the activated AurA reduces the affinity of interaction between the two proteins, perhaps contributing to the relocation of HEF1 away from the centrosome, or perhaps contributing to preferred interaction of AurA with other partner proteins in the context of the centrosome. AurA activity is known to be regulated by several other protein partners, including TPX2, Ajuba, and PP2,. In cells depleted for HEF1, AurA does not become activated, suggesting the association with HEF1 is functionally important. In cells with HEF1 overexpressed, and able to associate with the centrosome, the stoichiometry of HEF1 is significantly increased, allowing the protein to continue to interact with AurA in spite of phosphorylation by AurA, and thus promoting elevated AurA activity. For both AurA and HEF1, the centrosomal amplification and multipolar spindles seen with overexpression of the proteins is a secondary consequence of cytokinetic failure, with the exact mechanism yet to be defined, but may involve regulation by these proteins of a common effector.
HEF1-depleted cells have abnormally split centrosomes, which accumulate reduced levels of gamma-tubulin in G215,16,26
, have abnormally reduced accumulation of ninein, c-Nap-1, and other proteins, and are deficient in organizing microtubules at mitosis. These defects are likely to be independent of HEF1-AurA signaling, as AurA depletion does not result in centrosomal splitting (Supplemental Figure 5 B, C, D
), and may be direct (at the centrosome) or indirect. Our data suggest that HEF1 may normally act to restrain the activity of Nek2 12,20
, as HEF1 coimmunoprecipitates with Nek2, and Nek2 is hyperactivated in cells with depleted HEF1 or dominant negative HEF11-405
(), with increased Nek2 activation previously reported as sufficient to induce splitting. HEF1 may have additional activities required for centrosomal cohesion, as hyperactivation of Nek2 is not sufficient to completely remove c-Nap-1 from centrosomes in interphase cells 12
, while accumulation of ninein is an early step in the maturation of daughter centrosomes to mother centrosomes, and has not been described as influenced by Nek2. Such a role for HEF1 is separable from any secondary effect due to defects in cell attachment, as different domains of HEF1 caused splitting versus cell rounding. Intriguingly, recent protein interaction studies of the ancestral HEF1/p130Cas homolog in Drosophila 27
have suggested this protein associates with a component of the gamma-tubulin ring complex (γ-TuRC), which is important for microtubule nucleation 28
. It is also interesting that we have previously described the cleavage of HEF1 at amino acid 363 by caspases to produce a p55 species 3,13
. Although we originally found this p55 species in both mitotic and apoptotic cells, our ongoing work has suggested that the initial idea of HEF1 cleavage at mitosis may have arisen at least in part from contamination of drug-synchronized mitotic populations with apoptotic cells (results not shown). However, the fact that HEF11-353
does not associate with centrosomes or AurA in cells, while the slightly larger HEF11-405
does, implies that cleavage of HEF1 at this site may have a functional significance in disrupting centrosomal function during cell death.
Definition of HEF1 as a component of the AurA activation machinery is an important finding, providing evidence of a new channel for cross signaling between cell adhesion and mitosis. Further, AurA and Nek2 overexpression and hyperactivation has been observed in many tumors, and is associated with genomic instability 29–31
. It was shown for Cas proteins32
that upregulation of p130Cas and/or HEF1 correlates with poor prognosis in breast cancer. It has additionally been shown by us and others that upregulation of Cas proteins influences the transcription of a number of gene pathways associated with cancer development, enhancing activation of the MAPK pathway and promoting matrix metalloproteinase production (22,33
and others): it is entirely possible that altered Cas protein levels also induce transcriptional changes that influence genomic stability. Our data imply that beyond their well-defined functions in regulation of susceptibility to apoptosis and cell migration, HEF1 and potentially other members of the Cas family may make additional contributions to the processes of cell transformation through regulation of mitosis.