# The regulation of phosphoprotein phosphatases in the nucleus

> **NIH NIH R01** · UNIVERSITY OF CONNECTICUT SCH OF MED/DNT · 2024 · $70,595

## Abstract

ABSTRACT
An estimated 70% of all eukaryotic cellular proteins are regulated by phosphorylation. Strict temporal and
spatial control are essential for the fidelity of this process, as derailed signaling cascades lead to disease. While
the importance of phosphorylation is clear, knowledge gaps remain in the mechanisms that regulate key
proteins involved in this process, especially phosphoprotein phosphatases (PPP). Our long-term goal is to
understand the structural and functional mechanisms that control PPP activity in health and disease. Here, we
focus on the function of protein phosphatase 1 (PP1) and PP2A, both of which have major roles in cell division
and cancer. Our aims are designed to define the mechanisms of PP1- and PP2A:B55-based substrate
recruitment to obtain a systems biology understanding of the proteomes and phosphatomes directed by these
enzymes. For the PP2A family of enzymes, it is established that substrates are recruited by their variable B-
subunits. Here, we investigate PP2A:B55, the most abundant PP2A holoenzyme in cells and the primary
enzyme responsible for dephosphorylating CDK1 targets to initiate mitotic exit. Consistent with this, at mitotic
entry, PP2A:B55 activity is inhibited. This is achieved by two B55-specific inhibitors: FAM122A and ARPP19.
To molecularly define how these inhibitors block PP2A:B55 activity and to elucidate the molecular basis of B55
substrate recruitment. We have also developed a unique PP1 regulator (PhosTAP), which we show can be
used to fully define the PP1 interactome and phosphatome. Due to its 100% specificity and exceptional affinity
for only PP1, this novel PP1 PhosTAP can also be leveraged to specifically recruit PP1 to its point of action
within the cell, in a manner similar to that used by PROTACs for targeted degradation. Critically, both projects
require the purification of exceptionally challenging to produce samples (typically multimers) that strictly require
mammalian expression. It is only mammalian expression that provides the rigorous, reproducible samples
needed for the proposed structural and biophysical studies. Here, we request the costs for a new FPLC system
to replace an outdated system that is no longer functional. Our proposed experiments require exceptionally
pure samples. Obtaining these samples requires multiple purification steps using diverse chromatographic
media and highly functional sophisticated chromatographic systems. Each prep requires ~2.5 days on these
systems, clearly demonstrating the need to replace our non-functional system in order to successfully complete
all aims. Because these holoenzymes have critical roles in multiple human diseases, especially cancer, the
proposed work will establish these holoenzymes specifically, and PPPs generally, as potent and specific drug
targets.

## Key facts

- **NIH application ID:** 11052222
- **Project number:** 3R01GM144379-02S1
- **Recipient organization:** UNIVERSITY OF CONNECTICUT SCH OF MED/DNT
- **Principal Investigator:** Rebecca Page
- **Activity code:** R01 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2024
- **Award amount:** $70,595
- **Award type:** 3
- **Project period:** 2023-05-01 → 2027-04-30

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/11052222

## Citation

> US National Institutes of Health, RePORTER application 11052222, The regulation of phosphoprotein phosphatases in the nucleus (3R01GM144379-02S1). Retrieved via AI Analytics 2026-05-22 from https://api.ai-analytics.org/grant/nih/11052222. Licensed CC0.

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