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

  • Jacob P. Turowec
    Affiliations
    From the Department of Biochemistry, Schulich School of Medicine and Dentistry, Western University, London, Ontario N6A 5C1, Canada;
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  • Stephanie A. Zukowski
    Affiliations
    From the Department of Biochemistry, Schulich School of Medicine and Dentistry, Western University, London, Ontario N6A 5C1, Canada;
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  • James D.R. Knight
    Affiliations
    Samuel Lunenfeld Research Institute, Mount Sinai Hospital, Toronto, Ontario M5G 1X5, Canada;
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  • David M. Smalley
    Affiliations
    Department of Pharmacology, The University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27514
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  • Lee M. Graves
    Affiliations
    Department of Pharmacology, The University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27514
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  • Gary L. Johnson
    Affiliations
    Department of Pharmacology, The University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27514
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  • Shawn S.C. Li
    Affiliations
    From the Department of Biochemistry, Schulich School of Medicine and Dentistry, Western University, London, Ontario N6A 5C1, Canada;
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  • Gilles A. Lajoie
    Affiliations
    From the Department of Biochemistry, Schulich School of Medicine and Dentistry, Western University, London, Ontario N6A 5C1, Canada;
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  • David W. Litchfield
    Affiliations
    From the Department of Biochemistry, Schulich School of Medicine and Dentistry, Western University, London, Ontario N6A 5C1, Canada;
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  • Author Footnotes
    * J.P.T. was supported by a Doctoral Banting and Best Canada Graduate Scholarship and a Canada Graduate Scholarship Michael Smith Foreign Study Supplement. J.D.R.K. was supported by a post-doctoral fellowship from the Heart and Stroke foundation of Canada. S.A.Z. was supported by the CIHR Strategic Training Program in Cancer Research and Technology Transfer and an Ontario Graduate Scholarship. This work was funded by grants from the Canadian Institutes of Health Research (to D.W.L.), the National Sciences and Engineering Research Council (to G.A.L.), the Canadian Cancer Society (to S.S.L.), the National Institutes of Health (Grant No. GM101141), and the University Cancer Research Fund (UNC-CH) (to G.L.J. and L.M.G.). The UNC Michael Hooker Proteomics Center was supported by the NCI, National Institutes of Health (Grant No. CA016086).
    This article contains supplemental material.
Open AccessPublished:February 20, 2014DOI:https://doi.org/10.1074/mcp.M113.037374
      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 (
      • Cohen G.M.
      Caspases: the executioners of apoptosis.
      ). The ability of cancer cells to evade apoptosis is considered a driving feature that imparts a selective cellular advantage allowing cells to persist inappropriately (
      • Hanahan D.
      • Weinberg R.A.
      The hallmarks of cancer.
      ). 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 (
      • Hunter T.
      Signaling—2000 and beyond.
      ,
      • Taylor R.C.
      • Cullen S.P.
      • Martin S.J.
      Apoptosis: controlled demolition at the cellular level.
      ). 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 (
      • Olsson M.
      • Zhivotovsky B.
      Caspases and cancer.
      ,
      • Kurokawa M.
      • Kornbluth S.
      Caspases and kinases in a death grip.
      ).
      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 (
      • Kurokawa M.
      • Kornbluth S.
      Caspases and kinases in a death grip.
      ,
      • Duncan J.S.
      • Turowec J.P.
      • Vilk G.
      • Li S.S.
      • Gloor G.B.
      • Litchfield D.W.
      Regulation of cell proliferation and survival: convergence of protein kinases and caspases.
      ,
      • Tozser J.
      • Bagossi P.
      • Zahuczky G.
      • Specht S.I.
      • Majerova E.
      • Copeland T.D.
      Effect of caspase cleavage-site phosphorylation on proteolysis.
      ,
      • Desagher S.
      • Osen-Sand A.
      • Montessuit S.
      • Magnenat E.
      • Vilbois F.
      • Hochmann A.
      • Journot L.
      • Antonsson B.
      • Martinou J.C.
      Phosphorylation of bid by casein kinases I and II regulates its cleavage by caspase 8.
      ,
      • Walter J.
      • Schindzielorz A.
      • Grunberg J.
      • Haass C.
      Phosphorylation of presenilin-2 regulates its cleavage by caspases and retards progression of apoptosis.
      ,
      • Walter J.
      • Grunberg J.
      • Schindzielorz A.
      • Haass C.
      Proteolytic fragments of the Alzheimer's disease associated presenilins-1 and -2 are phosphorylated in vivo by distinct cellular mechanisms.
      ,
      • Hu Y.
      • Yao J.
      • Liu Z.
      • Liu X.
      • Fu H.
      • Ye K.
      Akt phosphorylates acinus and inhibits its proteolytic cleavage, preventing chromatin condensation.
      ). 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 (
      • Duncan J.S.
      • Turowec J.P.
      • Vilk G.
      • Li S.S.
      • Gloor G.B.
      • Litchfield D.W.
      Regulation of cell proliferation and survival: convergence of protein kinases and caspases.
      ). 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 (
      • Duncan J.S.
      • Turowec J.P.
      • Duncan K.E.
      • Vilk G.
      • Wu C.
      • Luscher B.
      • Li S.S.
      • Gloor G.B.
      • Litchfield D.W.
      A peptide-based target screen implicates the protein kinase CK2 in the global regulation of caspase signaling.
      ).
      Figure thumbnail gr1
      Fig. 1Workflow 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 TAILS
      The abbreviations used are: TAILS, terminal amino isotopic labeling of substrates; caspase, cysteine-dependent aspartate-directed protease; λ phosphatase, phosphatase derived from bacteriophage λ; DNP, 2,4-dinitrophenol; PARP1, poly(ADP-ribose) polymerase.
      1The abbreviations used are: TAILS, terminal amino isotopic labeling of substrates; caspase, cysteine-dependent aspartate-directed protease; λ phosphatase, phosphatase derived from bacteriophage λ; DNP, 2,4-dinitrophenol; PARP1, poly(ADP-ribose) polymerase.
      (terminal amino isotopic labeling of substrates) (
      • Kleifeld O.
      • Doucet A.
      • auf dem Keller U.
      • Prudova A.
      • Schilling O.
      • Kainthan R.K.
      • Starr A.E.
      • Foster L.J.
      • Kizhakkedathu J.N.
      • Overall C.M.
      Isotopic labeling of terminal amines in complex samples identifies protein N-termini and protease cleavage products.
      ). 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. (
      • Dix M.M.
      • Simon G.M.
      • Wang C.
      • Okerberg E.
      • Patricelli M.P.
      • Cravatt B.F.
      Functional interplay between caspase cleavage and phosphorylation sculpts the apoptotic proteome.
      ) 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.

