Date of Award

2014-01-01

Degree Name

Doctor of Philosophy

Department

Biological Sciences

Advisor(s)

Robert A. Kirken

Abstract

Homeostasis of the immune system is required for proper defense against pathogenic insult. Cells of the innate and adaptive compartments provide a strictly regulated response to clear infections while allowing for self-tolerance. Dysregulation of the components of the immune system can lead to immunodeficiency, autoimmunity, and cancer. Key players of the immune response are T, B, and NK cells, which become strongly activated by IL2 through its receptor. The β subunit of the receptor becomes tyrosine, serine, and threonine phosphorylated upon induction with IL2. Phosphorylation of tyrosine residues has been extensively studied, however, the putative regulatory role of serine and threonine phosphorylation on IL2 signaling has yet to be characterized. The first objective of this research was to identify novel phosphorylation sites on IL2Rβ. Using immunoprecipitation combined with LC-MS/MS five novel IL2Rβ phosphorylation sites, S268, T394, T450, S484, and S512 were identified. In an attempt to characterize T450 and S512 phosphosites, phosphospecific antibodies were generated and found to be reliable new tools to elucidate the role of T450 and S512 in IL2 signal transduction. Using these phosphospecific antibodies, the phosphorylation status of T450 in response to physiological stimuli was investigated. The phosphorylation of IL2Rβ T450 occurred in multiple cell types, including primary human PBMCs, with profiles indicating a general mechanism of IL2Rβ activation. Kinase/phosphatase inhibition studies along with siRNA or purified phosphatases allowed for the identification of ERK1/2 as the kinases that phosphorylate the receptor while PP1 was the phosphatase that mediates its dephosphorylation. Finally, IL2 induced IL2Rβ T450 was found to have a positive regulatory role as demonstrated in a HEK293 reconstitution system using WT or amino acid substitution within IL2Rβ. We assessed the effect of IL2 induced T450 phosphorylation in the assembly of the IL2R complex and the activation of key signaling molecules by immunoprecipitation and Western blot analysis. Phosphorylation of T450 was found to be important for IL2R complex formation, recruitment of JAK3, full activation of STAT5 and AKT. These results suggest a new target to modulate T-cells with therapeutic potential.

Language

en

Provenance

Received from ProQuest

File Size

120 pages

File Format

application/pdf

Rights Holder

Blanca Estela Ruiz-Medina

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