- Short communication
- Open Access
GSK3β N-terminus binding to p53 promotes its acetylation
© Eom and Jope; licensee BioMed Central Ltd. 2009
- Received: 29 January 2009
- Accepted: 05 March 2009
- Published: 05 March 2009
The prevalence in human cancers of mutations in p53 exemplifies its crucial role as a tumor suppressor transcription factor. Previous studies have shown that the constitutively active serine/threonine kinase glycogen synthase kinase-3β (GSK3β) associates with the C-terminal basic domain of p53 and regulates its actions. In this study we identified the GSK3β N-terminal amino acids 78–92 as necessary for its association with p53. Inhibitors of GSK3 impaired the acetylation of p53 at Lys373 and Lys382 near the GSK3β binding region in p53, indicating that GSK3β facilitates p53 acetylation. We also found that acetylation of p53 reduced its association with GSK3β, as well as with GSK3α. These results indicate that the N-terminal region of GSK3β binds p53, this association promotes the acetylation of p53, and subsequently acetylated p53 dissociates from GSK3.
- Nuclear Localization Sequence
- GSK3 Inhibitor
- Acetylation Site
- Camptothecin Treatment
- GSK3 Isoforms
Glycogen synthase kinase-3 (GSK3) phosphorylates more than 40 substrates, so its actions must be controlled in a substrate-specific manner to avoid spurious phosphorylation of unintended substrates upon fluxes in the activity of GSK3 . This must be accomplished by synchronous regulation of GSK3 binding to substrates or substrate-containing protein complexes and regulation of GSK3 activity, such as the activity-regulating serine-phosphorylation of GSK3. Thus, the association of GSK3 in protein complexes is likely as critical as post-translational modifications in controlling the actions of GSK3. This has been well-described for the Wnt signaling pathway where GSK3 must be bound to axin to phosphorylate axin-bound β-catenin . This substrate specificity implies the existence of different GSK3 recognition motifs for various binding partners, but GSK3 binding domain studies have been confined to Wnt signaling proteins [3–5]. Thus, little is known about binding domains in GSK3, and it is usually depicted as three domains, a small N-terminal domain, a slightly larger C-terminal domain, and a predominant middle kinase domain. Additionally, a nuclear localization sequence was recently identified .
To understand better the protein-protein interactions of GSK3β, we investigated the residues required for GSK3β to bind the tumor suppressor p53 . GSK3β forms a complex with nuclear p53 to promote p53-induced apoptosis, and the C-terminal p53 basic domain is necessary for this interaction [8–10]. GSK3β also interacts with p53 in the nucleus during cellular senescence , and GSK3β binds p53 in mitochondria . Although the interaction between GSK3β and p53 has been confirmed in several studies, the functional consequences are controversial, possibly because of the many other regulatory influences on p53 and the context- and cell-specific regulation and actions of p53. GSK3 has been reported to phosphorylate Ser33-p53  or Ser315-p53 and Ser376-p53 [13, 14], and to regulate the intracellular localization of p53 [10, 13, 14]. In this study we identified the domain of GSK3β necessary for its association with p53. Furthermore, we found that GSK3 promotes the acetylation of p53, and that p53 acetylation reduces its association with GSK3β.
We investigated if acetylation of p53 regulates its association with GSK3 since p53 acetylation sites are within, or close to, the residues of p53 required for its association with GSK3. SH-SY5Y cells were treated with 1 μM camptothecin for 3 hr to increase p53 levels, in the absence or presence of 1 μM TSA plus 5 mM nicotinamide to inhibit the deacetylation of p53. Each isoform of GSK3 was immunoprecipitated and p53 was found to co-immunoprecipitate with both GSK3 isoforms. Inclusion of TSA/nicotinamide with camptothecin treatment increased p53 acetylation on K373 and K382 but not on K320, and there was a large inhibition of the association of p53 with both GSK3 isoforms compared with their association with p53 in the absence of TSA/nicotinamide (Figure 4C). These results indicate that acetylation of p53 at K373 and K382, proximal to the GSK3β binding region, inhibits the association of GSK3 with p53.
p53 and GSK3 each has critical roles in determining cell survival [1, 7]. Therefore, it is particularly interesting to determine mechanisms regulating interactions between the two proteins. In this study we identified a novel protein-interacting region near the N-terminus of GSK3 that is critical for its interaction with p53. Furthermore, we found that GSK3 promotes the acetylation of p53 at sites (K373 and K382) near the GSK3β-binding region of p53, which spans residues 364–373. Highly acetylated p53, in turn only poorly associates with GSK3. These findings indicate that a region near the N-terminus of GSK3 associates with the C-terminal basic domain of p53 that contains several sites that can be acetylated, GSK3 promotes the acetylation of p53, and acetylated p53 dissociates from GSK3.
Little is known about domains in GSK3 that are critical for its interactions with other proteins despite the importance of these interactions for controlling and directing the actions of GSK3 . Since the interaction between GSK3β and p53 is important for controlling the actions of both proteins , we examined regions in GSK3 that are required for this interaction. Previously overlapping but non-identical binding sites in the carboxy lobe of GSK3β were identified as critical for associating with proteins in the Wnt signaling pathway, axin and GBP/FRAT [3–5]. Both axin and GBP bind a channel in GSK3β formed by an α-helix (residues 262–273) and an extended loop (residues 285–299), so their binding is mutually exclusive, but the interacting residues of GSK3β differ for axin and GBP . Unlike the Wnt signaling proteins, this entire C-terminal region of GSK3 was completely dispensable for its interaction with p53, which required GSK3β residues 78–92. Although the binding domain contains residues K85/K86 that are necessary for its kinase activity, kinase-dead GSK3β with these residues mutated to alanine associated with p53 equivalently to wild-type GSK3β indicating that GSK3β activity does not affect the interaction with p53, which has also been shown for kinase-dead GSK3β binding to components of the Wnt signaling system . Thus, these results demonstrate that different domains of GSK3β are utilized to enable GSK3β to regulate the Wnt and p53 systems. Differential protein-associating domains of GSK3β likely contribute to enabling GSK3 to selectively regulate phosphorylation of its substrates, which number over forty.
Several effects of GSK3 on p53 have been reported, including facilitation of its transcriptional activity and apoptosis [8–10], p53 phosphorylation [12–14], and p53 intracellular localization and trafficking . Since the region encompassing residues 364–373 in the C-terminal basic domain of p53 that is required for its association with GSK3β contains several sites that can be acetylated, we examined if GSK3 regulates p53 acetylation and if p53 acetylation regulates its association with GSK3. Both of these were found to occur, as GSK3 inhibitors substantially reduced the acetylation of p53 at two sites near the region of p53 required for its association with GSK3β, residues K373 and K382, but not at a more distant acetylation site, K320, and increased acetylation of p53 decreased its association with GSK3. Although the functional roles of p53 acetylation remain controversial, several reports suggest that acetylation increases p53 stability and promotes co-activator recruitment, leading to transcriptional activation of target genes . Our findings indicate that the N-terminal region of GSK3 associates with the basic domain of p53 and this facilitates p53 acetylation which leads to dissociation of p53 from GSK3.
This research was supported by a grant from the National Institutes of Health (MH38752).
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