Journal: PLoS ONE

Article Title: Analysis of STAT1 Activation by Six FGFR3 Mutants Associated with Skeletal Dysplasia Undermines Dominant Role of STAT1 in FGFR3 Signaling in Cartilage

doi: 10.1371/journal.pone.0003961

Figure Lengend Snippet: Full-length wild-type FGFR3 or its activating mutants N540K, G380R, R248C, Y373C, K650M and K650E were expressed in CHO cells, activated by brief FGF2 treatment and purified by immunoprecipitation as described in . Immunocomplexes were subjected to kinase assay with recombinant STAT1 as a substrate. Cells transfected with GFP vector serve as negative control for immunoprecipitation. Samples utilizing recombinant FGFR3 tyrosine kinase domain (TK) or those with omitted ATP serve as positive or negative control for kinase assay, respectively. Note that only K650M and K650E-FGFR3 mutants cause STAT1 phosphorylation, as evidenced by western blotting with antibody recognizing STAT1 only when phosphorylated at Y701 (P-Y701-STAT1). FGFR3 and STAT1 western blottings serve as controls for kinase or substrate quantity. Note the appearance of both immature and mature (glycosylated) FGFR3 forms expressed by CHO cells.

Article Snippet: Briefly, kinase reactions were performed for 30 minutes at 30°C in 50 μl of kinase buffer (60 mM Hepes-NaOH pH 7.5, 3 mM MgCl 2 , 3 mM MnCl 2 , 3 μM Na 3 VO 4 ) supplemented with 2.5 μg polyethylene glycol, 10 μM ATP and 300 ng of recombinant human STAT1 (Active Motif, Carlsbad, CA) as a substrate.

Techniques: Purification, Immunoprecipitation, Kinase Assay, Recombinant, Transfection, Plasmid Preparation, Negative Control, Phospho-proteomics, Western Blot

Journal: PLoS ONE

Article Title: Analysis of STAT1 Activation by Six FGFR3 Mutants Associated with Skeletal Dysplasia Undermines Dominant Role of STAT1 in FGFR3 Signaling in Cartilage

doi: 10.1371/journal.pone.0003961

Figure Lengend Snippet: HeLa, 293T and RCS cells, transfected with vectors expressing wild-type FGFR, activating FGFR3 mutants (N540K, G380R, R248C, Y373C, K650M and K650E), and kinase-inactive mutant K508M were grown for 48 hours, treated with FGF2 for 15 minutes and analyzed for given molecules by western blotting. The left and right panels represent independent experiments. Note that only K650M and K650E-FGFR3 cause strong activatory STAT1(Y701) phosphorylation whereas the other mutants cause weak (HeLa) or no activation (293T and RCS). This contrasts with ERK1/2 activation, which is induced by all six mutants in 293T and RCS cells (samples without FGF2 treatment). The P-Y701-STAT1 signal in HeLa cells was quantified by densitometry and graphed. Also note the differences in FGFR3 maturation, where HeLa and RCS cells but not 293T cells express mostly immature forms (lower arrow) of K650M and K650E-FGFR3. STAT1, ERK1/2 and ACTIN western blottings serve as loading controls. Cells transfected by GFP vector serve as negative control for transfection.

Article Snippet: Briefly, kinase reactions were performed for 30 minutes at 30°C in 50 μl of kinase buffer (60 mM Hepes-NaOH pH 7.5, 3 mM MgCl 2 , 3 mM MnCl 2 , 3 μM Na 3 VO 4 ) supplemented with 2.5 μg polyethylene glycol, 10 μM ATP and 300 ng of recombinant human STAT1 (Active Motif, Carlsbad, CA) as a substrate.

Techniques: Transfection, Expressing, Mutagenesis, Western Blot, Phospho-proteomics, Activation Assay, Plasmid Preparation, Negative Control

Journal: PLoS ONE

Article Title: Analysis of STAT1 Activation by Six FGFR3 Mutants Associated with Skeletal Dysplasia Undermines Dominant Role of STAT1 in FGFR3 Signaling in Cartilage

doi: 10.1371/journal.pone.0003961

Figure Lengend Snippet: (A) RCS chondrocytes transfected with vectors expressing wild-type FGFR3, activating FGFR3 mutants (N540K, G380R, R248C, Y373C, K650E and K650M), and kinase-inactive mutant K508M were grown for 48 hours and analyzed for the indicated molecules by western blotting. Note differential STAT1 and ERK activation by the activating FGFR3 mutants. Cells transfected with empty plasmid (pcDNA3) serve as negative control for transfection. (B) RCS chondrocytes were transfected as described in (A), grown for 72 hours and counted. Note the inhibition of RCS growth by wild-type FGFR3 as well as the activating mutants, as compared to cells transfected either by kinase-inactive K508M-FGFR3 or an empty plasmid. The data represent an average from four individually transfected wells with indicated standard deviation. The cell count difference compared between cells transfected with wild-type FGFR3 and empty plasmid, as well as the cell count difference between cells transfected with wild-type FGFR3 and N540K, G380R, R248C, Y373C, K650M and K650E mutants, were statistically significant (Student's t -test, p <0.01). (C) The experiment shown on (B) was repeated five times to eliminate the variance associated with differential transfection efficiency. The differences in percentages of growth compared between cells transfected with wild-type FGFR3 and empty plasmid, and between cells transfected with wild-type FGFR3 and N540K, G380R, R248C, Y373C, K650M and K650E mutants, were statistically significant (Student's t -test, p <0.01).

Article Snippet: Briefly, kinase reactions were performed for 30 minutes at 30°C in 50 μl of kinase buffer (60 mM Hepes-NaOH pH 7.5, 3 mM MgCl 2 , 3 mM MnCl 2 , 3 μM Na 3 VO 4 ) supplemented with 2.5 μg polyethylene glycol, 10 μM ATP and 300 ng of recombinant human STAT1 (Active Motif, Carlsbad, CA) as a substrate.

Techniques: Transfection, Expressing, Mutagenesis, Western Blot, Activation Assay, Plasmid Preparation, Negative Control, Inhibition, Standard Deviation, Cell Counting

Journal: PLoS ONE

Article Title: Analysis of STAT1 Activation by Six FGFR3 Mutants Associated with Skeletal Dysplasia Undermines Dominant Role of STAT1 in FGFR3 Signaling in Cartilage

doi: 10.1371/journal.pone.0003961

Figure Lengend Snippet: (A) The N540K, G380R, R248C, Y373C, K650M and K650E-FGFR3 mutants used in this study all cause FGFR3-skeletal dysplasias and signal through ERK MAP kinase in contrast to STAT1, that is activated mostly by the K650M and K650E-FGFR3 mutants. (B) According to the study compiling the clinical data of 591 patients suffering from FGFR3-related skeletal dysplasia , the STAT1-activating K650M and K650E account for as little as 4.9% of cases. It is therefore unlikely that activation of STAT1 plays a central role in FGFR3-related skeletal dysplasias as currently believed, but rather represents a signaling feature unique to small subset of patients carrying the K650M and K650E mutations.

Article Snippet: Briefly, kinase reactions were performed for 30 minutes at 30°C in 50 μl of kinase buffer (60 mM Hepes-NaOH pH 7.5, 3 mM MgCl 2 , 3 mM MnCl 2 , 3 μM Na 3 VO 4 ) supplemented with 2.5 μg polyethylene glycol, 10 μM ATP and 300 ng of recombinant human STAT1 (Active Motif, Carlsbad, CA) as a substrate.

Techniques: Activation Assay