# Revealing substrates and phosphoproteome level function of human STE20 kinases

> **NIH NIH R01** · YALE UNIVERSITY · 2020 · $117,717

## Abstract

Project summary
Protein phosphorylation is one of the most common and critical post-translational modifications governing
signaling cascades in humans. Phosphorylation of protein kinases governs their activity and regulation. The
importance of regulation by phosphorylation is further emphasized by the fact that protein kinases comprise
nearly 2% of the human proteome and numerous kinases have been implicated in processes that control cell
proliferation, motility, and apoptosis in healthy and diseased human cells. While identification of
phosphorylation sites within the human proteome has dramatically progressed in recent years, our
understanding of phosphorylation cascades is limited due to a distinct lack of knowledge of which kinases are
responsible for each phosphorylation event and the specific arrangement of phosphorylation sites leading to an
active kinase that phosphorylates its target substrate. Establishing direct connections of all human kinases to
the phosphoproteome and revealing a systems-level diagram of human signaling networks also remain
defining challenges. Since phosphorylation plays a central role in protein-protein interactions through phospho-
binding domains, new approaches that can address these questions in a comprehensive and unbiased fashion
are needed. Studying protein phosphorylation has been limited by the inability to generate phosphoproteins
with the specificity of natural systems. Genetically encoded non-standard amino acids (NSAAs) have recently
enabled site-specific incorporation of phosphoserine into proteins. We showed that a genomically recoded
organism (GRO), in which all TAG stop codons were converted to TAA and the deletion of RF-1, converted
TAG to an open sense codon dedicated for incorporating phosphoamino acids. Importantly, this technological
breakthrough enables site-specific expression of human phosphoproteins in an engineered bacterial system
(i.e., GRO containing phosphoserine orthogonal translation system, OTS). Furthermore, it provides a platform
technology to address questions probing the connectivity of the human kinome and the functional landscape of
phospho-binding domains. Here, we aim to further develop and apply this technology to generate optimized
platforms to address functional questions surrounding the phosphoserine component of the human
phosphoproteome (Aim 1). These new, enhanced platforms will enable studies to identify STE20 kinase
substrates that will directly inform future research into multiple human disease pathways as well as define a
general strategy to elucidate human kinase substrates (Aim 2). Finally, we aim to identify phosphorylation
sites that are drivers of protein-protein interactions in general, followed by, a systematic screen of the STE20
substrates in a coordinated effort to assign biological function to a portion of the human phosphoproteome
(Aim 3).

## Key facts

- **NIH application ID:** 10171453
- **Project number:** 3R01GM117230-05S1
- **Recipient organization:** YALE UNIVERSITY
- **Principal Investigator:** Farren J. Isaacs
- **Activity code:** R01 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2020
- **Award amount:** $117,717
- **Award type:** 3
- **Project period:** 2015-09-25 → 2021-03-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10171453

## Citation

> US National Institutes of Health, RePORTER application 10171453, Revealing substrates and phosphoproteome level function of human STE20 kinases (3R01GM117230-05S1). Retrieved via AI Analytics 2026-05-23 from https://api.ai-analytics.org/grant/nih/10171453. Licensed CC0.

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