EXPLORATION OF KINASE-REGULATED CELLULAR SIGNALING IN CHLAMYDOMONAS REINHARDTII

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  • March 21, 2019
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  • Werth, Emily
    • Affiliation: College of Arts and Sciences, Department of Chemistry
Abstract
  • Post-translational modifications (PTMs) on proteins to form functional protein products are a key level of cellular signaling regulation. Because of this, there has been an immense effort in the proteomics community to improve quantitative enrichment, acquisition, and bioinformatics strategies for the analysis of PTMs to probe metabolic pathways. The identification of dynamic protein phosphorylation events, a vital PTM, is especially important for understanding kinase/ phosphatase-regulated signaling pathways, and is the focus of this dissertation. The aim of this dissertation is to develop and apply phosphoproteomic strategies in the alga Chlamydomonas reinhardtii to characterize the role of protein phosphorylation on cellular regulation in a diverse array of signaling networks. Techniques for algal cell culturing, protein extraction, quantitative enrichment, acquisition and bioinformatics processing developed and adapted for Chlamydomonas are discussed (Chapter 2). Using these techniques, a quantitative workflow for a dual enrichment strategy to target intact protein kinases via capture on immobilized multiplexed inhibitor beads with subsequent proteolytic digestion of unbound proteins and peptide-based phosphorylation enrichment was developed (Chapter 3). This workflow obtained quantitative coverage on 115 protein kinases and 2,304 phosphopeptides. Application of the quantitative phosphoproteomic pipeline was employed to study the effect of Target of Rapamycin (TOR) kinase inhibition on the Chlamydomonas phosphoproteome in wild-type (Chapter 4) and extension into a rapamycin hypersensitive mutant line (Chapter 5). From the wild-type study, three TOR inhibitors with varying mechanisms of inhibition were used to obtain quantitative coverage on 2,547 unique phosphosites with 258 phosphosites differentially changing following inhibition. This approach identified Chlamydomonas homologs of TOR signaling-related proteins such as RPS6 and LARP1 that had decreased phosphorylation upon TORC1 inhibition. Additionally, this led to follow-up experiments guided by our phosphoproteomic findings showing that carotenoid levels are affected by TORC1 inhibition, the first evidence that carotenoid production is under TOR control. From the rapamycin hypersensitive mutant study, the workflow obtained quantitative coverage on 2,699 phosphosites with 316 sites changing following rapamycin treatment. This study showed similarities with the sites modulated in the wild-type study described in Chapter 4 while also providing another distinct group of phosphosites not previously interrogated.
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  • Hicks, Leslie
  • Graves, Lee
  • Ehrmann, Brandie
  • Glish, Gary
  • Jorgenson, James
Degree
  • Doctor of Philosophy
Degree granting institution
  • University of North Carolina at Chapel Hill
Graduation year
  • 2018
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