Sef Inhibits Fibroblast Growth Factor Signaling by Inhibiting FGFR1 Tyrosine Phosphorylation and Subsequent ERK Activation

1
Sef Inhibits Fibroblast Growth Factor Signaling
by Inhibiting FGFR1 Tyrosine Phosphorylation and
Subsequent ERK Activation
Authors Dmitry Kovalenko, Xuehoi Yang, Robert J.
Nadeau, Lauren K. Harkins, and Robert
Friesel The Journal of Biological
Chemistry April 18, 2003
Presentation Jaymie Krone March 11, 2005
2
Outline

1. Overview of FGFs and Sef
2. The Big Picture
3. The Questions
4. Q1 Results and Conclusions
5. Q2
6. Q3
7. Q4
8. The Answers

3
Overview of Fibroblast Growth Factors

• Family of polypeptides with 23 members
• Regulate
• Cell proliferation
• Migration
• Differentiation
• Embryonic pattern formation

4
Overview of Fibroblast Growth Factors
Pathway

• FGF complex binds and FGFRs dimerize

•   Autophosphorylation on tyrosine residues
  (docking sites for SH2 domain)

• FRS2 binds and is tyrosine-phosphorylated

• FRS2 binds to SH2 domain on the adaptor protein
  Grb2 and the protein-tyrosine phosphatase SHP2

• Grb2 attracts SOS

• SOS activates Ras pathway

5
Overview of Fibroblast Growth Factors

• Previous Studies- FGF signaling
• Is essential for cell proliferation and survival
  in mouse embryo before implantation
• Is essential for cell migration during
  gastrulation
• Has a role in development of brain, limb buds,
  and lung, as well as many other tissues and
  organs

6
Overview of Fibroblast Growth Factors

• Previous Studies
• Sprouty family of genes negatively regulate FGF
  signaling
• Spry acts between RTKs and Ras/Raf/ERK
• The FGF synexpression group
• a group of genes that share a complex expression
  pattern
• FGF8, FGF3, Spry, and Sef

7
Overview of Fibroblast Growth Factors

• Sef
• Similar expression to FGF genes
• transmembrane protein whose embryonic expression
  is similar to other members of the FGF
  synexpression group
• expression is regulated by FGF
• Studies with Zebrafish and Xenopus
• Sef inhibits FGF signaling
• Sef co-immunoprecipitates with FGFR1 and FGFR2

8
The Big Picture
FGF signaling
The Big Question
What inhibits FGF signaling?
The Little Picture
Sef
The Little Question
How does Sef inhibit FGF signaling?
9
Questions
Hypothesis Overexpression of mSef inhibits
tyrosine phosphorylation of FGFR
Q1 Does mSef associate with FGF1, and if so,
which structural domains of the mSef protein
mediate the interaction? Q2 Does mSef
overexpression inhibit FGF induced ERK
activation? Q3 Does mSef mediated inhibition of
the Raf/Mek/ERK pathway occur at the level of
Ras, or upstream of it? Q4 Does mSef affect
FGFR1 tyrosine phosphorylation?
10
Question 1 Figure 1

• Procedure
• prepared full length and deletion mutations of
  mSef
• V5- and His-tagged mSef or mSef deletion
  constructs were expressed in cells with wildtype
  FGFR1 for 24 hours
• cells treated with FGF1

Figure 1A
11
Question 1 Figure 1

• Procedure
• cell lysates immunoblotted directly with anti-V5
  (Sef)

• cell lysates immunoprecipitated with anti-FGFR
  antibodies and then immunoblotted with anti-His
  or anti-FGFR antibodies

Figure 1B
12
Question 1 Figure 1

• Results
• Full length mSef and mSefIC co-immunoprecipitated
  with FGFR1 (but mSefECTM and mSefEC did not)

• both unstimulated and FGF-stimulated FGFR1
  co-immunoprecipitated with mSef

Figure 1B
13
Question 1 Figure 1
Figure 1C Figure 1D
Procedure Same as before, only instead of normal
FGFR1, constitutively activated FGFR1 (Figure 1C)
and a kinase-inactive form of FGFR1 (Figure 1D)
are used Results same
14
Question 1

• Does mSef associate with FGF1, and if so, which
  structural domains of the mSef protein mediate
  the interaction?
• Yes, mSef associates with FGF1
• The cytoplasmic domain of mSef mediated the
  association between FGFR1 and mSef
• Also learned that the association between mSef
  and FGFR1 is independent of FGFR1 tyrosine kinase
  activation

15
Question 2 Figure 2

• Procedure
• cells were transduced with either AdGFP
  (adenovirus mediated green fluorescent protein)
  or AdmSef
• cell lysates Immunoblotted with antibodies for
  pERK (activated ERK), pRaf, and pMEK

Figure 2A
16
Question 2 Figure 2

• Results
• Overall levels of ERK were unaffected

• Only AdmSef resulted in a reduction in the
  levels of activated ERK (by 86)

