In this study, we found that OGT and OGA are in a transient complex during late M phase with the mitotic kinase Aurora B and with PP1. The Aurora kinase inhibitor ZM447439 ablated Aurora B staining at the midbody and colocalization with OGT. Furthermore, overexpression of OGT or OGA or the addition of ZM or the OGA inhibitor GlcNAc-thiazoline disrupted the mitotic GlcNAcylation and phosphorylation of the cytoskeletal protein vimentin. Together, these data suggest that OGT and OGA form complexes with mitotic kinases and phosphatases and that this complex controls the balance of phosphorylation and GlcNAcylation on mitotic substrates, such as vimentin, leading to proper segregation of the proteins into the daughter cells ().
Figure 8. An O-GlcNAc/phosphate complex forms in M phase to regulate the posttranslational state of proteins. A model representing the protein complex of Aurora kinase B (AKB), Protein Phosphatase 1 (PP1), O-GlcNAc transferase (OGT), and O-GlcNAcase (OGA) is proposed; (more ...)
Aurora B forms a tight complex with survivin and inner centromere protein (INCENP; Carmena and Earnshaw, 2003
), which then rides along the mitotic tubulin network interacting with substrates and other proteins (Carmena and Earnshaw, 2003
). PP1 interacts with Aurora B to negatively regulate kinase activity (Sugiyama et al., 2002
; Emanuele et al., 2008
). OGT interacts with PP1 (Wells et al., 2004
) as well as with OGA (Whisenhunt et al., 2006
). Therefore, at mitosis, a transient complex forms in which Aurora B, OGT, OGA, and PP1 interact synergistically to modulate the posttranslational state of given substrates. What is unclear from the coprecipitation studies is whether these enzymes are acting together in one large complex or are existing as mini-complexes of OGT-Aurora B, PP1-OGA, etc, or whether INCENP and survivin bridge the enzymes together by indirect interactions.
OGT contains a large N-terminal tetratricopeptide repeat domain (TPR), which targets OGT to complexes andsubstrates (Kreppel and Hart, 1999
; Jinek et al., 2004
). The transcriptional corepressor protein mSin3A targets OGT to complexes through the TPR (Yang et al., 2002
) and OGT-interacting protein 106 kDa (OIP-106) interacts with this domain on OGT to target OGT to RNA polymerase II (Iyer et al., 2003
; Iyer and Hart, 2003
). Interestingly, OIP106 interacts with the microtubule-based motor protein kinesin (Brickley et al., 2005
). This interaction might target an OIP106, OGT, and Aurora B complex to kinesin for transport. Therefore, a possible role of the OGT interactions could be in substrate or in complex targeting. Recently, OGT was found to interact with p38 MAP kinase during cellular stress (Cheung and Hart, 2008
). The kinase directed OGT to neurofilament H, an intermediate filament protein similar to vimentin, increasing the proteins GlcNAcylation state and solubility (Cheung and Hart, 2008
). Therefore, one can envision Aurora B interacting with OGT and targeting OGT to vimentin () or other potential mitotic substrates. Clearly, OGT substrate targeting is of critical importance in understanding the function of this enzyme, because a single catalytic subunit modifies hundreds of different proteins.
Aurora kinase activity is needed for the proper localization of OGT to the midbody at mitosis. Reduction in Aurora activity caused a significant decrease in midbody localization of OGT. Although Aurora B was still present at the midbody after ZM treatment, a more dispersed staining pattern was evident. OGT or OGA overexpression or treatment with GT did not alter the localization of Aurora B to the midbody. Therefore, Aurora B localization was not predicated on either the amount of OGT or OGA or on levels of cellular GlcNAcylation.
The actions of the inhibitors or overexpressed proteins altered the overall protein levels of Aurora B, OGT, and OGA. The protein levels of OGT and OGA are in equilibrium with total O-GlcNAc levels. Cellular concentrations of each enzyme change reciprocally with increased or decreased GlcNAcylation. For example, as GlcNAcylation increases, OGT decreases and OGA increases. (Slawson et al., 2006
). Interestingly, Aurora B protein levels were reduced with increased O-GlcNAc. At this time, the data do not clearly indicate whether Aurora B is an in vivo substrate for OGT and, therefore, a potential target for O-GlcNAc cycling. This decrease in Aurora B could be caused by changes in Aurora B protein stability, translation, or transcription. Conversely, Aurora B inhibition increased OGT protein levels to that of asynchronous cell levels. The expression changes seen with Aurora B and OGT could be symptoms of delayed cell cycle progression in which the cells are aberrantly mitotic or still at G2/M.
