The experiments described in this paper show that human securin can associate with the fully assembled, enzymatically active PP2A holoenzyme. We identified through a yeast two-hybrid screen, using hSecurin as bait, the B55δ regulatory subunit of PP2A. Pull-down and coimmunoprecipitation assays demonstrated that hSecurin interacts specifically with the PP2A complex both in vitro and in vivo and that this association does not depend on the cell cycle phase. Among the proteins shown to associate with PP2A and regulate its activity are the small t antigens encoded by SV40 and polyomavirus, the adenovirus E4orf4 protein, casein kinase II, and Hox II (19
). In some cases, the interaction results in a negative regulation of PP2A activity. Although we have not determined its effect on the specific activity of PP2A, hSecurin clearly does not block its enzymatic activity.
Our discovery that hSecurin associates with enzymatically active PP2A provided the impetus for testing whether phosphorylated hSecurin might serve as a substrate of PP2A. Our data strongly support the conclusion that this is the case. Experiments performed in vitro using a commercial source of PP2A and phosphorylated hSecurin as a substrate revealed that PP2A is able to dephosphorylate the various forms of phosphorylated hSecurin obtained from okadaic acid-treated cells but not the main phosphorylated form of hSecurin obtained from mitotically arrested nocodazole-treated cells. Thus, the ability of PP2A to dephosphorylate hSecurin is probably dependent on the specific phosphorylated residue.
The observation that okadaic acid treatment of cells results in the destabilization of hSecurin suggests that PP2A may regulate hSecurin stability. Okadaic acid has been shown to preferentially inhibit PP2A at the low concentration used in these experiments. However, we cannot rule out that okadaic acid does not inhibit to some degree the other major serine-threonine phosphatases PP1, PP2B/calcineurin, and PP2C or other less-well-characterized phosphatases (11
). Nevertheless, two observations strongly implicate PP2A in the regulation of hSecurin stability. First, decreasing cellular PP2A activity, as a result of using RNA interference against the catalytic subunit of PP2A, leads to a reduction in hSecurin levels. Second, increasing the cellular levels of the PP2A regulatory subunit B55δ promotes the accumulation of hSecurin by reducing its turnover, and which is very meaningful, this B55δ effect requires the phosphatase activity of PP2A. Moreover, the effect of PP2A on the stabilization of hSecurin is not a consequence of the influence of PP2A on the cell cycle progression, since cyclin B levels remain unchanged in either cells overexpressing B55δ or cells with reduced levels of PP2A-cs. In conclusion, we propose that the association of PP2A to hSecurin leads to hSecurin stabilization via its dephosphorylation.
It has been well documented that PP2A activity is important for the regulation of the stability of the proto-oncogenic transcription factor c-Myc. The fine tuning of c-Myc expression occurs at the protein level through modulation of its stability (3
). The half life of c-Myc polypeptides is determined largely by phosphorylation of two specific residues found in all mammalian Myc family members, which results in its degradation via the ubiquitin proteasome pathway (21
). PP2A was shown to be required to dephosphorylate c-Myc at serine 62, leading to its proteolysis (56
). Another example of proteins in which PP2A regulates their stability is the Pim family of protein kinases. The Pim kinases associate with the catalytic subunit of PP2A in vivo and are substrates of PP2A phosphatase in vitro. Overexpression of PP2A decreases the steady-state levels of the Pim proteins, and the inhibition of PP2A activity by okadaic acid enhances the stability of Pim-1 (33
In an attempt to determine the effect of okadaic acid treatment, and thus PP2A action, on the phosphorylation status of hSecurin, we found that not only was hSecurin hyperphosphorylated, but the various hyperphosphorylated forms were extremely unstable, being rapidly degraded by the proteasome. Knowing that the APC/C E3 ubiquitin ligase targets hSecurin for destruction during anaphase and mitotic exit, we naturally studied its involvement in okadaic acid-mediated instability of hSecurin. To be fully active, APC/C requires two additional subunits that regulate its target specificity, Cdc20 and Cdh1, thus forming two distinct complexes (APC/CCdc20
). Target specificity is due to the ability of Cdc20 and Cdh1 to recognize two specific destruction sequences on APC/C substrates, namely the D-box and KEN-box motifs (18
). hSecurin degradation is catalyzed by both Cdc20 and Cdh1, is mediated by a D-box and a KEN-box, and is inhibited only when both sequences are mutated (59
). Our results demonstrate that degradation of phospho-hSecurin, produced during okadaic acid treatment, is not dependent on the APC/C. First, the nondegradable form of hSecurin (with both D- and KEN-box motifs mutated) is not resistant to okadaic acid-mediated degradation. Second, the depletion of both APC/C activators, Cdc20 and Cdh1, by siRNA was not able to block the degradation of phospho-hSecurin. We thus turned to investigate the involvement of other ubiquitin ligases in this degradation process. Knowing that the action of the SCF ubiquitin ligase complex is tightly coupled to substrate phosphorylation status, we decided to study whether the SCF plays a role in the degradation of phospho-hSecurin. The SCF ligase has a broad function in many physiological processes by ubiquitinating proteins involved in, for example, cell cycle regulation, transcription, and signal transduction (22
). Recruitment of substrates to the SCF complex occurs via one of a variety of F-box protein subunits, which function as molecular adaptors (8
). F-box proteins interact via the F-box motif with the Skp1 subunit, which can bridge the F-box to the cullin (35
), which in turn serves as a scaffold to bring the catalytic RING finger protein to the substrate (50
). Substrate recognition by the F-box protein is mediated in most cases by phosphorylation of target proteins (22
To test the possible involvement of the SCF in mediating hSecurin degradation in okadaic acid-treated cells, we made use of a dominant-negative N-terminal Cul1 mutant that interferes with the degradation of SCF substrates (54
). Upon expression of this mutant by transient transfection, hSecurin was substantially stabilized in okadaic acid-treated cells. This result provides evidence that the SCF is involved in the proteolysis of phospho-hSecurin and indicates that the abundance of hSecurin in the cell is regulated not only by the APC/C during metaphase and mitotic exit, but also by the SCF at other cell cycle stages. Such a dual mode of degradation has been described for the Cdc25A phosphatase, an activator of cell cycle progression. Both the APC/C and the SCF are involved in Cdc25A turnover. While the APC/C degrades Cdc25A during mitotic exit, SCF regulates its abundance in the S and G2
phases. Moreover, in response to DNA damage or to stalled replication, the activation of the ATM and ATR protein kinases results in Chk1 and Chk2 activation and Cdc25A hyperphosphorylation that stimulate SCF-mediated ubiquitination of Cdc25A and its proteolysis (6
). In this context, it is worth mentioning that hSecurin is also degraded in response to irradiation (43
). The new findings reported here raise additional important questions that will need to be addressed in the future. These include the nature of the phosphorylation events that trigger hSecurin degradation and the role of SCF ubiquitination in regulating hSecurin levels during a normal cell cycle as well as in response to DNA damage. Unraveling the mechanisms involved in regulating hSecurin stability is of additional interest in that it may contribute to explaining the cause of the high hSecurin levels detected in many tumors, since no mutations in its promoter or in its open reading frame have been found to date, and it may further our understanding of how such high levels lead to oncogenesis.