Novel Mechanisms Governing Autoregulation of the Src Family Kinase Fyn and its Crosstalk with Protein Kinase A
Date of Award
Doctor of Philosophy (PhD)
Bryan A. Ballif
Phosphorylation is a post-translational modification important for regulating protein activity and protein binding capacity. It is used in many different signaling pathways within the cell. Src Family Kinases and Protein Kinase A (PKA) are two prototyptical non-receptor tyrosine and serine/ threonine kinases, respectively, which are found in canonical signaling pathways. These two kinases are critical for signaling in essentially every cell of a multicellular organism, and are particularly important in development, cell migration and proliferation. Although both proteins have been intensely studied for many decades, an understanding of the molecular mechanisms which govern their regulation and the regulation that they effect on other proteins are still being elucidated.
Fyn, like its related Src Family Kinase members, has previously been shown to be regulated by two tyrosine phosphorylation events at residues Y420 and Y531. Y420 is located in the kinase (Src Homology 1(SH1)) domain and it is a highly-characterized intermolecular autophosphorylation site that increases the activity of the kinase. Y531 is located near the C-terminus and is phosphorylated by C-terminal Src kinase (Csk). Phosphorylation of Y531 allows it to bind to R176 in the SH2 domain in an intramolecular fashion. In this conformation Fyn has only basal activity. Since these sites are essential for regulating the activity of the kinase, we hypothesized that four novel sites of tyrosine phosphorylation in Fyn could also importantly regulate the protein. Three of the novel sites lie in the SH2 domain, and one is located in the kinase domain. Mass spectrometry, in vitro kinase assays, as well as western blot analysis aided in uncovering that these novel Fyn phosphorylation sites fine tune the activity and substrate binding of the protein.
PKA has been implicated in a multitude of signaling pathways and is particularly important in cell growth, proliferation, and migration. Fyn and PKA have classically been considered to be in separate signaling pathways. However, research over the past several decades has provided evidence that there is crosstalk that exists between the two pathways. The SFK Fyn and PKA can phosphorylate each other, thereby regulating each other's activity. Based on these data, we hypothesized the existence of downstream effectors of this relatively uncharacterized pathway. It was hypothesized that the presence of Fyn could lead to PKA activation and to differences in PKA binding partners. Through the use of co-immunoprecipitations, Stable Isotope Labeling of Amino Acids in Cell Culture (SILAC) and quantitative mass spectrometry, many proteins were found to increase their binding to PKA in the presence of Fyn. Several proteins were selected and further biochemically validated. These data suggest that the presence of Fyn could allow for PKA to more importantly interact with discrete pools of proteins within the cell to effectuate its signal transduction. Together these studies provide understanding on critical and fundamental processes by which all cells function.
Number of Pages
Weir, Marion, "Novel Mechanisms Governing Autoregulation of the Src Family Kinase Fyn and its Crosstalk with Protein Kinase A" (2016). Graduate College Dissertations and Theses. 592.