Screening for potential nuclear substrates for the plant cell death suppressor kinase Adi3 using peptide microarrays

Autor: Timothy P. Devarenne, In-Cheol Yeo
Jazyk: angličtina
Rok vydání: 2020
Předmět:
0106 biological sciences
Microarrays
Information Theory
RNA polymerase II
01 natural sciences
Biochemistry
Database and Informatics Methods
Solanum lycopersicum
Transcription (biology)
Phosphorylation
Post-Translational Modification
Peptide sequence
Plant Proteins
0303 health sciences
Multidisciplinary
biology
Cell Death
Chemistry
Kinase
In Vitro Kinase Assay
Enzymes
Bioassays and Physiological Analysis
Engineering and Technology
Medicine
Sequence Analysis
Signal Peptides
Research Article
Computer and Information Sciences
Bioinformatics
Science
Research and Analysis Methods
03 medical and health sciences
Amino Acid Sequence Analysis
Plant Cells
Amino Acid Sequence
Kinase activity
Protein kinase A
Transcription factor
BLAST algorithm
030304 developmental biology
Enzyme Assays
Cell Nucleus
Background Signal Noise
Biology and Life Sciences
Proteins
Microarray Analysis
Mutation
Signal Processing
biology.protein
Enzymology
Peptides
Biochemical Analysis
Protein Kinases
010606 plant biology & botany
Zdroj: PLoS ONE, Vol 15, Iss 6, p e0234011 (2020)
PLoS ONE
ISSN: 1932-6203
Popis: The tomato AGC protein kinase Adi3 is a Ser/Thr kinase that functions as a negative regulator of programmed cell death through cell death suppression (CDS) activity in the nucleus. In this study, to understand the mechanism of Adi3 CDS, peptide microarrays containing random Ser- and Thr-peptide phosphorylation substrates were used to screen for downstream phosphorylation substrates. In the microarray phosphorylation assay, Adi3 showed promiscuous kinase activity more toward Ser-peptides compared to Thr-peptides, and a preference for aromatic and cyclic amino acids on both Ser- and Thr-peptides was seen. The 63 highest phosphorylated peptide sequences from the Ser-peptide microarray were selected as queries for a BLAST search against the tomato proteome. As a result, 294 candidate nuclear Adi3 substrates were selected and categorized based on their functions. Many of these proteins were classified as DNA/RNA polymerases or regulators involved in transcription and translation events. The list of potential Adi3 substrates was narrowed to eleven and four candidates were tested for phosphorylation by Adi3. Two of these candidates, RNA polymerase II 2nd largest subunit (RPB2) and the pathogen defense related transcription factor Pti5, were confirmed as Adi3 phosphorylation substrates by in vitro kinase assays. Using a mutational approach two residues, Thr675 and Thr676, were identified as Adi3 phosphorylation sites on RPB2. This study provides the foundation for understanding Adi3 CDS mechanisms in the nucleus as well as other cellular functions.
Databáze: OpenAIRE
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