Sef Inhibits Fibroblast Growth Factor Signaling by Inhibiting
Sef Inhibits Fibroblast Growth Factor Signaling by Inhibiting FGFR 1 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
Outline I. Overview of FGFs and Sef II. The Big Picture III. The Questions IV. Q 1 Results and Conclusions V. Q 2 VI. Q 3 VII. Q 4 VIII. The Answers
Overview of Fibroblast Growth Factors • Family of polypeptides with 23 members • Regulate: • Cell proliferation • Migration • Differentiation • Embryonic pattern formation
Overview of Fibroblast Growth Factors Pathway • FGF complex binds and FGFRs dimerize • Autophosphorylation on tyrosine residues (docking sites for SH 2 domain) • FRS 2 binds and is tyrosinephosphorylated • FRS 2 binds to SH 2 domain on the adaptor protein Grb 2 and the protein-tyrosine phosphatase SHP 2 • Grb 2 attracts SOS • SOS activates Ras pathway
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
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 • FGF 8, FGF 3, Spry, and Sef
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 FGFR 1 and FGFR 2
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?
Questions Hypothesis: Overexpression of m. Sef inhibits tyrosine phosphorylation of FGFR Q 1: Does m. Sef associate with FGF 1, and if so, which structural domains of the m. Sef protein mediate the interaction? Q 2: Does m. Sef overexpression inhibit FGF induced ERK activation? Q 3: Does m. Sef mediated inhibition of the Raf/Mek/ERK pathway occur at the level of Ras, or upstream of it? Q 4: Does m. Sef affect FGFR 1 tyrosine phosphorylation?
Question 1: Figure 1 Procedure prepared full length and deletion mutations of m. Sef • • V 5 - and His-tagged m. Sef or m. Sef deletion constructs were expressed in cells with wildtype FGFR 1 for 24 hours • cells treated with FGF 1 Figure 1 A
Question 1: Figure 1 Procedure • cell lysates immunoblotted directly with anti-V 5 (Sef) • cell lysates immunoprecipitated with anti-FGFR antibodies and then immunoblotted with anti-His or anti-FGFR antibodies Figure 1 B
Question 1: Figure 1 Results • Full length m. Sef and m. Sef. IC coimmunoprecipitated with FGFR 1 (but m. Sef. ECTM and m. Sef. EC did not) • both unstimulated and FGF-stimulated FGFR 1 coimmunoprecipitated with m. Sef Figure 1 B
Question 1: Figure 1 C Figure 1 D Procedure: Same as before, only instead of normal FGFR 1, constitutively activated FGFR 1 (Figure 1 C) and a kinase-inactive form of FGFR 1 (Figure 1 D) are used Results: same
Question 1 Does m. Sef associate with FGF 1, and if so, which structural domains of the m. Sef protein mediate the interaction? • Yes, m. Sef associates with FGF 1 • The cytoplasmic domain of m. Sef mediated the association between FGFR 1 and m. Sef • Also learned that the association between m. Sef and FGFR 1 is independent of FGFR 1 tyrosine kinase activation
Question 2: Figure 2 Procedure • cells were transduced with either Ad. GFP (adenovirus mediated green fluorescent protein) or Adm. Sef • cell lysates Immunoblotted with antibodies for p. ERK (activated ERK), p. Raf, and p. MEK Figure 2 A
Question 2: Figure 2 Results • Overall levels of ERK were unaffected • Only Adm. Sef resulted in a reduction in the levels of activated ERK (by 86%) • p. MEK was inhibited by m. Sef by 61% • p. Raf was reduced by m. Sef by 59% Figure 2 A
Question 2: Figure 2 Procedure • cells were transduced with either Ad. GFP or Adm. Sef and made quiescent (at rest) for 36 hrs • Cells exposed to FGF 2 • A tritiated thymidine incorporation asses was performed Result • A decrease in FGF-stimulated DNA synthesis Figure 2 A
