Page 105 - Haematologica May 2020

BCR-FGFR1 regulated by dimerization and chaperonin Hsp90
extracellular ligand binding domain of FGFR1 is removed in the fusion proteins. All transfected constructs displayed cell viability in the presence of IL-3 (Figure 3C and F). Thus, these data demonstrate that the BCR-FGFR1 fusion protein requires the N-terminal contribution of BCR for IL-3-independent proliferation as assayed in 32D cells.
LC-MS/MS analysis identifies novel phosphorylation sites
The strong tyrosine phosphorylation signal seen in BCR-FGFR1 and BCR-FGFR1-K656E lysates though immunoblotting (Figure 1) led to the inquiry of whether there were any novel phosphorylation sites within these fusion proteins. To investigate this question, HEK293T cell lysate expressing either FGFR1 or BCR-FGFR1 deriva- tives were immunoprecipitated and analyzed by LC- MS/MS as described.16
Both BCR-FGFR1 and BCR-FGFR1-K656E exhibit high phosphorylation levels on key tyrosine residues in the FGFR1 kinase domain, whereas BCR-FGFR1-K514A exhibited only slight phosphorylation (Figure 4). The lack of phosphorylation on the activation loop tyrosine residues in BCR-FGFR1-K514A indicates that FGFR1 kinase activity is critical for activation of the BCR-FGFR1 fusion.
Figure 2. Cell transformation of NIH3T3 cells by BCR-FGFR1. Representative plates from a focus assay are shown, with transfected constructs indicated. The graph shows the number of foci scored, normalized for transfection efficiency and calculated as a percentage of transforma- tion relative to FGFR3-TACC3 –/+ standard error of the mean (SEM). Assays were performed a minimum of three times per each DNA con- struct.
Additional serine, threonine, and tyrosine phosphoryla- tion sites were also detected in BCR-FGFR1 or BCR- FGFR1-K656E (Figure 4B-C) which have not been previ- ously reported. To determine the role of these novel phos- phoacceptor sites within the BCR domain of BCR-FGFR1 fusion proteins, phosphorylated serine or threonine residues were mutated to alanine, and phosphorylated tyrosine residues in BCR were mutated to phenylalanine. Each construct was assayed for focus formation. In addi- tion to these mutations, a BCR(Y177F)-FGFR1 mutant was also assayed as it mutates the Grb2 binding site, previous- ly shown to reduce activation of the BCR-FGFR1 fusion protein.19,20
The Y177F Grb2 mutation in BCR-FGFR1 shows a 50% decrease in transforming ability when compared to NIH3T3 cells expressing BCR-FGFR1. However, all addi- tional mutations in BCR phosphoacceptor sites displayed little to no difference in transforming ability (Table 1). The mutant BCR(T359A/S367A/S369A/S377A)-FGFR1, which mutates phosphorylated residues within the ABL SH2 binding domain present in BCR, was also transforming. Taken together, LC-MS/MS data and cell transformation assays suggest that tyrosine phosphorylation on activation loop residues within the FGFR1 kinase domain is critical for BCR-FGFR1 activation. BCR-FGFR1 relies on an active
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