Cancer is the second leading cause of death and costs billions annually. The paper examines the role of PA28γ in regulating cell fate decisions through the ERK and Akt pathways. Murine embryonic fibroblasts deficient or proficient in PA28γ were treated with staurosporine, an inhibitor of protein kinases. PA28γ deficient cells showed hypersensitivity to staurosporine, with decreased viability and ERK phosphorylation but no change in Akt expression. This suggests PA28γ enhances ERK signaling to modulate cell fate, demonstrating its role in survival pathways.

1. Abstract
Cancer is the second leading cause of death worldwide, costing
over $88 billion in 2011 in the United States alone. Proteasome
activator 28γ (PA28γ) expression is increased in several types of
cancer and is associated with poor prognosis and aggressiveness of
certain cancers. The expression of extracellular signal-regulated kinase
(ERK) pathway, which is regulated by the Akt pathway, is also
increased in over 50% of cancers, making it an important target for the
therapeutic treatment of cancer. Staurosporine, a cell permeable
protein kinase inhibitor with a dose-dependent nature, leads to
inhibition of multiple kinase targets, including ERK and Akt. To
elucidate the role of PA28γ on the Akt and ERK pathways, murine
embryonic fibroblasts (MEFs) were treated with staurosporine to
analyze variations in Akt/ERK expression as well as cell fate decisions.
PA28γ deficient cells demonstrated hypersensitivity to staurosporine
treatment, as analyzed by flow cytometry, caspase, and viability
assays. Furthermore, western blotting indicated differential expression
of phosphorylated ERK1/2 between PA28γ proficient and deficient
cells, suggesting an active role of PA28 in regulating ERK signaling,
thereby modulating cell fate decisions. Akt expression was not altered
by staurosporine treatment in PA28γ MEFs. Future studies aim to
clarify the mechanism of Akt phosphorylation as well as further classify
the role of PA28γ in connecting ERK and Akt signaling to cell fate
decision-making.
Introduction
Staurosporine competes for binding at ATP-binding sites on
kinases, leading to inhibition of many ATP-dependent protein-
signaling pathways resulting in apoptosis by activating caspase-3
(Yue et al., 1998). Staurosporine’s mechanism of action makes it a
potentially effective anti-cancer treatment, since cancer cells
require multiple signaling pathways to be altered in order to induce
apoptosis. In acute myeloid leukemia cells, treated with 1µM
staurosporine for 24-hours, cell proliferation was blocked at the
G2/M phase of the cell cycle and apoptosis was effectively
induced (Antonsson and Persson, 2009). Staurosporine treatment
was shown to decrease both ERK phosphorylation and Akt
phosphorylation.
The Extracellular Regulated Kinase, ERK1/2, pathway is the
primary Mitogen Activated Protein Kinase (MAPK) pathway
responsible for cell cycle progression and mitosis in many cell
types. Akt, or protein kinase B (PKB), is a serine/threonine kinase
that interacts with mediators of cell fate decisions to regulate
survival signaling, inhibit apoptosis, and promote and cell cycle
progression. Under stress conditions, activation of these two
pathways is normally inhibited, halting aberrant cell growth. In
cancers, Akt and Ras, upstream activators of ERK, are commonly
mutated to hyperactivate signaling in these pathways, leading to
uncontrollable growth and proliferation (Crowell et al., 2007).
PA28γ is a known regulator of the cell cycle and cell fate decision-
making pathway through key proteins such as GSK-3β, p53, and
p21. PA28γ expression is often increased in many cancers and
may have a role in maintaining unregulated cell growth and
mitosis. Since PA28γ is known to regulate other cell survival and
apoptotic signaling mediators, we hypothesized PA28γ has a role
in the stress survival signaling pathways.
Figure 2. PA28γ deficient MEFs experienced decreased viability
in response to staurosporine treatment. A) C57BL/6 and B)
PA28γ-/- MEFs were treated with staurosporine for 24 hours.
Viability was assessed by ViaCount® cell staining on a flow
cytometer. n=4, ±S.E.
Dilan S. Shah, Karisma Y. Sheth, Victoria J. Campbell and Lance F. Barton
Austin College, Biology Department, Sherman, TX
Elucidating the Role of PA28γ on Akt & ERK1/2 Signaling Pathways and
Cell Fate Decisions Following Staurosporine Treatment
0%
20%
40%
60%
80%
100%
120%
Control 0.1µM 0.2µM 0.4µM 0.8µM 1.0µM
cellfateoutcome(%)
Staurosporine
0%
20%
40%
60%
80%
100%
120%
Control 0.1µM 0.2µM 0.4µM 0.8µM 1.0µM
cellfateoutcome(%)
Staurosporine
A.