Question 2 Does m. Sef overexpression inhibit FGF induced ERK activation? • Yes, m. Sef overexpression inhibits ERK activation • This, in turn, reduces FGF stimulated DNA synthesis
Question 3: Figure 3 Procedure • cells were transduced with either Ad. GFP or Adm. Sef • cells stimulated with FGF 2, PDGF, EGF, or CS • cell lysates immunoblotted for p. ERK, and V 5 Results • ERK phosphorylation by PDGF, EGF, and CS activated pathways are not inhibited by m. Sef Figure 3 A
Question 3: Figure 3 Procedure • cells were transfected with ERK with either activated FGFR 1 or activated Ras • cell lysate was immunoprecipitated with anti-HA antibodies (hemagglutinin) • cell lysate was immunoblotted for p. ERK, HA, FGFR, and V 5 Results • m. Sef decreases ERK phosphorylation induced by active FGFR 1 but not active Ras Figure 3 B
Question 3: Figure 3 Procedure • cells were transduced with either Ad. GFP or Adm. Sef and stimulated with FGF 2 • a pull-down with GST-RBD was done (Ras binding domain) • cell lysate immunoblotted for Ras Results • m. Sef inhibits FGF-induced activation of Ras Figure 3 C
Question 3: Figure 3 Procedure • cells were transduced with either Ad. GFP or Adm. Sef and stimulated with FGF 2 • cell lysate immunoblotted with antibodies to p. Akt, and V 5 Results • m. Sef inhibits FGF induced activation of Akt (but not PDGF induced) Figure 3 D
Question 3: Figure 3 Procedure • cells were transfected with ERK and FGFR 1 and either m. Sef or m. Sef. IC and stimulated with FGF 2 • cell lysates immunoprecipitated for HA and immunoblotted for p. ERK, HA, V 5, and FGFR 1 Results • m. Sef. IC reduces activation of ERK (by 69%) but not as much as m. Sef (by 93%) Figure 3 E
Question 3 Does m. Sef mediated inhibition of the Raf/Mek/ERK pathway occur at the level of Ras, or upstream of it? • Signaling from Ras is intact in m. Sef overexpressed cells • m. Sef does not decrease ERK phosphorylation induced by active Ras • m. Sef inhibits FGF-induced activation of Ras • m. Sef inhibits multiple FGF-induced signaling pathways and the inhibition occurs upstream of Ras
Question 4: Figure 4 Procedure • cells were transduced with either Ad. GFP or Adm. Sef and stimulated with FGF 2 • cell lysate was immunoprecipitated for FGFR • cell lysate was immunoblotted for p. Y (phosphotyrosine), FGFR, and V 5 Results • m. Sef reduces FGF-induced tyrosine phosphorylation of FGFR by about 70% Figure 4 AB
Question 4: Figure 4 Procedure • same as before, except immunoprecipitated and immunoblotted for FRS 2, a substrate of FGFR 1 Results • m. Sef reduces FGF-induced tyrosine phosphorylation of FRS 2 by about 70% Figure 4 CD
Question 4 Does m. Sef affect FGFR 1 tyrosine phosphorylation? • Yes, m. Sef reduces FGFR 1 tyrosine phosphorylation by about 70% • m. Sef also reduces FRS 2 phosphorylation
The Answers Q 1: Does m. Sef associate with FGF 1, and if so, which structural domains of the m. Sef protein mediate the interaction? Yes, at the cytoplasmic domain Q 2: Does m. Sef overexpression inhibit FGF induced ERK activation? Yes, by 86% Q 3: Does m. Sef mediated inhibition of the Raf/Mek/ERK pathway occur at the level of Ras, or Upstream upstream of it? Q 4: Does m. Sef affect FGFR 1 tyrosine Yes, by 70% phosphorylation?
The Little Question How does Sef inhibit FGF signaling? The Answer: By reducing FGFmediated tyrosine phosphorylation of FGFR 1 and FRS 2 Sef interferes with FRS 2 binding to Grb 2
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. . .
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.
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