Resolving multi-site protein phosphorylation by phosphoryl-AMPylation

/Abstract Protein phosphorylation is the most common post-translational modification in eukaryotic cells and impacts nearly every cellular process. Despite the ubiquity of phospho-regulation and its long history of study, there remain technical challenges to accurately detecting protein phosphorylation events, particularly in multi-site phosphorylation. For example, it remains impossible to predict how or if multi-site phosphorylation will shift a proteins migration on SDS-PAGE and, as a result, detecting phosphorylated proteins by western blot requires a priori knowledge to raise a phospho-specific antibody. As SDS-PAGE is arguably the most widely used, accessible, and cost-effective means of protein analysis, advancing this technology to enable the consistent and quantifiable detection of protein phosphorylation would benefit nearly all fields of cell biology. This exploratory project will exploit a novel mechanism for chemically modifying protein phospho-sites for the purpose of developing new methods to study protein phosphorylation. Specifically, we will take advantage of the newly characterized Legionella pneumophila effector protein LnaB, which installs adenosine monophosphate (AMP) specifically onto the phosphate group of phosphorylated serine, threonine, and tyrosine. These proof-of-principle studies will determine whether LnaB can accommodate ATP analogs with an alkyne adduct (ATP-alkyne), thus facilitating click-chemistry with azide-containing molecules for the modular modification of protein phospho-sites. Towards this end, we will first identify LnaB orthologs with optimal catalytic efficiency and broad specificity phosphoryl-AMPylase activity. Subsequently, we will test whether ATP analogs with alkyne adducts (on either the adenine base or ribose) are suitable substrates for LnaB. As a proof-of-principle application, we will use this method to quantify multi-site phosphorylation. Specifically, we will install “mass tags” – azide-containing peptides of defined molecular weight – on phosphorylated proteins to quantitatively shift their mass in a manner that is linearly dependent on the number of phosphates. By combining this with SDS-PAGE, we will determine whether we can quantify multi-site phosphorylation of recombinant proteins and detect both phosphorylated and non- phosphorylated proteins with a single antibody in complex samples by western blot analysis, thus directly quantifying fraction phosphorylated. Our long-term goal is to develop a modular set of reagents for modifying phospho-proteins for unique downstream applications, including counting multi-site phosphorylation by SDS- PAGE, fluorescent labeling, streamlined western blot detection, and, in the future, modulation of specific phospho-regulatory axes to alter signaling pathways for therapeutic purposes. Project Number: 1R21GM163082-01 | Fiscal Year: 2026 | NIH Institute/Center: National Institute of General Medical Sciences (NIGMS) | Principal Investigator: Matthew Parker | Institution: UT SOUTHWESTERN MEDICAL CENTER, DALLAS, TX | Award Amount: $449,900 | Activity Code: R21 | Study Section: Macromolecular Structure and Function B Study Section[MSFB] View on NIH RePORTER: https://reporter.nih.gov/project-details/11286990