Here we report two main findings. First, the translational regulator, PUM2, is a cell cycle-regulated protein and a novel substrate of Aurora-A. Second, PUM2 can interact with Aurora-A to protect Aurora-A from APC/CCdh1-mediated protein ubiquitination/degradation and to enhance the kinase activity of Aurora-A. This reveals the moonlight role of PUM2 in mitotic control, apart from its role in translational regulation during interphase.
Befitting that Aurora-A kinase plays a central role in cell division, the substrates of this kinase include various cell cycle regulatory proteins such as TPX2 
, TACC3 
, HURP 
, Eg5 
, CDC25B 
, Ajuba 
and PAK1 
. Together, these substrates are presumed to be responsible for at least some of the cell cycle activities that have been ascribed to Aurora-A. It is imperative to identify any additional substrates and interacting proteins that may exist to complete the description of the role of Aurora-A in the regulation of mitosis. PUM2, a novel Aurora-A substrate that has been identified in this study, has a well-known role in the regulation of translation. Interestingly, our results suggest an additional novel role of PUM2 in serving as an upstream regulator of Aurora-A through physical interaction. The molecular mechanism of PUM2-mediated kinase regulation acts to both activate and stabilize Aurora-A. PUM2 could affect the cell cycle progression through regulation of Aurora-A.
Overexpression of the PUM2 mutant, which failed to interact with Aurora-A, and depletion of PUM2 both led to the destabilization of Aurora-A (). This was made evident by the low susceptibility of Aurora-A to ubiquitination in cells in which PUM2 was overexpressed (). Moreover, the carboxy-terminal D-box of Aurora-A was shown to be the region responsible for PUM2 binding (), suggesting that the binding of PUM2 might protect Aurora-A from being attack by APC/CCdh1
(). In a recent report, the mRNAs associated with PUM2 were systematically identified by the recovery of endogenously formed ribonucleoprotein complexes and the analysis of associated RNAs with DNA microarrays 
. This study indicated that Aurora-A mRNA was not included in the transcripts that were reproducibly associated with PUM2. This precluded possible effects of PUM2 on Aurora-A mRNA translation. Moreover, it has been reported that APC/CCdh1
is kept inactive in early mitosis and becomes active by the phosphorylation of cyclin-dependent kinase (cdk) only from late mitosis to G1 phase 
. Why there is PUM2 to protect Aurora-A from being attack by APC/CCdh1
in early mitosis? It is reported that timely destruction of the anaphase inhibitor, securin, by APC/CCdh1
is regulated by the nucleocytoplasmic transport factors Rae1 and Nup98 
. Rae1 and Nup98 would form a complex with APC/CCdh1
and securin during prometaphase. This suggested that the Rae1-Nup98 complex does not inhibit the destruction of securin by preventing the interaction between APC/CCdh1
and securin, but by preventing the ubiquitination of APC/CCdh1
-bound securin. It has been proposed that when securin is restricted in the Rae1-Nup98 complex, it would prime cells for the sudden degradation of securin at the metaphase-anaphase transition 
. In this study, we found that PUM2 could affect the ubiquitination of Aurora-A by forming a complex with Aurora-A. PUM2 might control the destruction of Aurora-A through a similar mechanism in regulating the degradation of securin, although it remains to be determined whether APC/CCdh1
is in the PUM2-Aurora-A complex in early mitosis.
The mechanism by which PUM2 regulates the cell cycle progression.
In addition to its role in enhancing protein stability, the physical interaction of PUM2 and Aurora-A is also required for the promotion of Aurora-A kinase activity (). It has been shown that PUM2 binds to the C-terminal region of Aurora-A, the same region that has been identified as the interaction site for TPX2 with Aurora-A 
. This suggests that PUM2 might mediate the kinase activity of Aurora-A through the same mechanism as TPX2. However, only substrate phosphorylation activity of Aurora-A was increased in the presence of PUM2, not its auto-phosphorylation activity (). This suggests that the phenomenon observed might be due to another mechanism. PUM2 is a relatively large protein containing 1064 amino acids (theoretical molecular weight 114 kDa). Therefore, we speculated that the proper phosphorylation and accumulation of PUM2 at the centrosome might serve as a scaffold for the recruitment of other Aurora-A activators such as TPX2, PAK1 and Ajuba to regions that are proximal to the centrosomes, thereby triggering the rapid increase in the activity of Aurora-A (). Moreover, it is interesting to note that PUM2 was localized in the centrosome from S-phase to metaphase. When the cells entered anaphase, PUM2 could not be detected in the centrosome, consistent with the time at which Aurora-A becomes unstable and with less kinase acitvity (). This indicated that PUM2 might be essential for mitotic entry by means of the regulation of Aurora-A. Through recruiting Aurora-A activators and protecting Aurora-A from attack by APC/CCdh1
, PUM2 might enhance the quantity and activity of Aurora-A dramatically, thereby causing mitotic entry. It will be interesting to learn if PUM2 competes or cooperates with the other, previously identified activators or destabilization factors of Aurora-A, and if Aurora-A interacts with more than one regulator at a time.
It has been shown that PUM2 is involved in the regulation of cell division as the other two factors, Maskin and CPEB, that are involved in regulating the translation of Xenopus
maternal cyclin B1 mRNA. Besides, several studies also revealed that interphase components might be used to regulate mitosis, and many mitotic key regulators also have important functions during interphase. First, RanGTP, a protein that has a well-established function in nuclear trafficking in interphase, uses similar principles to regulate spindle assembly in mitosis as it does during interphase 
. Another interphase component, Lamin B, which is important for nuclear envelope assembly and in maintaining nuclear shape, has also been reported to play a role in mitosis. It can assemble into a matrix-like network during mitosis, and the formation of this mitotic matrix then tethers a number of spindle assembly factors, followed by the stimulation of microtubule assembly 
. Moreover, the Aurora-A activators PAK1, HEF and Ajuba are present at focal adhesions and serve as downstream effectors of integrin during the process of cell migration. It is possible that disassembly of focal adhesion molecules during mitosis releases the protein complexes and results in the activation of Aurora-A 
In conclusion, all components in the PUM2-CPEB-Maskin protein complex are not only involved in translational regulation in interphase but also have roles in the progression of mitosis. This finding delineates an economical design that employs components that regulate both the progression of interphase and mitosis.