1. Tumor protein D52 represents a negative regulator of ATM protein levels
Yuyan Chen, Alvin Kamili, Jayne R Hardy, Guy E Groblewski, Kum Kum Khanna, and Jennifer A Byrne.
Speaker: Ching-Ho, Chang
Advisor: Dr. Guor-Mour, Her
Date: 2015/04/22

2. Introduction
Genomic stability and integrity are maintained through sophisticated cellular DNA damage response (DDR) network.
Alter chromatin organization
Switch on cell cycle checkpoint
Activate DNA repair
Modulate numerous cellular processe
Defects in DDR

3. DNA repair ability of tumor cells
prevents the accumulation of lethal DNA damage from
cytotoxic agents and ionizing radiation (IR)
Leading to tumor resistance
DDR plays a central role in tumorigenesis and cancer therapy
DNA double-strand breaks (DSBs)
Activation of ATM protein

4. ATM protein Full Name: (ataxia telangiectasia mutated) protein
Phos- phatidylinositol 3-kinase-related protein kinase (PIKK) family.
It phosphorylates several key proteins that initiate activation of the DNA damage checkpoint, leading to cell cycle arrest, DNA repair or apoptosis. .
A-T symptomatology

5. P
CHK2
ATM
DNA damage
ATM P
p53
Chromatin
changes
Autophosphorylation P
Substrate
phosphorylation P
Brca1 P P
Nbs1
Focus formation
p53
Main component in the G1–S cell cycle checkpoint.
CHK2
Regulates several cell cycle checkpoints.
Loss of functional ATM is associated with both increased genomic instability and cancer risk.

6. Certain kinds of leukemias and lymphomas, all characterized by
high rates of inactivating mutations, and/or deletion of ATM (Hall, 2004).
Reduced ATM protein levels have been reported in :
Glioblastoma (Tribius, Pidel and Casper, 2001)
Breast cancer (Angèle et al.,2003)
Colorectal cancer (Grabsch et al.,2006)
Gastric cancer (Kang et al.,2008)

7. Studies have recently indicated a possible role of
TPD52 in regulating DDR
TPD52 transcript levels show the highest positive correlationwith G2 radiosensitivity (Sims et al., 2007)
TPD52 knockdown in multiple tumor cell lines significantly reduced their sensitivity to radiation treatment. (Niu et al., 2010)
DDR
TPD52

8. TPD52 Full Name: tumor protein D52
Another name: D52; N8L; PC-1; PrLZ; hD52
Location: chromosome 8q21.13
Human TPD52 isoforms are∼200 amino acid residues
in length and contain a number of sequence motifs.
Interactions
Proteolytic signal
Byrne, Frost, Chen & Bright 2014.

9. Related paralogous genes of TPD52
TPD52 was initially cloned through differential screening of a breast carcinoma
cDNA library.
Overexpression of TPD52 in many different types of malignancies. (Shehata et al.,2008)

10. TPD52 expression levels
Normal tissue
(Pierre et al., 2014)
Cancer

11. TPD52 function
Plasma membrane-based exocytic, endocytic functions
Calcium ion binding
Ca2+
Tissue invasion
Promote proliferation
B-cell maturation

12. TPD52 functions in cell line models
Cellular survival, proliferation, migration, and invasion

13. Results

14. Overexpression of TPD52 DNA repair capacity IR DNA damage vector
HA-tagged
TPD52
vector
DNA damage
SKBR-3 cells

15. F.1 Overexpression of TPD52 compromised DNA repair capacity
in SK-BR-3 cells
2 Gy 72h

16. F.2
Ectopic expression of TPD52 downregulated ATM mediated DNA repair signaling
γH2AX foci
Early markers of DSB sites.
Important factor associated
with radiosensitivity. C
ATM mediated DNA repair signaling
H2AX P
γH2AX

17. Whether knockdown of endogenous TPD52 could
improve cellular responses to DSBs

18. F.3 Transient knockdown of endogenous TPD52 in SK-BR-3 cells increased
ATM-mediated DNA repair signaling. C

19. To further address the effects of TPD52 expression
on ATM protein levels
ATM
ATM
TPD52
ATM
ATM
ATM

20. F.4
Stable TPD52 expression decreased Atm protein but not transcript levels in mouse BALB/c 3T3 cells.

21. To determine whether the reduction of Atm levels alters cellular responses to DSBs

22. F.5
Stable TPD52 expression disrupted γH2AX focus formation and sensitized cells to DNA damage.

23. F.6 Stable TP D52 expression sensitized cells to γ-ray irradiation.
TPD52 expression downregulates Atm protein levels, which contributes to the defective cellular DDR.

24. Interactions between TPD52 and ATM
Pull-down assays
GTH (Glutathione)
TPD52
Sepharose
Bead
GST (Glutathione S transferase)
cell lysate
Western blot

25. F.7
TPD52 directly interacted with ATM
SDS
109
134

26. F.8 ATM and TPD52 directly interact in vitro and form a complex.
ATM directly interacted with TPD52.
1.5-fold
ATM and TPD52 directly interact in vitro and form a complex.
TPD52
ATM
1-247&
Whether ATM and TPD52 form a complex.

27. TPD52 directly interacted with ATM.
TPD52 decreased Atm protein but not transcript levels in 3T3 cell lines.
TPD52 directly interacted with ATM.
Whether TPD52 was involved in ATM degradation
Identified a potential PEST motif located at ATM residues R832-K892
vector
TPD52
expressing
50 μM
MG132
proteasome inhibitor

28. F.9
Proteasome inhibitor MG132 did not increase Atm protein levels in BALB/c 3T3 cell lines

29. Summary TPD52 as a novel negative regulator of ATM protein levels.
Increased TPD52 expression might widely used mechanism of
inactivating ATM in different types of cancer.
Increased TPD52 expression might contribute to tumor
development by facilitating genomic instability.
Elevated TPD52 levels in tumor may represent a marker of sensitivity to DNA damaging agents.