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Open Access Publications from the University of California

Chemoproteomic tools for studying kinase signaling

  • Author(s): Levin, Rebecca
  • Advisor(s): Shokat, Kevan M
  • Burlingame, Alma L
  • et al.

Protein kinases are key signaling proteins responsible for maintaining cellular homeostasis. These enzymes catalyze the transfer of the γ-phosphate of adenosine triphosphate (ATP) to a serine, threonine or tyrosine residue of a substrate protein. The introduction of this additional negative charge may control the function of the substrate, by altering conformation or altering sites of protein binding. Over 500 kinases serve diverse roles in integrating signals from extracellular stimuli, regulating the cell cycle, and regulating gene expression. The field of mass spectrometry based proteomics has made identifying and quantifying sites of phosphorylation on a proteome-wide basis relatively facile, with hundreds of thousands of sites of phosphorylation identified to date. However, there remains a disconnect between the few known substrates, and thus function, of any given kinase, and the hundreds of thousands of known sites of phosphorylation across the proteome. In this thesis, we describe efforts to close this gap through the engineering of a kinase to accept a bio-orthogonal analog of ATP as a substrate, and thus trace its substrates via mass spectrometry. We apply the technique to Cdk9, identifying a novel regulatory function in transcript termination, and AMPK, identifying evidence for the involvement of AMPK in mediating cell motility and adhesion. Furthermore, we demonstrate that GTPase Rab1 is a substrate of innate immunity kinase TAK1, and show phosphorylation of the dynamic switch region of Rab1, and perhaps Rab GTPases as a family, can regulate its function. In the second half of this thesis, we address efforts to further understand the interconnectivity of the kinome, as many kinases function in tightly orchestrated signaling cascades, with kinases phosphorylating substrate kinases. Through the use of the Multiplexed Kinase Inhibitor Bead (MIB) methodology, we investigate the how mis-regulation of these cascades drives oncogenesis. Through the use of the MIBs approach, which enriches for activated kinases and is coupled to quantitative proteomics, we identify mechanisms of resistance to targeted kinase therapeutics as well as identify non-mutated kinase drivers of tumorgenesis. Lastly, we describe increased MIBs sensitivity through the use of parallel reaction monitoring (PRM), a targeted proteomic approach.

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