# Engineered orthogonal signaling systems for selective phosphorylation of protein kinase substrates

> **NIH NIH R21** · YALE UNIVERSITY · 2024 · $391,531

## Abstract

ABSTRACT
Protein kinases function in cell signaling through regulated phosphorylation of specific substrates. Current
methods allow one to comprehensively identify the substrates of a given kinase, yet we lack general methodology
for determining which substrates are critical for specific functions of that kinase. Likewise, approaches for probing
the functional impact that phosphorylation has upon such key substrates are lacking. Here, we propose to
develop technology allowing for directed phosphorylation of a single protein kinase substrate at defined sites.
With this approach, we engineer a kinase mutant that can only phosphorylate a designer allele of one of its
substrates. To accomplish this goal we will leverage knowledge of kinase phosphorylation site specificity gained
through our recent comprehensive analysis of the human serine-threonine kinome and consequent
understanding of the structural determinants of kinase selectivity. We will establish the feasibility of this
technology using the tumor suppressor kinase LKB1 as a model system. LKB1 has a well-defined substrate
repertoire, phosphorylating and activating a set of 14 downstream protein serine-threonine kinases exclusively
on Thr residues. We will engineer LKB1 so that it instead phosphorylates Ser residues and does not act on its
endogenous substrates. We will then engineer compensating mutations in one of its key substrates, the AMP-
activated protein kinase (AMPK), to restore phosphorylation and activation by mutant LKB1. Human cancer cell
lines co-expressing these alleles will be analyzed for LKB1-dependent activation of AMPK to the exclusion of
other LKB1 substrates. To determine whether this system faithfully recapitulates endogenous signaling, we will
examine phosphorylation of established substrates downstream of AMPK, and we will globally map changes to
the phosphoproteome in response to AMPK activation. At the outcome of these studies, we will have established
a system in which a single substrate of a kinase is phosphorylated with identical dynamics as the native
substrate. Future studies will expand this approach into other LKB1 substrates, facilitating studies of how LKB1
functions as a tumor suppressor. We will ultimately apply this technology generally to other protein kinases
implicated in cancer whose critical substrates are currently unknown.

## Key facts

- **NIH application ID:** 10952454
- **Project number:** 1R21CA293623-01
- **Recipient organization:** YALE UNIVERSITY
- **Principal Investigator:** BENJAMIN E TURK
- **Activity code:** R21 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2024
- **Award amount:** $391,531
- **Award type:** 1
- **Project period:** 2024-08-06 → 2026-07-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10952454

## Citation

> US National Institutes of Health, RePORTER application 10952454, Engineered orthogonal signaling systems for selective phosphorylation of protein kinase substrates (1R21CA293623-01). Retrieved via AI Analytics 2026-06-12 from https://api.ai-analytics.org/grant/nih/10952454. Licensed CC0.

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