Authors: Dmitry Kovalenko, Xuehoi Yang, Robert J. Nadeau, Lauren K. Harkins, and Robert Friesel

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 • Overview of FGFs and Sef • The Big Picture • The Questions • Q1 Results and Conclusions • Q2 • Q3 • Q4 • 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 • Results • mSef decreases ERK phosphorylation induced by active FGFR1 but not active Ras Figure 3B

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 • Results • mSef inhibits FGF-induced activation of Ras Figure 3C

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 • Results • mSef inhibits FGF induced activation of Akt (but not PDGF induced) Figure 3D

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 • Results • mSefIC reduces activation of ERK (by 69%) but not as much as mSef (by 93%) Figure 3E

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.