• pMEK was inhibited by mSef by 61

• pRaf was reduced by mSef by 59

Figure 2A
17
Question 2 Figure 2

• Procedure
• cells were transduced with either AdGFP or
  AdmSef and made quiescent (at rest) for 36 hrs

• Cells exposed to FGF2

• A tritiated thymidine incorporation asses was
  performed

• Result
• A decrease in FGF-stimulated DNA synthesis

Figure 2A
18
Question 2

• Does mSef overexpression inhibit FGF induced ERK
  activation?
• Yes, mSef overexpression inhibits ERK activation
• This, in turn, reduces FGF stimulated DNA
  synthesis

19
Question 3 Figure 3

• Procedure
• cells were transduced with either AdGFP or
  AdmSef
• cells stimulated with FGF2, PDGF, EGF, or CS
• cell lysates immunoblotted for pERK, ERK, and V5

• Results
• ERK phosphorylation by PDGF, EGF, and CS
  activated pathways are not inhibited by mSef

Figure 3A
20
Question 3 Figure 3

• Procedure
• cells were transfected with ERK with either
  activated FGFR1 or activated Ras
• cell lysate was immunoprecipitated with anti-HA
  antibodies (hemagglutinin)
• cell lysate was immunoblotted for pERK, HA,
  FGFR, and V5

Figure 3B

• Results
• mSef decreases ERK phosphorylation induced by
  active FGFR1 but not active Ras

21
Question 3 Figure 3

• Procedure
• cells were transduced with either AdGFP or
  AdmSef and stimulated with FGF2
• a pull-down with GST-RBD was done (Ras binding
  domain)
• cell lysate immunoblotted for Ras

Figure 3C

• Results
• mSef inhibits FGF-induced activation of Ras

22
Question 3 Figure 3

• Procedure
• cells were transduced with either AdGFP or
  AdmSef and stimulated with FGF2
• cell lysate immunoblotted with antibodies to
  pAkt, Akt, and V5

Figure 3D

• Results
• mSef inhibits FGF induced activation of Akt (but
  not PDGF induced)

23
Question 3 Figure 3

• Procedure
• cells were transfected with ERK and FGFR1 and
  either mSef or mSefIC and stimulated with FGF2
• cell lysates immunoprecipitated for HA and
  immunoblotted for pERK, HA, V5, and FGFR1

Figure 3E

• Results
• mSefIC reduces activation of ERK (by 69) but
  not as much as mSef (by 93)

24
Question 3

• Does mSef mediated inhibition of the Raf/Mek/ERK
  pathway occur at the level of Ras, or upstream of
  it?
• Signaling from Ras is intact in mSef
  overexpressed cells
• mSef does not decrease ERK phosphorylation
  induced by active Ras
• mSef inhibits FGF-induced activation of Ras
• mSef inhibits multiple FGF-induced signaling
  pathways and the inhibition occurs upstream of Ras

25
Question 4 Figure 4

• Procedure
• cells were transduced with either AdGFP or AdmSef
  and stimulated with FGF2
• cell lysate was immunoprecipitated for FGFR
• cell lysate was immunoblotted for pY
  (phosphotyrosine), FGFR, and V5

Figure 4AB

• Results
• mSef reduces FGF-induced tyrosine
  phosphorylation of FGFR by about 70

26
Question 4 Figure 4

• Procedure
• same as before, except immunoprecipitated and
  immunoblotted for FRS2, a substrate of FGFR1

• Results
• mSef reduces FGF-induced tyrosine
  phosphorylation of FRS2 by about 70

Figure 4CD
27
Question 4

• Does mSef affect FGFR1 tyrosine phosphorylation?
• Yes, mSef reduces FGFR1 tyrosine phosphorylation
  by about 70
• mSef also reduces FRS2 phosphorylation

28
The Answers
Q1 Does mSef associate with FGF1, and if so,
which structural domains of the mSef protein
mediate the interaction? Q2 Does mSef
overexpression inhibit FGF induced ERK
activation? Q3 Does mSef mediated inhibition of
the Raf/Mek/ERK pathway occur at the level of
Ras, or upstream of it? Q4 Does mSef affect
FGFR1 tyrosine phosphorylation?
Yes, at the cytoplasmic domain
Yes, by 86
Upstream
Yes, by 70
29
The Little Question
How does Sef inhibit FGF signaling?
The Answer By reducing FGF-mediated tyrosine
phosphorylation of FGFR1 and FRS2 Sef interferes
with FRS2 binding to Grb2
30
Wait. Is this the answer?
The authors point out that previous zebrafish
experiments indicate that Sef disrupts FGF
signaling at the level of MEK, which conflicts
with their own results...
31
Wait. Is this the answer?
But another, more recent paper seems to agree
with the zebrafish experiments. It shows that
human Sef binds to activated MEK and inhibits the
dislocation of the MEK-ERK complex.
Sef is a Spatial Regulator for Ras/MAP Kinase
Signaling. Torii, S., et al. Developmental
Cell, July 2004.