B.
go roos
Hypotheses
• PA28γ-/- MEFs will experience resistance to apoptosis following
staurosporine treatment due to PA28γ’s known role in the apoptotic
signaling pathway.
• Staurosporine treatment will cause phospho-ERK1/2 expression to
decrease.
• Staurosporine treatment will decrease Akt phosphorylation but not in
PA28γ deficient MEFs, due to PA28γ’s role as a mediator of cell fate
decision making.
Conclusions
• PA28γ-/- MEFs experience hypersensitivity to staurosporine treatment
indicating PA28γ plays a role in survival signaling.
• Activation of ERK1/2 is decreased in PA28γ-/- MEFs, supporting a role
for PA28γ enhancing the ERK1/2 signaling pathway to affect cell fate
decisions.
• Staurosporine treatment does not activate Akt signaling cascades in
this model.
Acknowledgements
We would like to thank Joel H. Barrett, Rose C. Massey, Vidur Marwaha,
and Brandon Dang for fruitful conversation and all other past and
present Barton lab members whose work has made our project possible.
We also thank the Austin College Biology department for providing
facilities and funding for the research and Dr. Larry Driver for travel
support.
Figure 4: Neither Akt expression nor activation is affected by
staurosporine treatment or PA28γ expression. A) Western blot
images of total Akt expression in C57BL/6 and PA28γ-/- samples.
Banding intensities were normalized against β-actin. B) Table of
expression results for various forms of Akt. n=2 or 3.
Figure 3. Activation of ERK1/2 by phosphorylation is
decreased in PA28γ-/- MEFs following 24 hour staurosporine
treatment. A) Total ERK1/2 expression remained relatively
constant in all samples. Banding intensity was normalized against
β-actin using ImageStudio®. B) Activation of ERK1/2 by
phosphorylation increased in C57BL/6 MEFs with increasing
staurosporine doses (0-1µM) but not in PA28γ-/- MEFs.
Densitometry values for ERK 1 and ERK2 values were summed to
yield ERK1/2. n=2, ±standard deviation.
References
• Antonsson, A., Persson, J. (2009). Induction of Apoptosis by Staurosporine Involves the
Inhibition of Expression of the Major Cell Cycle Proteins at the G2/M Checkpoint
Accompanied by Alterations in Erk and Akt Kinase Activities. International Journal of
Cancer Research and Treatment, 29(8), 2893-2898.
• Crowell, J. A., Steele, V. E., & Fay, J. R. (2007). Targeting the AKT protein kinase for
cancer chemoprevention. Molecular Cancer Therapeutics, 6(8), 2139-2148.
• Yue, T.L., Wang, C., Romanic, A.M., Kikly, K., Keller, P., DeWolf, W.E. Jr., Hart, T.K.,
Thomas, H.C., Storer, B., Gu, J.L., Wang, X., Feuerstein, G.Z. (1998). Staurosporine-
induced apoptosis in cardiomyocytes: A potential role of caspase-3. Journal of Molecular
and Cellular Cardiology, 30, 495–507.
0
0.02
0.04
0.06
0.08
0.1
0.12
0.14
0.16
Control
0.1µM
0.2µM
0.4µM
0.8µM
1.0µM
ERK1/2
signal
intensity
Staurosporine
PA28γ+/+
PA28γ-­‐/-­‐
0
0.005
0.01
0.015
0.02
0.025
0.03
0.035
0.04
0.045
0.05
Control
0.1µM
0.2µM
0.4µM
0.8µM
1.0µM
Phospho-­‐ERK1/2
signal
intensity
Staurosporine
PA28γ+/+
PA28γ-­‐/-­‐
B.
Figure 1. Proposed cell signaling pathway involving PA28γ,
ERK1/2, and Akt.
Protein
Results
Total
Akt
No
difference
between
genotypes
P
-­‐Thr308
Akt
Detectable
only
at
1
μM
staurosporine
No
difference
between
genotypes
P
-­‐Ser473
Akt
No
detectable
phosphorylation
pERK2
pERK1
pERK2
pERK1
PA28γ+/+
PA28γ-/-
B.
Staurosporine
PA28γ+/+
PA28γ-/-
A.
Staurosporine
ERK2
ERK1
ERK2
ERK1
A.
PA28γ+/+
PA28γ-/-
Staurosporine
Viable Apoptotic Dead
Viable Apoptotic Dead
Future Directions
• Inhibitors of the MEK/ERK pathway will be used to tease out the role of
the ERK1/2 pathway in staurosporine response and to clarify PA28γ’s
role in ERK1/2 activation.
Akt
ERK1/2
PI3K
PIP2
PIP3
PTEN
Ras
Raf
MEK
Cyclin D1
Cell cycle
progression
Apoptosis
BAD
MDM2
p53
Caspase 9
DNA repair
?
PA28γ
?