PH domain-only protein PHLDA3 is a p53-regulated
repressor of Akt
Tatsuya Kawase1, Rieko Ohki2(1Tokyo University of Science, 2National Cancer Center Research Institute)
Figure 1 PHLDA3 is induced by p53
(A) HCT116 p53 (+/+) and HCT116 p53 (-/-) cells were subjected to γ-ray
irradiation or 5-FU. Cells were harvested at the indicated times following
treatment. RNA was purified from each sample and analyzed by quantitative real-
time PCR. The mRNA levels of PHLDA3 and p21 were standardized by mRNA
levels of GAPDH.
(B) Western blotting was performed to detect PHLDA3 protein expression.
Indicated cells were treated with 5-FU for 20 hrs. Note that neither PHLDA3 nor
p21 protein is induced in cell lines carrying mutant p53.
Figure 4 PHLDA3 interferes with Akt activation by inhibiting Akt binding to
(A) Indicated cells were infected with Ad-LacZ, Ad-p53 or Ad-PHLDA3 at moi 35,
and analyzed 18 hrs post-infection, when apoptotic cells were not yet detected. Akt
and Erk activities were assessed by analyzing phosphrylated Akt and Erk1/2 by
(B) Akt subcellular localization in 293T cells with the constitutive active PI3K/Akt
pathway. 293T cells were transfected with GFP-PH-Akt together with DsRed,
DsRed-wtPHLDA3 or DsRed-mtPHLDA3, and GFP-PH-Akt subcellular
localization was analyzed. Arrows indicate Akt localized at the plasma membrane.
(C) PHLDA3 inhibits PH-Akt binding to PIP2and PIP3. Binding of GST-PH-Akt to
immobilized PIP was assessed by protein-lipid overlay assay. Nitrocellulose
membranes spotted with serially diluted PIP2and PIP3were incubated with the
indicated proteins. While GST did not interfere with Akt binding to PIP, PHLDA3
significantly interfered. Bound Akt was detected with anti-Akt PH domain antibody.
Figure 5 PHLDA3 is required for p53-dependent apoptosis
(A) PHLDA3 induction by Ad-p53 in MM468 cells stably expressing control shRNA
or shRNA targeting PHLDA3. Cells were infected with Ad-LacZ or Ad-p53 at moi
15. Induction of PHLDA3 protein was verified by anti-PHLDA3 polyclonal and
monoclonal antibodies. Expressions of p53, p21 and Noxa were also analyzed.
(B, C) Downregulation of PHLDA3 results in decreased p53-dependent apoptosis and
elevated Akt phosphorylation. Ctrl-sh, PH3-sh1 and PH3-sh2 cells were infected with
Ad-p53 at moi 3 (in B) or moi 1 (in C). Apoptotic cells as determined by cells with
sub-G1 DNA content were analyzed 43 hrs post-infection (B). In C, cells were treated
with z-VAD-fmk to protect Akt from p53-dependent Akt degradation. Cells were
harvested 50 hrs post-infection, and analyzed by Western blotting.
(D) Ctrl-sh or PH3-sh2 cells were transfected with GFP-PH-Akt, and treated with
EGF for 5 min 24 hrs post-transfection. Representative images of cells are shown.
Figure 6 PHLDA3 suppresses anchorage-independent cell growth
(A) Control and myr-Akt-expressing cells (1x104cells) were plated in soft agar and
cultured for 4 weeks. Colonies were analyzed by Image J software. Colonies were
counted from 3 plates and the mean numbers of colonies +/- SD are shown.
(B) Ctrl-sh, PH3-sh1 and PH3-sh2 cells were plated and analyzed as described above.
(C) Novel Akt inhibiting pathway downstream of p53 mediated by PHLDA3.
