Novel Tyrosine Phosphorylation Sites Fine Tune the Activity and Substrate Binding of Src Family Kinases

Abstract

Src Family tyrosine Kinases (SFKs) are critical regulators of cellular processes including proliferation, migration and growth. SFKs are positively and negatively regulated by phosphorylation at two key tyrosine residues. We and others, over the course of hundreds of recent phosphoproteomic studies, have identified novel phosphotyrosines on SFKs from a variety of cells and tissues. These novel sites are located in important functional domains leading us to test the hypothesis that these sites can alter SFK enzymatic activity and binding. We used site-directed mutagenesis to generate point mutants in the SFK Fyn; phosphomimetic and non-phosphorylatable alleles exhibited altered kinase activity or affinity for a known binding partner, ESDN. Expression of SFKs in yeast followed by mass spectrometry analysis showed these sites can be the result of autophosphorylation. Finally, we generated transgeneic worms expressing various Fyn alleles in mechanosensory neurons and observed significantly altered neuronal migration patterns. We propose a model whereby SFK autophosphorylation at these novel tyrosine phosphorylation sites fine tunes SFK regulation of the wide array of cellular processes SFKs control. This project was supported by NSF grant IOS 1021795 and by the Vermont Genetics Network-grant 8P20GM103449 from the NIH INBRE Program of the NIGMS.