Author ORCID Identifier

https://orcid.org/0009-0001-9690-6688

Date of Award

Spring 4-14-2026

Document Type

Thesis (Ph.D.)

Department or Program

Biochemistry and Cell Biology

First Advisor

Arminja N. Kettenbach

Abstract

Reversible phosphorylation is a crucial post-translational modification (PTM) that modulates a variety of signaling pathways intracellularly, with aberrant protein phosphorylation being a hallmark of many human diseases. Kinases, which catalyze phosphorylation of their substrates, have been extensively studied in the context of the signaling pathways they regulate and their effects on cellular processes. In contrast, phosphoprotein phosphatases (PPPs), which catalyze the removal of phosphate groups from substrate proteins, have only recently come to the research spotlight. For a long time, protein phosphatases were deemed unspecific housekeeping enzymes, but they are now known to display exquisite substrate specificity. Phosphoprotein phosphatase 1 (PP1) and phosphoprotein phosphatase 3 or calcineurin (PP3, CN), the subjects of investigation of this thesis, belong to the family of PPPs that mediate the majority of Ser/Thr dephosphorylation events in the cell.

Each PPP recognizes its substrates via PPP-specific short linear motifs (SLiMs) present on the interaction surface of their regulators. We focus on dissecting the binding determinants of the PP1 catalytic isoform β (PP1cβ) towards a group of regulators that contain a distinct PP1 SLiM. The four mammalian PP1 catalytic isoforms (α, β, γ1, and γ2) share a greater than 80% sequence identity, with most amino acid differences found in the N- and C-termini extremities. Despite their high homology, PP1c isoforms exhibit distinct spatiotemporal distributions and functions, driven by differences in their affinities for targeting subunits that determine intracellular localization and substrate specificity. Using affinity purification and mass spectrometry, we identify that the C-terminus and the threonine 197 (Thr197) residue in the PP1cβ sequence drive the isoform-specific interaction of the PP1cβ isoform and PP1 regulators that contain the MyPhoNE SLiM. This investigation elucidates the importance of a secondary PP1-specific SLiM (MyPhoNE) and validates the integral role of the PP1cβ-myosin phosphatase target subunit 1 (MYPT1) interaction in the subsequent dephosphorylation of Polo-like kinase 1 (PLK1) in early mitosis.

We then pivot to calcineurin (CN), with the goal of identifying novel CN regulators and examining the effects of the de novo E282K missense mutation on its interactome and substrateome. Using mutagenesis of CN-specific SLiMs present in potential CN regulators and mass spectrometry, we test the predictive power of SLiMs in expanding the CN interactome. Lastly, we characterize the effect of the missense CN E282K mutation on the CN interactome and the substrate dephosphorylation profile via proteomics and phosphoproteomics. Developmental and epileptic encephalopathy 91 (DEE91) has been linked to the CN variant E282K and is characterized by epileptic encephalopathy, intellectual disabilities, dysmorphism, hypotonia, and autism spectrum disorder. We show that CN E282K alters the CN substrate dephosphorylation preferences from basic to acidic, leading to global changes in CN-mediated signaling.

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