PED/PEA-15 is a recently identified protein featuring a broad antiapoptotic function (5
). PED/PEA-15 inhibits the apoptotic signal of FasL, tumor necrosis factor alpha, and TRAIL (7
) and also blocks apoptosis following the activation of several SAPKs (6
). Previous work in our own as well as in other laboratories has shown that phosphorylation by PKC plays an important role in enabling the antiapoptotic function of PED/PEA-15 (7
In the present paper we demonstrate that PED/PEA-15 phosphorylation by Akt also regulates PED/PEA-15 control of cell apoptosis.
We have shown that Akt phosphorylates PED/PEA-15 in vitro as well as in intact cells. PED/PEA-15 sequence analysis revealed a low-stringency Akt phosphorylation site, Ser116
. Hence, in vitro, Akt phosphorylates the wild-type PED/PEA-15 but not a mutant PED/PEA-15 featuring the Ser116
→Gly substitution. Akt also phosphorylates twofold more effectively a synthetic peptide featuring the sequence of PED/PEA-15 surrounding Ser116
than two other equally sized PED/PEA-15 peptides including either Ser104
. In addition, active Akt does not phosphorylate the PEDS116→G
mutant when expressed in 293 cells, whereas it does phosphorylate wild-type PED/PEA-15. Finally, Western blotting studies with a specific phospho-Ser116
PED/PEA-15 antiserum identified Ser116
as the major Akt phosphorylation site of PED/PEA-15 in vivo and in vitro. Akt substrate serines are usually embedded in RXRXXS consensus sequences (1
). In contrast, the 5-amino-acid region of PED/PEA-15 upstream from Ser116
exhibits only a single Arg residue. The same structural feature has been reported for the Akt Ser substrates of CREB (10
) and of the 14-3-3ζ scaffold protein (27
). In the case of PED/PEA-15, we showed that PED/PEA-15 directly binds to Akt. The binding is independent of Akt activity, as it effectively occurs to both constitutively active and inactive Akt mutants as well as to wild-type Akt. In addition, PED/PEA-15 binds Akt with an affinity similar to that exhibited for ERK1, a known PED/PEA-15 ligand (14
) (data not shown). It is possible, therefore, that in the cell PED/PEA-15 recruits Akt and that this facilitates PED/PEA-15 phosphorylation by the kinase.
When expressed in 293 cells, the S116
→G mutant PED/PEA-15 showed an almost twofold decrease in antiapoptotic function compared to wild-type PED/PEA-15. In 293 cells, PED inhibition of apoptosis requires PED activation of ERKs (5
). However, reduced antiapoptotic function of the Ser116
→G mutant did not appear to depend on changes in its interaction with ERKs, as ERK activation levels were not different in cells expressing the mutant and in wild-type cells (data not shown). Interestingly, a pharmacological blocking of wild-type PED/PEA-15 phosphorylation at Ser116
was accompanied by a significant increase in 293 cell apoptosis, even in the presence of serum. These findings suggest that, at least in the 293 cells, PED may switch from an unphosphorylated proapoptotic form to a phosphorylated antiapoptotic form. Hence, expression of the Ser116
→Gly mutant induces apoptosis, likely contributing to the reduced protection from apoptosis observed in 293PEDS116→G
compared to the 293PEDWT
cells. Kubes et al. reported that endothelin-activated CaMKII also phosphorylates PED/PEA-15 at Ser116
). Consistent with our findings, endothelin exerts antiapoptotic action in different cell types (9
). It is unlikely that CaMKII serves as a downstream mediator of Akt action on PED/PEA-15 phosphorylation in the intact cell, as blocking of CaMKII does not inhibit Akt phosphorylation of PED/PEA-15 at Ser116
. More likely, therefore, PED/PEA-15 represents a common target for multiple kinases transducing survival signals triggered by growth factors and cytokines, supporting an important role of PED/PEA-15 in the cellular regulation of apoptotic programs.
Earlier works indicated that PED/PEA-15 phosphorylation at Ser116 facilitates subsequent phosphorylation by PKC at Ser104. In the present report, we show that the single substitution Ser104→G of PED/PEA-15 also reduced the antiapoptotic activity of PED/PEA-15, but only by 30%. These findings indicate that impaired phosphorylation at Ser104 may account, in part, for reducing PED/PEA-15 antiapoptotic action when phosphorylation of Ser116 is disabled. Consistent with this possibility, blocking of PKC only slightly increased apoptosis in 293S116→G cells.
Despite the relevance of phosphorylation at Ser116, other factors seemed to be involved in regulating PED/PEA-15 antiapoptotic function in 293 cells. Hence, apoptosis induced by serum starvation was less in cells expressing the S116→G mutant than in cells expressing no PED/PEA-15 at all. Akt regulation of PED function is not unique to the 293 cells, however. In fact, we report that blocking of Akt in human glioma cells expressing high levels of PED/PEA-15 simultaneously decreases PED/PEA-15 phosphorylation at Ser116 and rescues sensitivity to the apoptotic cytokine TRAIL.
Akt regulates a number of cellular functions including cell survival (25
). Different substrates are phosphorylated by Akt and converted into survival proteins (3
). This function, in turn, is accomplished by regulating protein-protein interaction (12
), by affecting protein localization (3
) or protein stability (24
). In the present work, we show that Akt phosphorylation increases the stability of PED/PEA-15 in the cell. Hence, PED/PEA-15 levels are low in cells subjected to serum starvation, when Akt activity is inhibited, and in cells subjected to pharmacological blocking of the PI 3-K/Akt pathway. PED/PEA-15 cellular levels increase upon expression of a constitutively active Akt mutant. In addition, we report that the S116
→G PED/PEA-15 mutant features reduced stability compared to that of wild-type PED/PEA-15. At variance with wild-type PED/PEA-15, the S116
→G mutant is unaffected by Akt. Thus, at least in part, Akt regulates PED protein stability and antiapoptotic function by phosphorylating PED/PEA-15 at Ser116
. PED/PEA-15 degradation is blocked by treatment of the cells with the proteasome inhibitor lactacystin, suggesting that PED/PEA-15 intracellular levels are regulated by the ubiquitin pathway (data not shown). Whether this hypothesis holds and whether PED/PEA-15 ubiquitination is, in turn, inhibited by Akt phosphorylation is currently being investigated in our laboratory.
The PED/PEA-15 gene is amplified in human breast cancer (20
) as well as in other tumors and PED/PEA-15 overexpression may have a role in skin carcinogenesis (S. Santopietro and J. Portella, personal communication). Akt is also upregulated in a number of human cancers (26
). In this work, we demonstrated that PED/PEA-15 is a substrate for Akt and obtained evidence that Akt phosphorylation and stabilization of PED/PEA-15 is a previously unrecognized mechanism involved in Akt survival signaling. Simultaneous increases in PED/PEA-15 cellular levels and Akt activity might function cooperatively in tumorigenesis and/or tumor progression in humans.