Anti-14-3-3γ (specific) antibody was obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Antibodies to flag the β-actin were purchased from Sigma (St. Louis, MO, USA). Anti-p53 (DO-1) and GFP antibodies were obtained from Oncogene (La Jolla, CA, USA) and BD Pharmingen (San Diego, CA, USA), respectively. Cycloheximide and MG132 were purchased from Calbiochem (La Jolla, CA, USA).

cDNA encoding human 14-3-3γ was cloned behind the CMV promoter in pCMV-Tag2 (with flag tag at N-terminal) expression vector (Stratagene, La Jolla, CA, USA). Human non-small cell lung carcinoma cell line H358 (p53 null) from the American Type Culture Collection (ATCC, Rockville, MD, USA) was cultured in Dulbecco’s modified Eagle’s medium (DMEM; Gibco-BRL) supplemented with 10% fetal bovine serum. Transfection was conducted using LipoTAXI mammalian transfection kit (Stratagene, La Jolla, CA, USA) according to the manufacturer’s instructions. In brief, 3×10⁵ H358 cells were seeded in 60-mm dish and the cells cultured until they reached a confluence of 50–70%. The cells were washed with serum/antibiotic-free DMEM medium prior to transfection. Plasmid pCMV-flag-14-3-3γ (5 μg) purified with Perfectprep Plasmid midi (Eppendorf, Hamburg, Germany) and 50 μl of LipoTAXI reagent diluted in 900 μl of serum/antibiotic-free DMEM medium were mixed gently and incubated at room temperature for 30 min. After adding 1.5 ml of serum/antibiotic-free DMEM medium the mixture was placed onto the cells. Transfection was performed at 37°C in an incubator under 5% CO₂ for 5 h and then further 2.5 ml DMEM containing 20% serum was added. After incubation overnight the DNA mixture was replaced by fresh, complete medium and the cells were incubated for 48 h and split into three 10-cm plates. When the cells grew to 95% confluence, 0.5 μg/ml of G418 (Invitrogen, Carlsbad, CA, USA) was added to medium to select the clones. After three weeks, 24 clones were collected and expression of 14-3-3γ protein was assessed by immunoblotting using anti-Flag antibody (Sigma).

Ad-GFP, Ad-p53 (wild-type) and Ad-p53 (mutant, R175H) expressed GFP, wild-type and mutant p53 were kindly provided by Dr Cyrus Vaziri (Boston University School of Medicine) and Dr Bernard Futscher (Arizona Cancer Center, The University of Arizona), respectively. HCT116 cells with p53 knocked out were plated in 60-mm culture dishes (in DMEM containing 10% fetal bovine serum) and infected by adding virus directly to the medium when the cultures had attained a confluence of ~60%. After 24-, 32- and 48-h infection, the cells were harvested and endogenous 14-3-3γ protein level was analyzed by immunoblotting. H358 cells stably expressing flag-14-3-3γ were also infected by adenovirus-GFP, wild-type and mutant p53 for 24 h and then treated with cycloheximide alone or together with proteasome inhibitor MG132. Eight and 16 h later, cells were lysed and exogenous flag-14-3-3γ was analyzed by immunoblotting.

Human 14-3-3γ fragments (1:1-78 amino acid, N-terminal; 2:79-131 amino acid, central part; 3:132-184 amino acid, central part; and 4:185-247 amino acid, C-terminal) were amplified by PCR and inserted in-frame into pGEX2T vector (Amersham Pharmacia Biotech) for expression of GST fusion proteins. All constructs were sequenced to make sure the sequences were correct. The recombinant glutathione-S-transferase (GST) tagged proteins were expressed in E. coli strain DH5α with 0.5 mM isopropyl-β-L-thiogalactopyranoside (IPTG) at 37°C for 3 h. Cells were collected and lysed in B-PER bacterial protein extraction reagent (Pierce). After incubation on ice 30 min, the samples were centrifuged and the supernatants were purified using glutathione sepharose 4B beads (Amersham Pharmacia Biotech).

