# Redox regulation of protein kinase function: biochemical mechanisms and cellular consequences

> **NIH NIH R35** · NORTH CAROLINA AGRI & TECH ST UNIV · 2024 · $362,500

## Abstract

SUMMARY. My research program focuses on understanding the organization and regulation of
phosphorylation-dependent signaling networks in health and disease. In particular, we are interested in
deciphering the biochemical and cellular mechanisms of crosstalk between phosphorylation-dependent
signaling and other signaling pathways at the level of posttranslational modification of protein kinases. One
attractive modification is reversible oxidation, mediated by reactive oxygen species (ROS). ROS are emerging
as critical second messengers in signaling pathways related to many pervasive diseases, including diabetes,
cancer, and cardiovascular disease. A growing body of evidence suggests that protein kinases are directly
regulated by redox modifications. For instance, the reversible oxidation of Cys residues in redox-sensitive
kinases has been shown to influence their activity (both positively and negatively), subcellular localization, and
protein-protein interactions. In many cases, the modified Cys is conserved among other members in the same
kinase family. This raises the intriguing possibility that reversible oxidation may be a general means of regulating
kinase function inside cells. To explore this possibility further, we are examining the impact of H2O2-depedent
oxidation on the global substrate selection of several mitogen-activated protein kinase (MAPK) and AGC family
members using functional protein microarrays and complementary biochemical, biophysical, mutational,
computational, and cell-based strategies. Our data suggest that H2O2-dependent oxidation shifts the substrate
preference of many kinases, leading to distinct substrate profiles in the oxidized and reduced states. For
instance, with support of an NIGMS SC1 award (5SC1GM130545), we are currently studying the biochemical
mechanisms underlying the H2O2-induced shifts in the substrate selection of the canonical AGC and MAPK
family members, PKA-Cα and ERK2. These studies suggest that reversible oxidation of PKA-Cα and ERK2
increases their affinity for some substrates while decreasing or having little effect on others. Interestingly,
different types of redox modification (e.g., diamide-mediated oxidation, H2O2-dependent oxidation, or
glutathionylation) led to different activity profiles toward the same model substrates. With support from the
proposed MIRA, we propose to build on these studies by asking questions about 1) the effects of redox
modification of kinase substrate selection in various cellular contexts and their impact on signaling outcomes
related to disease; 2) the impact of redox modification on the spatiotemporal regulation of kinase activity profiles
in cells; and 3) how the global substrate profiles of other protein kinases, including understudied kinases in the
“dark kinome”, are affected by H2O2-dependent oxidation and/or different types of redox modifications. Together,
these studies will offer unique insights into mechanisms of crosstalk between redox- and phosphoryla...

## Key facts

- **NIH application ID:** 10847563
- **Project number:** 1R35GM153737-01
- **Recipient organization:** NORTH CAROLINA AGRI & TECH ST UNIV
- **Principal Investigator:** Robert Howard Newman
- **Activity code:** R35 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2024
- **Award amount:** $362,500
- **Award type:** 1
- **Project period:** 2024-08-01 → 2029-05-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10847563

## Citation

> US National Institutes of Health, RePORTER application 10847563, Redox regulation of protein kinase function: biochemical mechanisms and cellular consequences (1R35GM153737-01). Retrieved via AI Analytics 2026-05-24 from https://api.ai-analytics.org/grant/nih/10847563. Licensed CC0.

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