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).

Public Health Relevance

Protein kinases transmit most of the signaling events in human cells and deviations in their enzymatic activity result in disease. Here we will apply new genomic and proteomic technological approaches to study protein kinases with genetically encoded phosphorylated amino acids. These approaches will enable the synthesis of programmable activated kinases to decode the human phosphoproteome and reveal novel therapeutic targets.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
1R01GM117230-01
Application #
9007600
Study Section
Genomics, Computational Biology and Technology Study Section (GCAT)
Program Officer
Gerratana, Barbara
Project Start
2015-09-25
Project End
2020-07-31
Budget Start
2015-09-25
Budget End
2016-07-31
Support Year
1
Fiscal Year
2015
Total Cost
$326,616
Indirect Cost
$126,616
Name
Yale University
Department
Physiology
Type
Schools of Medicine
DUNS #
043207562
City
New Haven
State
CT
Country
United States
Zip Code
06510
Barber, Karl W; Muir, Paul; Szeligowski, Richard V et al. (2018) Encoding human serine phosphopeptides in bacteria for proteome-wide identification of phosphorylation-dependent interactions. Nat Biotechnol 36:638-644
Barber, Karl W; Rinehart, Jesse (2018) The ABCs of PTMs. Nat Chem Biol 14:188-192
Ma, Natalie Jing; Hemez, Colin F; Barber, Karl W et al. (2018) Organisms with alternative genetic codes resolve unassigned codons via mistranslation and ribosomal rescue. Elife 7:
Barber, Karl W; Miller, Chad J; Jun, Jay W et al. (2018) Kinase Substrate Profiling Using a Proteome-wide Serine-Oriented Human Peptide Library. Biochemistry 57:4717-4725
Gassaway, Brandon M; Petersen, Max C; Surovtseva, Yulia V et al. (2018) PKC? contributes to lipid-induced insulin resistance through cross talk with p70S6K and through previously unknown regulators of insulin signaling. Proc Natl Acad Sci U S A 115:E8996-E9005
Barbieri, Edward M; Muir, Paul; Akhuetie-Oni, Benjamin O et al. (2017) Precise Editing at DNA Replication Forks Enables Multiplex Genome Engineering in Eukaryotes. Cell 171:1453-1467.e13
Fan, Yongqiang; Evans, Christopher R; Barber, Karl W et al. (2017) Heterogeneity of Stop Codon Readthrough in Single Bacterial Cells and Implications for Population Fitness. Mol Cell 67:826-836.e5
Park, Hyun Bong; Perez, Corey E; Barber, Karl W et al. (2017) Genome mining unearths a hybrid nonribosomal peptide synthetase-like-pteridine synthase biosynthetic gene cluster. Elife 6:
Mohler, Kyle; Aerni, Hans-Rudolf; Gassaway, Brandon et al. (2017) MS-READ: Quantitative measurement of amino acid incorporation. Biochim Biophys Acta Gen Subj 1861:3081-3088
D'Lima, Nadia G; Khitun, Alexandra; Rosenbloom, Aaron D et al. (2017) Comparative Proteomics Enables Identification of Nonannotated Cold Shock Proteins in E. coli. J Proteome Res 16:3722-3731

Showing the most recent 10 out of 15 publications