Confluent cells (70%) were harvested and lysed in 0.5% Nonidet P-40 lysis buffer (50 mM Tris-Cl, pH 7.4, 0.25 M NaCl, 0.5% NP-40, 50 mM NaF and ptotease inhibitors). The pull-down assay was conducted by incubating cell extracts in lysis buffer with GST-14-3-3s or GST bound to 40 μl glutathione sepharose beads for 2 h at 4°C. After five washes with lysis buffer, the bound proteins were subjected to SDS-PAGE, transferred to nitrocellulose membrane, and immunoblotted with primary antibodies.

Frozen human lung cancerous (squamous cell carcinoma and adenocarcinoma) and normal tissues were obtained from the Department of Pathogenic Biology, Affiliated Hospital of Jiangsu University (Zhenjiang, China). Frozen tissues and cultured cells were lysed in NP-40 lysis buffer containing 50 mM Tris-Cl (pH 7.4), 0.15 M NaCl, 0.5% NP-40, 1 mM DTT, 50 mM sodium fluoride, and 2 μl/ml protease inhibitor cocktail (Sigma). Protein concentrations were determined using the Bio-Rad protein assay kit (Bio-Rad Laboratories, Hercules, CA, USA) and 50 μg protein was resolved by electrophoresis on a 10% SDS-PAGE gel. The proteins were then transferred onto a nitrocellulose membrane and non-specific binds were blocked by incubating with 5% non-fat milk in TBST buffer (0.01 M Tris-Cl, pH 8.0, 0.15 M NaCl, 0.5% Tween-20) at room temperature for 1 h. The membrane was subjected to the indicated antibodies and the proteins were detected by the SuperSignal West Pico detection system (Pierce, Rockford, IL, USA).

H358 cells were co-transfected with Flag-tagged 14-3-3γ expression vector and His-tagged ubiquitin expression vector. At 16 h after transfection, cells were infected with Ad-GFP, Ad-p53 (wt) and Ad-p53 (mt) for 32 h and then harvested. Cells were lysed in NP-40 lysis buffer (50 mM Tris-Cl, pH 7.4, 0.25 M NaCl, 0.5% NP-40, 50 mM NaF and ptotease inhibitors). Co-immunoprecipitation was conducted using Anti-Flag M2 affinity gel (Sigma). Briefly, 200 μg of total protein in 500 μl lysis buffer was incubated with 20 μl Anti-Flag M2 affinity gel at 4°C overnight with rotation and the beads were then washed four times with lysis buffer. The proteins were eluted by boiling in SDS-PAGE sample buffer and applied to SDS-PAGE gels. 14-3-3γ ubiquitin was detected by immunoblotting using the anti-ubiquitin antibody (Upstate).

The Chi-squared test was performed to determine the correlation between mutant p53 and 14-3-3γ protein expression and P<0.05 is considered significant.

Our previous study indicates that high levels of 14-3-3γ protein exist in human lung cancer tissues as compared with the normal lung (16). Importantly, overexpression of 14-3-3γ protein can cause genomic instability by inducing polyploidization in human lung cancer cells (34). p53, a tumor suppressor protein, is reported to interact with several 14-3-3 isoforms including 14-3-3γ and this interaction appears to be functionally significant (32,33). In an attempt to understand the functional relationship between p53 and 14-3-3γ in lung cancers we determined the expression of p53 and 14-3-3γ by western blotting. In total 80 lung cancer tissues and 35 lung normal tissues were used in this experiment. A typical representative expression profile was shown in Fig. 1. As expected, we could not detect any signals of p53 in the 35 normal tissues. Consistent with our previous study, only one of 35 normal tissues had a band of 14-3-3γ. This normal tissue was probably contaminated with cancer cells. However, out of 80 cancer tissues there were 35 (43.8%) with p53 overexpression of (Fig. 1). In these 35 specimens, 33 expressed 14-3-3γ protein. Statistical analysis between 14-3-3γ and p53 proteins expression showed that 14-3-3γ expression significantly correlated with overexpression of p53 protein (P=0.0001; Table I). This result indicated that 14-3-3γ expression in lung cancers was dependent on overexpression of p53.