Figure 2 PHLDA3 is a direct target gene of p53
(A) Genomic organization of PHLDA3 (chr1: 198,164,473-198,171,696) is shown
together with the plots of pairwise genomic alignments between human and mouse,
human and rat, and human and chicken. Genomic alignment plots were constructed
using VISTA. Based on genomic alignments, nucleotide conservation was calculated
with a 100 bp window, and conservation percentage is shown as a plot. The position
and nucleotide sequence of p53 responsive element are shown at the bottom. (B)
PHLDA3 promoter region with or without p53RE (shown as an oval) was cloned
upstream of firefly luciferase reporter gene with a minimal promoter, and luciferase
reporter assay was performed. Constructs contain indicated positions relative to the
transcription initiation site. Nucleotides that matched p53 consensus binding sites, 4 th
and 7 th nucleotides for promoter 2 mut. 1 and the 14 th nucleotide for promoter 2
mut.2, were mutated. Constructs were tested for transactivation by wild-type p53 and
p53-V143A. The assay was performed 24 hrs post-transfection. (C) HCT116 p53 (+/+)
cells were treated with 5-FU and ChIP-chip analysis was performed. Genomic locus of
PHLDA3 is shown together with the results obtained by p53 and tri-methyl K4/H3
ChIP-chip. Vertical axis shows the probability value (-log P), which reflects the fold
enrichment of ChIP-chip samples. Blue and red lines at the top indicate the p53-
consensus region computed from TRANSFAC analysis. One p53-consensus region
matched completely with p53RE obtained from our analysis (shown by a red line).
Figure 3 PHLDA3 induces apoptosis and represses Akt
(A) PHLDA3 induces caspase-dependent cell death. MM468 cells were transduced with
Ad-LacZ or N-terminally HA-tagged PHLDA3-expressing adenovirus (Ad-PHLDA3) at
moi 15. Cells were cultured with or without caspase-specific peptide inhibitor z-VAD-
fmk. Cells were harvested 48 hrs post-infection, and analyzed by FACS.
(B) Binding of GST-PHLDA3, GST-PH-Akt or GST to immobilized PIP was assessed
by protein-lipid overlay assay. Nitrocellulose membranes spotted with 100 pmol of
different phospholipids were used for the assay. Bound proteins were detected with anti-
GST antibody. Note that GST produced no signal under the conditions employed.
(C) 293T cells were transfected with GFP, GFP-wtPHLDA3, GFP-mtPHLDA3, GFP-
PH-Akt or GFP-PH-Grp1, and analyzed for GFP-positive cells 48 hrs post-transfection.
The apoptotic rate, measured by PI-positive cells (cells stained with PI without fixation),
is shown. Mean apoptotic rates +/- SD from three experiments are shown.
(D) PHLDA3 inhibits Akt activation. COS7 cells were transfected with the indicated
fusion proteins for 24 hrs, and subsequently stimulated with EGF for 5 min. Induction of
Akt phosphorylation was detected in control cells expressing GFP upon EGF treatment.
Akt activity after EGF treatment was analyzed by Western blotting. GFP fusion protein
levels were also analyzed by Western blotting.
(E) Cell spreading is not inhibited by PHLDA3. Cell spreading assay was perfomed.
LY294002 was added 10 min before re-plating. More than 150 cells were counted from
three fields, and the percentages of fully spread cells are shown. Note that PI3K inhibitor
LY294002 inhibited cell spreading under the conditions tested. Mean percentage +/- SD
from three fields is shown.
Figure 8 PHLDA3-derived peptides or PHLDA3-mimic small molecules are good candidates for anticancer drug.
Figure 7 PHLDA3 locus is lost and PHLDA3 expression is downregulated in LCNECs
(A) Chromosome copy number alterations analyzed by MCG cancer array-800 CGH. Note that the PHLDA3 gene was frequently lost in human lung endocrine tumors.
(B) Expression of PHLDA3 was analyzed by quantitative RT-PCR. Total RNAs were prepared from normal lung tissues (derived from patients A-E) and LCNECs (derived from patients 1T-12T). PHLDA3 mRNA levels were analyzed as in Figure 1A. In the right
column, the mean expression +/- SD of PHLDA3 expression in normal lungs and tumor samples is shown.
(C) LCNEC tumor sections were subjected to immunohistochemistry to detect activated Akt. Stronger positive brown signals were detected in tumor regions (T) compared to normal tissue regions (N).