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 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.Apoptosis is a cell death program integral to various biological processes such as tissue homeostasis and development (1). The ability of cancer cells to evade apoptosis is considered a driving feature that imparts a selective cellular advantage allowing cells to persist inappropriately (2). A major component of apoptotic evasion in cancer arises from the misregulation of two enzyme classes, protein kinases and caspases. Kinases transfer the γ-phosphate from ATP to proteins to alter substrate function, and caspases act as executioners of the apoptotic program by facilitating the demolition of cellular constituents by cleaving key structural and enzymatic proteins (3, 4). Attenuation of caspase activity arising through kinase-mediated post-translational modifications or genetic mutations or deletions can contribute to malignant phenotypes by blocking apoptotic progression (5, 6).Interestingly, numerous examples have implicated cross-talk between caspases and kinases as a major apoptotic regulatory mechanism, and anecdotal examples have been identified in which phosphorylation at P4, P2, and P1′ (see Fig. 1A for cleavage site nomenclature) has been shown to block cleavage and affect cellular phenotypes (6–12). Accordingly, phosphorylation-dependent regulation of caspase-mediated cleavage has been hypothesized as a global regulator of apoptotic progression, especially in the context of cancer, where hyperactive, oncogenic kinases may act to increase phosphosite occupancy within caspase cleavage motifs (7). Indeed, we previously tested this hypothesis using predictive peptide match programs and identified CK2 phosphorylation sites on caspase-3 that regulated its activation by caspase-8 and -9 (13).Open in a separate windowFig. 1.Workflow for the global, unbiased analysis of the integration of phosphorylation and caspase-mediated degradation. A, illustration of the cleavage site nomenclature for proteases. Caspases cleave the scissile bond between a P1 aspartic acid and the P1′ residue. B, HeLa cell lysates were treated with or without λ phosphatase and subjected to caspase treatment followed by dephosphorylation of the sample previously left phosphorylated. Primary amines on protein N termini and lysine residues were dimethylated using heavy (+34, open circles) or light (+28, black circles) formaldehyde. Samples were pooled and trypsinized, which exposed an amine on the N terminus of the internal tryptic peptide. These peptides are captured through reaction with an ∼80-kDa aldehyde-substituted polymer. Importantly, native protein N termini and neo-N termini generated by caspase cleavage are resistant to reaction with the polymer because their reactive amines have been blocked by dimethylation. Enrichment of the N-terminome then occurs via negative selection when the reacted polymer is filtered away using a 10-kDa cut-off spin column. LC-MS/MS analysis of isotopically dimethylated peptides then allows comparative analysis between caspase degradomes of phosphorylated and dephosphorylated lysates. Caspase substrates will be inferred through identification of those peptides with a P1 aspartic acid. In the event that there is no difference in caspase substrate proteolysis between phosphorylated and dephosphorylated samples, a peptide ratio of ∼1:1 will be observed in MS1 1]. Of interest are those peptide pairs that deviate from a 1:1 ratio 2].To build on our predictive strategy, we devised an unbiased, proteomic methodology to identify novel proteins for which phosphorylation regulates cleavage via caspases. We measured the caspase degradome in the context of a native phosphoproteome and compared it to the caspase degradome generated from lysates formerly dephosphorylated with λ bacteriophage phosphatase. To identify these events, we utilized the N-terminomic workflow TAILS¹ (terminal amino isotopic labeling of substrates) (14). Comparative analysis of the caspase degradomes from phosphorylated and dephosphorylated lysates revealed Yap1 and Golgin-160 as caspase substrates negatively regulated by phosphorylation.Surprisingly, we also identified a number of caspase substrates for which cleavage is promoted by phosphorylation, and during the course of our study, Dix et al. (15) demonstrated that phosphorylation at P3 can promote the cleavage of caspase peptide substrates. Our proteomic screen highlighted MST3 as a caspase substrate positively regulated by phosphorylation; however, in contrast to results obtained for MST3 protein in lysates, phosphorylation exerted a negative influence on the cleavage of an MST3 peptide, as was the case for other peptides modeled after Yap1 and Golgin-160. Collectively, these data suggest that although inhibitory effects of phosphorylation can arise through phosphorylation of residues proximal to the cleavage site, the positive effect of phosphorylation may stem from determinants other than those near the scissile bond. Subsequently, to test the effect of phosphorylation throughout the entirety of the caspase motif, we systematically walked phosphoserine through the length of model caspase-3, -7, and -8 substrate peptides and found that phosphorylation was generally inhibitory to caspase cleavage. Again, these observations suggest that positive effects of phosphorylation on the caspase cleavage of proteins observed in lysates likely arise through modulated ternary protein structure. Overall, our studies demonstrate that N-terminomics approaches can be tailored to identify novel, hierarchical events controlling the cleavage of caspase substrates.