p53 acts as a transcription factor to activate or silence a number of downstream target genes (27,28). To investigate the effect of p53 on 14-3-3γ protein level we introduced both wild-type p53 and mutant p53 (R175H) into human colon cancer cell line HCT116 in which endogenous wild-type p53 gene had been knocked out. We infected the cells with adenovirus containing GFP, wild-type p53 (wt) and mutant p53 (mt) cDNA for 24, 32 and 48 h, and then the endogenous 14-3-3γ protein level was examined by immunoblot analysis using a specific antibody against 14-3-3γ. The same blot was also probed with antibodies against GFP and p53 to detect the efficiency of adenovirus infection. Anti-human β-actin antibody was also used on the same blot to control for variations in loading. The results showed that after 32 h the wild-type p53 caused a considerable decrease (~50%) in 14-3-3γ protein level (Fig. 2). However, neither GFP nor mutant p53 (R175H) had any effect on levels of 14-3-3γ in HCT116 cells (p53^−/−). Hence, wild-type p53, but not mutant p53 (R175H) suppressed endogenous 14-3-3γ protein levels in colon cancer cells.

Like other genes, 14-3-3γ regulation may occur at multiple levels, including transcription, post-transcriptional regulation of mRNA, translation and post-translational modification (36). Our previous study demonstrated that one of mechanism of p53 suppression of 14-3-3γ is through binding its promoter and inhibiting 14-3-3γ transcription (35). In order to clarify whether p53 suppresses 14-3-3γ post-transcription we stably transfected 14-3-3γ with Flag tag into human lung cancer cell line H358 which is p53 null and then examine whether p53 suppresses this exogenous 14-3-3γ protein. H358 cells which constitutively express Flag-14-3-3γ protein were infected by adenovirus-GFP, wild-type p53 and mutant p53 (R175H) for 24 h and cycloheximide was added to inhibit protein synthesis. At 0, 8 and 24 h after cycloheximide treatment, the Flag-14-3-3γ protein levels were determined by immunoblot analysis using an antibody against Flag. Similarly to the effect of p53 on the endogenous 14-3-3γ in colon cancer cell line HCT116, wild-type p53 decreased 50% of Flag-14-3-3γ protein (Fig. 3). This result clearly indicates that p53 can negatively regulate 14-3-3γ by post-translational modification.

To determine whether p53 mediated 14-3-3γ reduction by stimulating proteasomal degradation of 14-3-3γ a specific inhibitor of proteasomal activity, MG132, was used and its effect on p53-mediated 14-3-3γ reduction was examined. The results showed that MG132 treatment completely abolished the p53-mediated reduction of 14-3-3γ protein (Fig. 4) which suggests that p53-mediated suppression of 14-3-3γ is accomplished by stimulating the process of proteasomal degradation of the protein.

It has been reported that wild-type of p53 can bind to 14-3-3γ (32,33). This interaction may lead to proteasomal degradation of 14-3-3 protein. However, the detailed binding domain of 14-3-3γ is still unclear. To further explore the possible region of 14-3-3γ that is involved in binding to p53, we made GST-14-3-3γ truncated fusion proteins including N-terminal, GST-14-3-3γ1 (1-78 amino acids); central regions, GST-14-3-3γ2 and 3 (79-131 and 132-184 amino acids); and C-terminal GST-14-3-3γ4 (185-247 amino acids) (Fig. 5A). HCT116 cells were lysed and pull-down assay experiment was performed to determine the interaction. Fig. 5B showed that the C-terminal region of 14-3-3γ bound to p53 very efficiently compared with other domains, demonstrating that p53 could interact with C-terminal of 14-3-3γ.

Since ubiquitination is required in the process of protein degradation by 26S protesome which can be blocked by MG132, we next determined whether p53 increases 14-3-3γ protein ubiquitin. H358 cells were co-transfected with Flag-tagged 14-3-3γ expression vector and His-tagged ubiquitin expression vector and then infected with Ad-GFP, Ad-p53 (wt) and Ad-p53 (mt) viruses. After immunoprecipitation by anti-Flag antibody, 14-3-3γ ubiquitination was evaluated, and Fig. 6 clearly shows that wild-type p53 stimulated 14-3-3γ ubiquitination in vivo. Taken together, these data indicated that p53 could bind to 14-3-3γ, and enhanced its ubiquitin and proteasome-mediated degradation.