title: An unbiased proteomic screen reveals caspase cleavage is positively and negatively regulated by substrate phosphorylation.
abstract: 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.
authors.affiliation: Department of Biochemistry, Schulich School of Medicine and Dentistry, Western University, London, Ontario N6A 5C1, Canada;
authors.forename: Jacob P
authors.initials: JP
authors.lastname: Turowec
authors.forename: Stephanie A
authors.initials: SA
authors.lastname: Zukowski
authors.forename: James D R
authors.initials: JD
authors.lastname: Knight
authors.forename: David M
authors.initials: DM
authors.lastname: Smalley
authors.forename: Lee M
authors.initials: LM
authors.lastname: Graves
authors.forename: Gary L
authors.initials: GL
authors.lastname: Johnson
authors.forename: Shawn S C
authors.initials: SS
authors.lastname: Li
authors.forename: Gilles A
authors.initials: GA
authors.lastname: Lajoie
authors.forename: David W
authors.initials: DW
authors.lastname: Litchfield
mesh_terms: D020169:Caspases / Q000737:chemistry* / Q000378:metabolism*; D006367:HeLa Cells; D006801:Humans; D007553:Isotope Labeling; D008565:Membrane Proteins / Q000378:metabolism; D008958:Models, Molecular; D010455:Peptides / Q000378:metabolism*; D010750:Phosphoproteins / Q000378:metabolism; D010766:Phosphorylation; D011499:Protein Processing, Post-Translational; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism; D059748:Proteolysis; D040901:Proteomics / Q000379:methods*; D000099317:Hippo Kinases
chemical_list: D020169:Caspases; D008565:Membrane Proteins; D010455:Peptides; D010750:Phosphoproteins; D017346:Protein Serine-Threonine Kinases; C109163:STK24 protein, human; D000099317:Hippo Kinases