1. Over-expression of EphB1 leads to Stat3 phosphorylation#227
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Danielle Swain, Richard Noring, Jake Knowles, Roberto Campos-González and Zev Gechtman. Cell Signaling Technology Inc. 3 Trask lane, Danvers, MA 01923
The Receptor Tyrosine Kinase (RTK) family com-
prises some important disease drivers. Over-ex-
pressed or mutated RTKs in disease may lead to
aberrant phosphorylation of intracellular signaling
molecules. Therefore it is desirable to monitor
simultaneously the activation levels of RTKs and
signaling nodes. Here we used a multiplexed
antibody array to monitor the phosphorylation of
28 RTKs and 11 intracellular signaling molecules
simultaneously. We have observed a strong signal
on a spot corresponding to phospho-Stat3 (Tyr705)
with lysates derived from CHO cells transiently
over-expressing EphB1. The strong Phospho-Stat3
signal was not seen in non-transfected cells or in
lysates made form cells transfected with several
other Eph family members. Western blotting with
phospho-specific antibodies showed an increase
in Stat3 phosphorylation on Tyr705 in CHO cells
upon EphB1 ectopic expression. The amounts of
Tyr705 Stat3 phosphorylation positively correlated
with the levels of EphB1 protein. Co-precipitation
experiments showed that EphB1 and Stat3 interact
both in CHO cells and MCF-7 breast cancer cells.
Phosphorylated Jak2 was not found in these im-
muno-precipitates suggesting an involvement of
another tyrosine kinase in Stat3 phosphorylation
in cells over-expressing EphB1. Consistently with
these observations, a potent Jak1 and Jak2 inhibitor
INCB018424 inhibited the basal but not the EphB1
induced Stat3 Tyr705 phosphorylation in transfected
CHO cells. These results suggest that EphB1
over-expression may bypass the requirement in Jak
kinase and lead to Stat3 activation either directly or
through another receptor-tethered kinase.
Abstract
• Evidence for interaction between Stat 3 and EphB1 was obtained by co-precipitation
experiments. EphB1 was overexpressed in CHO cells and was immuno-precipitated
using two different Stat3 antibodies directed against two distinct epitopes within
Stat3. The interaction between Stat3 and EphB1 could be either direct or indirect
(part of a complex).
• Overexpression of EphB1 leads to increase in Stat3 phosphorylation on Tyr705.
Increase in the phosphorylation of Stat3 on Tyr705 is typically associated with
its activation and translocation to the nucleus to modulate gene expression.
• The tyrosine kinase that is responsible for the increase in Stat3 Tyr705 phosphorylation
in cells overexpressing EphB1 is not of the Jak family. This is evidenced by the lack of
inhibition of EphB1 induced Stat3 Tyr705 phosphorylation by a Jak inhibitor compound
INCB018424.
Conclusion
Fig. 1: Heat map display of PathScan®
RTK array (fluorescent
readout) results. CHO cells were transiently transfected with
various RTKs.
Signal
Phospho-Stat3
0
3000
6000
9000
12000
15000
M-CSFR Axl Tie2 FGFR1 FGFR4RetTyro3RonEphB4EphB3EphB1EphA3EphA2EphA1
0
2000
4000
6000
8000
10000
M-CSFR Axl Tie2 FGFR1 FGFR4RetTyro3RonEphB4EphB3EphB1EphA3EphA2EphA1
Phospho-Src
Signal
0
2000
4000
6000
8000
10000
M-CSFR Axl Tie2 FGFR1 FGFR4RetTyro3RonEphB4EphB3EphB1EphA3EphA2EphA1
Phospho-Erk1/2
Signal
0
4000
8000
12000
16000
18000
M-CSFR Axl Tie2 FGFR1 FGFR4RetTyro3RonEphB4EphB3EphB1EphA3EphA2EphA1
Phospho-S6
Signal
Fig. 2: Specific signal associated with the spot corresponding to Phospho-Stat 3
(Tyr705) is seen upon overexpression of EphB1. CHO Cells were transfected with
expression vectors encoding various RTKs. Lysates were prepared and analyzed
using PathScan®
RTK array (Fluorescent Readout #7949). Signals associated with
spots corresponding to p-Stat3, p-Src, p-Erk1/2 and p-S6 are shown.
INCB018424, µM 0 0.03 0.1 0.5 2.5 10
CHOMock
pStat3
(Tyr705)
Stat3
pJak2
(Tyr1007/1008)
0 0.03 0.1 0.5 2.5 10
CHO EphB1
Fig. 5: EphB1 induces Stat3 (Tyr705) phosphorylation. CHO cells were either mock transfected
or transfected with EphB1 encoding construct. Cells were treated with various concentrations of
the Jak inhibitor compound INCB0184242 for 1.5 hrs. Lysates were analyzed by western blot with
various antibodies indicated in the figure.
– + – +
Total Lysate
pStat3 (Tyr705)pYBlot:
– + – +
IP Total Stat3 Total Lysate IP Total Stat3
EphB1
cDNA:
Fig. 3: Evidence for EphB1 and Stat3 interaction in CHO cells transfected
with EphB1-Flag. (Stat3 antibodies used in IP #9312 Rabbit polyclonal)
p-Stat3
Mock
A B CIP:
Blot:
Total Stat3 Flag
EphB1
A B C
Mock
A B C
EphB1
A B C
pY
Mock
A B C
EphB1
A B C
Fig. 4: Co-precipitation of ectopically expressed EphB1-Flag with endogenous Stat3 in CHO cells. IP
with EphB1 ectodomain mAb (A) Flag antibodies (B), Stat3 Rabbit mAb Antibodies #2358 (C).
EphB1