Quantitative mass spectrometric approaches to study the role of protein phosphorylation in cell signaling

Date of Completion

January 2008


Biology, Cell




Protein phosphorylation is the most common reversible post-translational modification to modulate protein function in eukaryotic cells. It is also an extensively employed mode of signal transduction. Protein phosphorylation events drive cell-cycle transitions and numerous responses such as cell survival, growth, and proliferation. Methods relying on the 32P radioisotope and phospho-tyrosine antibodies have been the cornerstone of cell biological research on protein phosphorylation. However, in recent years, mass spectrometry has increasingly been used both for the identification and quantification of in vivo protein phosphorylation. This dissertation presents results from two contrasting mass spectrometric approaches towards quantifying protein phosphorylation: (1) a targeted approach for the quantification of the multisite phosphorylation status of Cyclin Dependent Kinases during cell-cycle and apoptosis, and (2) a proteomic approach for the identification of T-Cell Receptor (TCR) inducible phosphorylation. In the second study, 10,710 unique phosphorylation sites on 3,084 proteins were identified, of which 696 sites showed TCR-responsive changes. The major finding is that the scope of phosphorylation in response to TCR stimulation is widespread and it extensively targets proteins involved in all of the salient T cell activation-associated phenomena: cell surface protein patterning, TCR endocytosis, actin-cup formation, inside-out integrin activation, microtubule polarization, alternative splicing, and cytokine transcription. ^