1. and Masanori Hatakeyama
Biological activity of the Helicobacter pylori virulence factor CagA is determined by variation in the tyrosine phosphorylation sites
Hideaki Higashi, Ryouhei Tsutsumi, Akiko Fujita, Shiho Yamazaki†, Masahiro Asaka, Takeshi Azuma†,
and Masanori Hatakeyama
14428– PNAS October 29 , vol no. 22
Speaker：林冠勳
Professor：吳夙欽
2002/11/05

2. Helicobacter pylori ( H. pylori )
Gram-negative bacterium
Cause chronic gastritis and gastroduodenal ulcers
Associate with the gastric cancer and MALT lymphoma
Infection half of the world’s population

3. Cytotoxin associated gene A (cagA)
Signal the nucleus to release I L-8
High immunogenicity
128 ~ 145 kDa protein
Inject into host cell by type IV secretion system
Tyrosine phosphorylation in host cell

4. Activation is dependent on a membrane-associated host cellular kinase that phosphorylates CagA at multiple sites
AGS cell infect
(Confocal laser scanning microscopy)
CagA
SHP-2
actin
10 mm.
hummingbird phenotype

5. hummingbird phenotype
CagA
NCTC11637 CagA :Western CagA-specific sequence WSS P
SHP-2 P
F32 CagA : Ester CagA-specific sequenceESS
hummingbird phenotype

6. Expression Vector Constructs
H.pylori standard strain NCTC11637
C-terminal hemagglutinin (HA)
or Flag-epitope-tagged.
cagA gene
pSP65SRα mammalian expression vector

7. SH2 protein tyrosine phosphatase 2 (SHP-2)
Cytoplasmic tyrosine phosphatase
SH2：src homology 2 domains
binding to protein
PTP：protein tyrosine phosphatase
catalysis of protein tyrosine dephosphorylation
N-SH2
C-SH2

8. SHP-2-Myc
SHP-2-SH2∆C-Myc : R138 and H169→ alanine
SHP-2-SH2∆N-Myc : Arg-32 (R32) and His-53 (H53) → alanine
SHP-2-SH2∆NC-Myc : R138 and H169, Arg-32 (R32) and His-53 (H53) → alanine
Human SHP-2 cDNA
Myc-epitope
pSP65SRα mammalian expression vector

9. CagA and SHP-2 expression vectors
Lipofectamine
calcium phosphate method
Human AGS gastric epithelial cells
monkey COS-7 cells
17 h after transfection
Hummingbird phenotype
lysed in lysis buffer
Immunoprecipitation
and Immunoblotting.

10. Identification of in Vivo Tyrosine Phosphorylation Sites in the EPIYA Containing Repeat Sequence of CagA
The EPIYA motifs present
in NCTC11637 CagA
X: EPIYA→EPIAA

11. Identification of tyrosine phosphorylation sites of CagA and
their roles in SHP-2 binding and morphological transformation
immunoprecipitates (IP)
total cell lysates (TCL)

12. Correlation Among Number of Tyrosine Phosphorylation Sites, SHP-2-Binding Activity, and Hummingbird Phenotype Induction of CagA.

13. AGS cells were transiently transfected with an expression
vector for WT or EPIYA mutant of CagA.
Cells showing the hummingbird phenotype were counted.

14. Involvement of the SH2 Domains of SHP-2 in CagA Binding

15. COS-7 cells were cotransfected with expression vectors for HA-tagged CagA and Flag-taggedCagA.
Cell lysates were subjected to immunoprecipitation (IP) with anti-Flag. Immunoprecipitates and total cell lysates (TCL) were immunoblotted (IB) with the indicated antibodies
A model of CagA–SHP-2 interaction.

16. EPIYA→EPIAA

17. The relative amounts of tyrosine phosphorylated CagA(black bars)
CagA-bound SHP-2 (white bars) were calculated with the values for WT CagA

18. Delineation of Amino Acid Residues That Determine SHP-2-Binding Affinity Among Distinctly Structured CagA Proteins
ABCcc :
ABCDcc :
ABC970DFcc :
ABD961FD :

19. AGS cells used in a then were examined for the
induction of the hummingbird phenotype.

20. type IV secretion system.