Ligand-independent activation of tyrosine kinase in fibroblast growth factor receptor 1 by fusion with beta-galactosidase.
To examine the biological role of fibroblast growth factor receptor 1 (FGFR1) oligomerization for its signal transduction, we construct an expression vector encoding a FGFR1-beta-galactosidase fusion protein. This vector is designed to fuse the 3'-portion of FGFR1 to beta-galactosidase. Transfection of this vector into FGFR-negative rat L6 myoblast cells results in ligand-independent inhibition of differentiation into myocytes, suggesting that FGFR1 within this fusion protein is constitutively activated. This can be confirmed by demonstrating that this fusion protein exhibits the tyrosine kinase activity and phospholipase C gamma 1 is tyrosine-phosphorylated even in the absence of ligand stimuli. Since the transfected cells also exhibit the enzyme activity of beta-galactosidase which is known to be active only in a tetramer form, this constitutive activation can be elicited by tetramerization of FGFR1. Furthermore, deletion of a region corresponding to C terminal 10 amino acids important for tetramerization of beta-galactosidase from this expression vector abolishes the constitutively active nature of FGFR1 with simultaneous loss of beta-galactosidase activity. Transfection of non-deleted expression vector into NIH3T3 cells results in acquisition of focus-forming activity while a deleted form of expression vector fails to show this activity even in the presence of basic FGF. These results would suggest that tetramerization of FGFR1 can produce a constitutively active form responsible for transformation of NIH3T3 cells.
Kouhara
H
H
Department of Medicine III, Osaka University Medical School, Japan.
Kurebayashi
S
S
Hashimoto
K
K
Kasayama
S
S
Koga
M
M
Kishimoto
T
T
Sato
B
B
D016475:3T3 Cells; D000818:Animals; D001483:Base Sequence; D004247:DNA / Q000096:biosynthesis; D004789:Enzyme Activation; D016222:Fibroblast Growth Factor 2 / Q000494:pharmacology; D005697:Galactosides / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D005822:Genetic Vectors; D051379:Mice; D008969:Molecular Sequence Data; D010766:Phosphorylation; D011505:Protein-Tyrosine Kinases / Q000096:biosynthesis*; D051381:Rats; D020794:Receptor Protein-Tyrosine Kinases; D051496:Receptor, Fibroblast Growth Factor, Type 1; D017468:Receptors, Fibroblast Growth Factor / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000378:metabolism*; D015398:Signal Transduction; D014162:Transfection; D014407:Tumor Cells, Cultured; D010738:Type C Phospholipases / Q000378:metabolism*
D005697:Galactosides; D017468:Receptors, Fibroblast Growth Factor; D011993:Recombinant Fusion Proteins; C007816:beta-galactoside; D016222:Fibroblast Growth Factor 2; D004247:DNA; C496349:Fgfr1 protein, mouse; C496350:Fgfr1 protein, rat; D011505:Protein-Tyrosine Kinases; D020794:Receptor Protein-Tyrosine Kinases; D051496:Receptor, Fibroblast Growth Factor, Type 1; D010738:Type C Phospholipases