Overexpression of 14-3-3ζ in cancer cells activates PI3K via binding the p85 regulatory subunit

Abstract

The ubiquitously expressed 14-3-3 proteins regulate many pathways involved in transformation. Previously, we found that 14-3-3ζ overexpression increased Akt phosphorylation in human mammary epithelial cells. Here, we investigated the clinical relevance and molecular mechanism of 14-3-3ζ-overexpression-mediated Akt phosphorylation, and its potential impact on breast cancer progression. We found that 14-3-3ζ overexpression was significantly (P=0.005) associated with increased Akt phosphorylation in human breast tumors. Additionally, 14-3-3ζ overexpression combined with strong Akt phosphorylation was significantly (P=0.01) associated with increased cancer recurrence in patients. In contrast, knockdown of 14-3-3ζ expression by small interfering RNA in cancer cell lines and tumor xenografts reduced Akt phosphorylation. Furthermore, 14-3-3ζ enhanced Akt phosphorylation through activation of phosphoinositide 3-kinase (PI3K). Mechanistically, 14-3-3ζ bound to the p85 regulatory subunit of PI3K and increased PI3K translocation to the cell membrane. A single 14-3-3-binding motif encompassing serine 83 on p85 is largely responsible for 14-3-3ζ-mediated p85 binding and PI3K/Akt activation. Mutation of serine 83 to alanine on p85 inhibited 14-3-3ζ binding to the p85 subunit of PI3K, reduced PI3K membrane localization and activation, impeded anchorage-independent growth and enhanced stress-induced apoptosis. These findings revealed a novel mechanism by which 14-3-3ζ overexpression activates PI3K, a key node in the mitogenic signaling network known to promote malignancies in many cell types.

Figure 1. Overexpression of 14-3-3ζ is associated with increased Akt phosphorylation

(a) Representative immunohistochemical (IHC) staining of phospho-Akt Ser473 (P-Akt) (IHC score: 0, negative; 1+, moderate positive; 2+, strong positive) and 14-3-3ζ (0/1+, weak positive; 2+, moderate positive; 3+, strong positive) in matched breast cancer specimens. Bar represents 50 µm. (b) Graphical representation of 14-3-3ζ expression correlated with levels of phospho-Akt Ser473 (P-Akt) by immunohistochemical staining in breast cancer specimens (n=121) (Chi square, P = 0.005). (c) Disease-free survival rates of patients examining combined 14-3-3ζ expression and phospho-Akt Ser473 expression. 14-3-3ζ scores 0, 1+, and 2+ were considered negative (−) and 3+ was considered positive (+). P-Akt scores 0 and 1+ were considered negative (−) and 2+ was considered positive (+). P value was determined by log rank analysis.

Figure 2. 14-3-3ζ binds to p85 and enhances PI3K membrane localization and activation

(a) MCF10A cells stably transfected with HA-14-3-3ζ (10Aζ) or vector as control (10A.vec) were immunoprecipitated (IP) with PY20 antibody and subjected to PI3K assays. Immunoblot (IB) of total p85 showed similar protein recovery following immunoprecipitation. (b) Immunoblot of membrane and cytoplasmic proteins from MCF10A transfectants. ErbB2 and alcohol dehydrogenase (ADH) were used as membrane and cytoplasmic markers, respectively. Relative intensity was quantified by densitometry using ErbB2 and ADH as loading controls and standardized to vector control. The membrane to cytoplasmic ratio was determined by dividing the corrected value obtained by densitometry of the membrane fraction by the corrected value obtained by densitometry of the cytoplasmic fraction. (c) Immunofluorescent staining of MCF10A cells stably transfected with vector (10A.vec) or HA-tagged 14-3-3ζ (10A.ζ). Cells were stained with anti-HA (14-3-3ζ) and p110 specific antibodies. DAPI staining represents nuclei. Arrows indicate areas of membrane localization. The scale bars represent 20µm. (d) MCF10A HA tagged 14-3-3ζ transfectants and their respective vector control cells were immunoprecipitated (IP) with anti-HA or IgG followed by immunoblot with p110, p85, and 14-3-3ζ antibodies. (e) MCF7 cells were immunoprecipitated (IP) with 14-3-3 or p85 antibodies to detect endogenous association of 14-3-3ζ and p85.

