Regulation of FGFR autophosphorylation and implications for downstream cell signaling

While extensive research has been done to delineate the components of different cellular signaling pathways upon receptor tyrosine kinase (RTK) activation, little is known about the regulation of receptor autophosphorylation. Using a combination of rapid chemical quench and high resolution mass spectrometry, we show that FGFR autophosphorylation occurs through a sequential, highly ordered process. Additional biochemical assays demonstrate that this ordered autophosphorylation is kinetically controlled and is due to differences in the rate of phosphotransfer at each of the tyrosine phosphorylation sites. Mutagenesis experiments further show that these differences in substrate recognition are imparted by both the primary sequence surrounding the phosphorylation site as well as the position of the tyrosine within the tertiary structure. In addition, we investigated the mechanism of loss-of-function mutations implicated in LADD syndrome and demonstrate that two of the mutations result in decreased catalytic activity, which in effect, results in diminished receptor tyrosine autophosphorylation. In light of the tremendous scope of FGFR signaling in development to maintenance of cellular homeostasis, novel chemical scaffolds are necessary to inhibit RTKs upon their dysregulation. We present a detailed structural analysis of a novel tyrosine kinase inhibitor in complex with FGFR1 and highlight key features of the small molecule inhibitor that could be modified to increase its efficacy. Taken together, data presented here suggests that receptor tyrosine kinase autophosphorylation occurs by an ordered process regulated intrinsically by properties of the kinase core. Thus, RTK autophosphorylation offers an additional level of both temporal and spatial control to RTK signaling which mediates the precise recruitment of downstream cellular signaling molecules and effector proteins.