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Association of a 85-kDa serine kinase with activated fibroblast growth factor receptor-4
S Vainikka1, V Joukov, P Klint
1Molecular/Cancer Biology Laboratory, Haartman Institute, University of Helsinki, Finland.
Abstract:
Fibroblast growth factors (FGFs) transduce a variety of biological signals via four distinct tyrosine kinase receptors. We have characterized the phosphorylation of FGF receptor 4 (FGFR-4) and its association with a putative substrate, p85, using transfected L6 myoblast and NIH3T3 fibroblast cell lines. FGFR-4 was phosphorylated in vivo and in vitro mainly on serine and threonine residues in several peptides and to a lower degree on tyrosine residues. When analyzed further by in-gel kinase assay, immunoprecipitates of ligand-activated FGFR-4 contained a serine autophosphorylated polypeptide doublet of 85 kDa. Analysis of the major autophosphorylation site Y754F mutant of FGFR-4 showed that binding of p85 and its serine phosphorylation were independent of receptor autophosphorylation at this site. Okadaic acid treatment increased the basal autophosphorylation activity of p85 but decreased FGFR-4 tyrosine phosphorylation. In contrast, orthovanadate treatment increased the tyrosine phosphorylation of FGFR-4. These data show that a serine kinase is associated with activated FGFR-4 and suggest a role for serine phosphorylation in FGFR-4 function.
Insights
Fibroblast growth factor receptor 4 (FGFR-4) is phosphorylated on serine and threonine residues. A serine kinase associates with activated FGFR-4, suggesting serine phosphorylation
Area of Science:
- Molecular Biology
- Cell Signaling
- Biochemistry
Background:
- Fibroblast growth factors (FGFs) mediate cellular signals through tyrosine kinase receptors.
- FGFR-4 is one of four distinct FGF receptors involved in signal transduction.
Purpose of the Study:
- To characterize the phosphorylation of FGFR-4.
- To investigate the association between FGFR-4 and its substrate, p85.
- To elucidate the role of serine phosphorylation in FGFR-4 function.
Main Methods:
- Utilized transfected L6 myoblast and NIH3T3 fibroblast cell lines.
- Performed in vivo and in vitro phosphorylation assays.
- Conducted in-gel kinase assays and analyzed FGFR-4 mutants (Y754F).
- Treated cells with okadaic acid and orthovanadate.
Main Results:
- FGFR-4 undergoes phosphorylation primarily on serine and threonine residues, with lower levels on tyrosine.
- Ligand-activated FGFR-4 immunoprecipitates revealed an 85 kDa serine autophosphorylated polypeptide doublet.
- p85 binding and serine phosphorylation are independent of FGFR-4 autophosphorylation at the Y754 site.
- Okadaic acid enhanced p85 autophosphorylation but reduced FGFR-4 tyrosine phosphorylation; orthovanadate increased FGFR-4 tyrosine phosphorylation.
Conclusions:
- A serine kinase is associated with activated FGFR-4.
- Serine phosphorylation plays a role in FGFR-4 signaling.
- FGFR-4 signaling involves complex cross-talk between tyrosine and serine kinases.