An Unbiased Proteomic Screen Reveals Caspase Cleavage Is Positively and Negatively Regulated by Substrate Phosphorylation

Post-translational modifications of proteins regulate diverse cellular functions, with mounting evidence suggesting that hierarchical cross-talk between distinct modifications may fine-tune cellular responses. For example, in apoptosis, caspases promote cell death via cleavage of key structural and...

Full description

Saved in:
Bibliographic Details
Published in:Molecular & cellular proteomics 2014-05, Vol.13 (5), p.1184-1197
Main Authors: Turowec, Jacob P., Zukowski, Stephanie A., Knight, James D.R., Smalley, David M., Graves, Lee M., Johnson, Gary L., Li, Shawn S.C., Lajoie, Gilles A., Litchfield, David W.
Format: Article
Language:English
Subjects:
Caspases - chemistry
Caspases - metabolism
HeLa Cells
Humans
Isotope Labeling
Membrane Proteins - metabolism
Models, Molecular
Peptides - metabolism
Phosphoproteins - metabolism
Phosphorylation
Protein Processing, Post-Translational
Protein-Serine-Threonine Kinases - metabolism
Proteolysis
Proteomics - methods
Citations: Items that this one cites
Items that cite this one
Online Access:Get full text
Tags: Add Tag
No Tags, Be the first to tag this record!
Description
Summary:Post-translational modifications of proteins regulate diverse cellular functions, with mounting evidence suggesting that hierarchical cross-talk between distinct modifications may fine-tune cellular responses. For example, in apoptosis, caspases promote cell death via cleavage of key structural and enzymatic proteins that in some instances is inhibited by phosphorylation near the scissile bond. In this study, we systematically investigated how protein phosphorylation affects susceptibility to caspase cleavage using an N-terminomic strategy, namely, a modified terminal amino isotopic labeling of substrates (TAILS) workflow, to identify proteins for which caspase-catalyzed cleavage is modulated by phosphatase treatment. We validated the effects of phosphorylation on three of the identified proteins and found that Yap1 and Golgin-160 exhibit decreased cleavage when phosphorylated, whereas cleavage of MST3 was promoted by phosphorylation. Furthermore, using synthetic peptides we systematically examined the influence of phosphoserine throughout the entirety of caspase-3, -7, and -8 recognition motifs and observed a general inhibitory effect of phosphorylation even at residues considered outside the classical consensus motif. Overall, our work demonstrates a role for phosphorylation in controlling caspase-mediated cleavage and shows that N-terminomic strategies can be tailored to study cross-talk between phosphorylation and proteolysis.
ISSN:1535-9476
1535-9484
DOI:10.1074/mcp.M113.037374