Figure 3. Phosphorylation of serine 83 on p85 defines a 14-3-3 binding motif

(a) MCF7 14-3-3ζ transfectants (MCF7ζ) were stably transfected with empty vector (vector), histidine/Xpress-tagged (His/Xpress) wild type p85 (p85^WT), or tagged p85 mutated in the 14-3-3 binding site encompassing S83 (p85^S83μ). Exogenous p85 was immunoprecipitated (IP) with Xpress or His antibodies followed by immunoblot with HA to detect exogenous 14-3-3ζ. Immunoprecipitation efficiency of Xpress or His-tagged p85 was determined by immunoblot with p85 antibody. Relative intensity was quantified by densitometry and standardized to p85^WT. (b) p85 phospho-serine 83 antibody (p85-P-S83) immunoprecipitates (IP) from MCF7ζ cells or total cell lysates from MCF7ζ (TCL) were untreated (−) or treated (+) with calf intestine alkaline phosphatase (CIAP) followed by immunoblot with p85-P-S83 and p85 antibodies. (c) 293T cells were transiently transfected with p85^WT or p85^S83μ. Lysates were immunoprecipitated with p85-P-S83 antibody followed by Xpress immunoblot to confirm p85 expression and loss of serine 83 phosphorylation on p85^S83μ. (d) top: in vitro kinase assays using purified protein kinase A (PKA) and either GST fused full length p85 (GST-p85) or GST fused p85 peptide spanning amino acids 50-109 (GST-p85Δ2) as substrate. Serine 83 was either wild type serine 83 (WT) or serine 83 mutated to alanine (SA). Bottom: Comassie blue staining of purified GST-p85, GST-p85Δ2 or GST showing equal amounts used in the kinase assay.

Figure 4. Serine 83 phosphorylation on p85 is necessary for PI3K activation and membrane translocation in 14-3-3ζ overexpressing cells

(a) Immunoblot of membrane and cytoplasmic proteins from MCF7ζ.p85 stable transfectants. Exogenous p85 localization was determined by Xpress antibody. ErbB2 and 14-3-3ζ were used as membrane and cytoplasmic markers, respectively. Relative intensity was quantified by densitometry using ErbB2 and 14-3-3ζ as loading controls and standardized to vector control for p110 or p85^WT for Xpress. The membrane to cytoplasmic ratio was determined by dividing the corrected value obtained by densitometry of the membrane fraction by the corrected value obtained by densitometry of the cytoplasmic fraction. (b) Exogenous p85 was immunoprecipitated with His antibody from the indicated MCF7ζ transfectants followed by PI3K assays (top). Akt phosphorylation was determined from whole cell lysates by immunoblot with phospho-Akt Ser473 (P-Akt) (bottom). Relative activity and intensity were quantified by densitometry and standardized to vector control. (c) MCF10A.vec (10A.vec) and MCF10Aζ (10Aζ) cells were transiently transfected with p85^WT or p85^S83μ. Exogenous His/Xpress-p85 was immunoprecipitated (IP) with His antibody followed by PI3K assays (upper panel). Immunoprecipitation efficiency of His-tagged p85 was determined by immunoblot (IB) with p85 antibody (lower panel). Relative activity was quantified by densitometry and standardized to p85^WT for each cell line. (d) Mouse embryonic fibroblasts (MEF) with knockout p85 (KO) were re-constituted with histidine/Xpress-tagged (His/Xpress) p85 wild type (85^WT), p85 14-3-3 binding mutant (85^S83μ) or empty vector (Vec). Cells were serum starved then stimulated with FBS for the indicated times. Akt activation was assessed by immunoblot for phospho-Akt Ser473 (P-Akt). Relative intensity was quantified by densitometry and standardized to KO/Vec for each cell line.

Figure 5. p85 serine 83 phosphorylation and 14-3-3ζ binding modulates transformation in 14-3-3ζ overexpressing cells

(a) Proliferation assay of MCF7ζ.p85^WT or p85^S83μ stable transfectants in 0.5% serum (linear regression, p=0.02) (b) MCF7ζ.p85^WT or p85^S83μ stable transfectants were serum starved for the indicated times. Apoptotic cells were identified by TUNEL staining (t test, *, p=0.003). (c) Soft agar colony formation of MCF7ζ.p85^WT, or p85^S83μ transfectants (t test, *, p=0.03). Error bars in (A–C) represent SEM. (d) MCF7 stable transfectants were grown in normal serum conditions. Cell lysates were immunblotted with the indicated antibodies. Akt activation was assessed by immunoblot for phospho-Akt Ser473 (P-Akt). Exogenous p85 levels were detected with Xpress antibody.