Our data highlighted a novel mechanism underlying the potential oncogenic function of 14-3-3ζ, whereby, 14-3-3ζ binding to p85 on the phospho-serine 83 motif in the N-terminal domain of p85 contributes to PI3K membrane localization, PI3K activation, and cancer cell proliferation and survival. 14-3-3 proteins impact multiple pathways implicated in tumorigenesis; however, it is not clear which 14-3-3ζ interacting proteins or regulated pathways may contribute to cellular transformation. The current study using the p85S83μ mutant demonstrated that 14-3-3ζ binding to the phospho-serine 83 motif on p85 plays an important role in activation of the PI3K/Akt pathway. We demonstrated that mutation of the 14-3-3-binding motif on p85 effectively reduced PI3K activation and impaired many general properties of cellular transformation. Although other mechanisms may contribute to 14-3-3ζ’s tumor-promoting function, our data demonstrated that 14-3-3ζ activation of PI3K could serve as a target for multiple cancer types.
Our data demonstrates an interaction between 14-3-3ζ and p85 in breast cancer cells. In addition, 14-3-3ζ was also found to interact with the p85 subunit of PI3K in platelets and 14-3-3τ was found to interact with the p110 subunit of PI3K in T cells; however, neither study defined an interaction site for 14-3-3 on either PI3K subunit (Bonnefoy-Berard et al., 1995
; Munday et al., 2000
). The sequence surrounding serine 231 is not highly conserved among species, and mutation of serine 231 and serine 154 in our study did not impact 14-3-3ζ binding to p85. In contrast, the serine 83 motif is highly conserved and mutation of this motif abrogated 14-3-3ζ binding, indicating that the serine 83 motif on p85 is likely the major motif mediating 14-3-3ζ binding. However, we do not rule out that other sites may also contribute to overall 14-3-3 binding. We have also demonstrated that 14-3-3ζ overexpression could activate PI3K in breast cancer cells. In contrast, 14-3-3τ overexpression reduced PI3K activity in activated T cells (Bonnefoy-Berard et al., 1995
). Interestingly, in vitro
experiments utilizing platelet cytosol determined the effect of recombinant 14-3-3ζ on PI3K activity was biphasic. At low concentrations, 14-3-3ζ inhibited PI3K, but at higher concentrations, 14-3-3ζ enhanced PI3K activity (Munday et al., 2000
). Signaling pathways are highly dynamic and have well established dose dependent responses. It is reasonable to speculate that activation of PI3K by 14-3-3ζ in cancer cells is a consequence of the 14-3-3ζ overexpression above a threshold level. Additional cofactors and stimuli in different cell types may also affect the interaction of 14-3-3ζ and p85, thereby changing PI3K activity and downstream biological processes.
Because the interaction between 14-3-3ζ and p85 is dependent on phosphorylation of serine 83 on p85, it is important to study the regulation of serine 83 phosphorylation. As we were investigating along this line, protein kinase A (PKA) was identified to phosphorylate serine 83 on p85 in response to thyroid hormone stimulation in non-transformed thyroid cells (Cosentino et al., 2007
; De Gregorio et al., 2007
), which supports our finding that serine 83 on p85 was phosphorylated in vivo
. Here, we confirmed that PKA could phosphorylate serine 83 on p85 and that serine 83 phosphorylation contributes to 14-3-3ζ-mediated transformation of cancer cells. These data suggest differential regulation of p85 serine 83 phosphorylation, and thus differential regulation of PI3K signaling, between non-transformed cells and 14-3-3ζ overexpressing cancer cells. Our data also raise questions as to whether 14-3-3ζ binding to serine 83 of p85 controls other cellular process and disease states dependent on PI3K. The PI3K pathway not only impacts cancer, but is also involved in immunity, obesity, diabetes, energy balance, angiogenesis, hematopoiesis, and stem-cell self-renewal, as well as the survival and function of neurons, heart, bone, muscle, and liver tissue (Katso et al., 2001
; Kim et al., 2005
; Vanhaesebroeck et al., 2005
). Further investigation is needed to determine whether the ubiquitously expressed 14-3-3ζ modulates these important biological functions by targeting and activating PI3K.
Development of clinically applicable, specific inhibitors of the PI3K/Akt pathway is a current focus in the area of targeted therapy. In our patient cohort, high levels of both 14-3-3ζ expression and Akt phosphorylation in tumors was associated with breast cancer recurrence (), indicating that the14-3-3ζ/PI3K interaction is important in breast cancer progression and interfering with this interaction may provide clinical benefit. Directly targeting 14-3-3ζ may lead to toxic side effects due to a potential role of 14-3-3 in normal cellular homeostasis. However, disrupting the interaction of 14-3-3ζ and p85 by targeting the 14-3-3/p85 interface may be a drugable target since our data demonstrated that mutation of serine 83 on p85 reduced transformation-related properties of cancer cells. Understanding the dynamic regulation of 14-3-3ζ interaction with p85 may allow development of effective therapies targeting this pathway.
14-3-3 binding to target proteins may act as a “molecular anvil” to induce a conformational change in the target protein, which may lead to enhanced interactions with other proteins, post-translational modifications, or altered subcellular localization (Yaffe, 2002
). We showed that 14-3-3ζ binding to phospho-serine 83 on the p85 subunit of PI3K increased PI3K membrane localization in cancer cells. The N-terminal domain of p85 is known to have a negative regulatory function on PI3K activity that is relieved by receptor tyrosine kinase interaction (Yu et al., 1998
). Similarly, 14-3-3ζ binding to serine 83 in the N-terminal domain may relieve the inhibitory function of p85, facilitate the interaction of PI3K with other adapter molecules, or enhance membrane localization and activity. This suggests 14-3-3ζ overexpression and serine 83 phosphorylation on p85 could represent alternative mechanisms to PI3K gene mutations or PTEN-loss to activate PI3K pathway in cancer. Interestingly, although MCF-7 cells harbor an activating mutation in exon 9 of p110 that makes PI3K activation independent of p85, our data showed that mutation of serine 83 on p85 could still reduce PI3K membrane localization and activation in MCF-7 cells. This suggested that serine 83 phosphorylation on p85 may define a novel regulatory mechanism of PI3K activation. This model implies that targeting 14-3-3ζ or pathways regulated by 14-3-3ζ may be effective anti-cancer strategies in patients whose tumors overexpress 14-3-3ζ.