Vimentin, a type III intermediate filament protein, undergoes a series of phosphorylations in the head domain during mitosis (Izawa and Inagaki, 2006
; Omary et al., 2006
), which destabilizes the filament and allows for the proper segregation of the filaments in the daughter cells (Izawa and Inagaki, 2006
). A mitotic GlcNAcylation site at Ser-54 (Wang et al., 2007
) maps directly next to the CDK1 phosphorylation site at Ser-55 (Tsujimura et al., 1994
). The mitotic GlcNAcylation site was determined from cells mitotically arrested with nocodazole followed by chemo-enzymatic tagging and enrichment, chemical derivatization (BEMAD, Beta-Elimination followed by Michael addition by DTT), and mapping by ion-trap mass spectrometer (Wang et al., 2007
). This mapping strategy does not preclude the possibility that vimentin is GlcNAcylated at other times of the cell cycle or after cellular stimulus or stress.
Several other cytoskeletal proteins such as neurofilaments or keratins contain O-GlcNAc in the head domain (Chou and Omary, 1993
; Dong et al., 1993
; Ku and Omary, 1995
). The type I intermediate filament proteins keratin 8 and 18 contain up to three GlcNAcylation sites and multiple phosphorylation sites in the head domain (Chou and Omary, 1993
; Haltiwanger and Philipsberg, 1997
). Using nocodazole to generate mitotic extracts in HT29 cells, keratins were found to have both increased GlcNAcylation and phosphorylation, although, this observation was not seen in HeLa cells treated identically (Chou and Omary, 1993
). Interestingly, using other synchronization reagents or isolation of floater mitotic cells for mitotic enrichment did not lead to the observed changes in keratin mitotic GlcNAcylation as seen with the anti-microtubule agents such as nocodazole (Chou and Omary, 1993
). These data suggest that disruption of the tubulin network is key to the observed increase in keratin GlcNAcylation. However, these other synchronization methods do not yield a population of mitotic cells as synchronized as nocodazole treatment (Chou and Omary, 1993
). We also saw an increase in a highly synchronous M phase population after nocodazole treatment compared with double thymidine block. Because in our vimentin experiments we also synchronized cells by nocodazole, we cannot determine if the changes in vimentin GlcNAcylation was due to disruption of the microtubulin network or M phase–specific changes. More detailed studies using different M phase enrichment techniques, varying concentrations of nocodazole, and possibly vimentin glyco-site specific antibodies are needed to address this question.
Vimentin GlcNAcylation was increased after OGT overexpression and OGA inhibition, but what remains unclear is how vimentin function is altered. The increased GlcNAcylation did not disrupt phosphorylation at the adjacent CDK1 site, but late M phase phosphorylation was altered. The decrease in pSer-82 was dependent on the overexpression of either OGT or OGA or GT inhibition of OGA. Because OGA overexpression caused a decrease in the late M phase Ser-82 phosphorylation, then the cells are likely exhibiting problems progressing through M phase and less likely that the GlcNAc is preventing Ser-82 phosphorylation by blocking the accessibility of the kinase for that site. Interestingly, OGT and OGA overexpression caused an increase in Ser-71 phosphorylation and an appearance of a higher molecular weight band but GT decreased phosphorylation. The GT data argues for reciprocity between glyco-phospho states on filament proteins such as seen with Neurofilament M (Deng et al., 2007
). In the case of vimentin, however, the M phase regulation is much more complicated than just a matter of GlcNAcylation and phosphorylation being antagonistic to each other, because overexpression of both OGT and OGA led to increased GlcNAcylation.
Importantly, ZM treatment skewed the population of pSer-55 toward G2/M phosphorylation levels instead of an M phase level, suggesting that Ser-55 phosphorylation occurs before GlcNAcylation. This observation explains why the two inhibitors in combination produce an effect phenotypically like that of ZM alone at this site. The actions of GT in locking the O-GlcNAc in place appeared after Ser-55 phosphorylation/dephosphorylation. Possibly, the Ser-55 acts as a priming phosphorylation site for an OGT/PP1 complex to interact with vimentin and add the O-GlcNAc to the adjacent serine while dephosphorylating Ser-55.
Alternatively, inhibitor treatment disrupted the cycling of the modifications on vimentin, locking the protein in specific posttranslational states. Staining of vimentin under these conditions showed a slight increase in filament structure. Potentially, increases in GlcNAcylated vimentin or disruption of the removal of O-GlcNAc from the protein might block filament disassembly. These data suggest that the timing of phosphorylation and GlcNAcylation of vimentin are connected and dependent on each other. Disruption of this timing could potentially disrupt the proper segregation of vimentin in the two daughter cells.
These observations suggest that O-GlcNAc processing by OGT and OGA is coupled to phosphorylation processing during M phase. Linking mitotic kinases and phosphatases with OGT and OGA generates a signaling complex that could potentially fine-tune the regulation of target proteins. This study demonstrates the potential for dynamic regulation of a large number of mitotic proteins by this complex. However, more targets of this complex need to be identified before a clear picture of mitotic regulation is possible. The use of large-scale proteomic methods to map mitotic GlcNAcylation sites would greatly aid in determining substrates for this complex.