1. Polo样激酶Plk3对p73转录活性的影响
及对其磷酸化位点的分析
桑梅香
河北医科大学第四医院/河北省肿瘤医院

2. Schematic representation of Polo and Polo-like kinases (Plks)
Kinase Domain
PBD1
PBD2
Mammals
(Homo sapiens)
Xenopus laevis
Drosophila melanogaster
Caenorhabditis elegans
Schizosaccharomyces pombe
Saccharomyces cerevisiae
Plk1
Plk2
Plk3
Plk4
603
685
646
907
Plx1
598
Polo
576
Plc1
649
683
Plo1
Cdc5
705

3. Functions of Polo-Box Domain and Kinase Domain of Plks
PBD1
PBD2
PB2 P S
Kinase
PB1

4. Polo-like kinases (Plks) and p53 family
DNA Damage
ATM(ATR)
Plk1 ?
p53
p73
p63 ?
DNA Damage
ATM(ATR)
Plk3

5. Co-localization of Plk3 and p73 in cells
DAPY HA-p FLAG-Plk Merge

6. Interaction between Plk3 and p73α
DBD OD
SAM TA
１　　
１　　 B
GST-p73α
GST
1-62
1-130
50-
37-
25-
kDa C
GST
1-130
1-62
GST-p73α
kDa
50-
75-
〔35S〕FLAG-Plk3

7. Plk3 inhibits p73α-mediated transcription activation
HA-p73α
FLAG-Plk
HA-p73α
FLAG-Plk
HA-p73α
FLAG-Plk
p73α
Plk3
p21WAF1
Bax
p53AIP1
GAPDH
HA-p73α
FLAG-Plk

8. Kinase-deficient Plk3 (K52R) fails to inhibit p73α-mediated transcription activation
HA-p73α
Plk3(K52R)
HA-p73α
Plk3(K52R)
HA-p73α
Plk3(K52R)
p73α
Plk3
p21WAF1
Bax
p53AIP1
GAPDH
HA-p73α
Plk3(K52R)

9. Plk3 inhibits p73α-mediated transcription activation
H1299 (p53 null)
pcDNA p p73/Plk p73/Plk3(K52R)

10. Plk3 inhibits the p73α-mediated apoptosis
HA-p73α+Plk3(K52R)
Actin
PARP
cleaved PARP ►
pcDNA3
HA-p73α
HA-p73α+Plk3
FLAG-Plk3
Plk3(K52R)
CDDP (10μM)

11. Plk3 phosphorylates p73 at its N-terminal region in vitro
DBD OD
SAM TA
１　　
１　　 B
GST-p73α
GST
1-62
1-130
50-
37-
25-
kDa C
GST
1-130
1-62
GST-p73α
Control

12. T86 is not the phosphorylation sites of p73 targeted
by Plk3
amino residues of p73 between :
AQFNLLSS TMDQMSSRAA SASPYTPEHA ASVPTHSPYA QPSSTFDTMS PAP
This short fragment include 11 Ser residus and 5 Thr residues. A
GST
1-130 (T86A)
1-62
GST-p73α
Control

13. Conclusions
Plk3 inhibits p73α-mediated transcription activation, whereas kinase-deficient Plk3 fails to inhibit p73α-mediated transcription activation.
Plk3 inhibits the p73α-mediated apoptosis through physical interaction and phosphorylation.

14. Thank You For Your Attention!