Date of Award

6-27-2022

Document Type

Thesis (Ph.D.)

Department or Program

Biochemistry and Cell Biology

First Advisor

Arminja Kettenbach

Abstract

Protein phosphorylation is a highly regulated mechanism of cell signaling control and its deregulation is implicated in disease. The kinases that catalyze the addition of phosphate groups onto their substrate proteins have been well studied, their signaling pathways mapped, and their effects on cell and organismal health observed. Knowledge of the phosphatases that reverse the reaction only recently began to come into focus. Phosphoprotein phosphatases (PPPs), long thought to be housekeeping enzymes, are now known to be exquisitely specific towards their substrates, but the exact nature of phosphatase regulation—both upstream and downstream of the phosphatase—is unclear.

PPPs recognize substrates through short linear motifs (SLiMs) on their substrates and interactors. Using affinity-purification and mass spectrometry, we identify over one hundred interactors for the PPP calcineurin and expand our understanding of calcineurin’s role in the cell. We discover a novel role for calcineurin at the nuclear pore complex and support its role as a regulator of nuclear import and export by identifying a dephosphorylation site on RanBP3 that controls nuclear export of SMAD2. Using our list of interactors, we redefine calcineurin’s LxVP SLiM and gain insight into calcineurin’s affinity towards its substrates.

We then develop two methods for identifying calcineurin-specific dephosphorylation sites, including using the SLiM-based inhibitor A238L, and find a preference for calcineurin to dephosphorylate substrates containing an [S/T]P motif. Analysis in multiple cell lines revealed cell-type specific calcineurin interactors and substrates and further work with the A238L inhibitor provided insight into calcineurin inhibition mechanisms.

Finally, we analyze strategies for normalizing the PPP enrichment strategy phosphatase inhibitor beads and mass spectrometry (PIB-MS). PPPs act as holoenzyme complexes, with catalytic subunits forming dimers and trimers with one or two of the hundreds of regulatory proteins responsible for directing the PPPs activity towards its substrates. Applying PIB-MS to breast cancer cell lines revealed differential holoenzyme complex formations between triple-negative breast cancer and non-triple-negative breast cancer subtypes, implying the subtypes have differential phosphatase signaling. We further discovered a link between PPP complexes and Hippo pathway activity in triple-negative breast cancer, expanding our understanding of PPPs in disease.

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