      DISCUSSION

      The convergence of caspase and kinase signaling has recently been described as a major mechanism in the regulation of apoptosis (
      • Kurokawa M.
      • Kornbluth S.
      Caspases and kinases in a death grip.
      ,
      • Duncan J.S.
      • Turowec J.P.
      • Vilk G.
      • Li S.S.
      • Gloor G.B.
      • Litchfield D.W.
      Regulation of cell proliferation and survival: convergence of protein kinases and caspases.
      ). We utilized a TAILS N-terminomic strategy to monitor alterations in the caspase degradome of HeLa extracts by comparing the native phosphoproteome to lysates formerly dephosphorylated with λ phosphatase. Through this novel approach we identified caspase-mediated proteolytic events that were both positively and negatively regulated by phosphorylation. We identified and validated three proteins from our screen; Yap1 and Golgin-160 were degraded less when phosphorylated, whereas phosphorylation promoted cleavage of MST3. Approximately one in three identified caspase substrates exhibited an effect of phosphorylation on cleavage (based on proteins for which negative effects of phosphorylation were equal to or greater than that of Yap1 or positive effects of phosphorylation were equal to or greater than that of MST3). In accordance with the work of Dix et al. (
      • Dix M.M.
      • Simon G.M.
      • Wang C.
      • Okerberg E.
      • Patricelli M.P.
      • Cravatt B.F.
      Functional interplay between caspase cleavage and phosphorylation sculpts the apoptotic proteome.
      ), our data suggest that the majority of proteins for which phosphorylation has an effect on cleavage undergo more extensive cleavage when phosphorylated. In fact, our TAILS analysis revealed that proteins in which phosphorylation appeared to promote cleavage outnumbered proteins in which phosphorylation prevented cleavage by more than 3-fold (15 proteins with more cleavage than MST3 in phosphorylated lysates versus 4 proteins with less cleavage than Yap1 in phosphorylated lysates). Overall, these data demonstrate the potential for widespread influence of phosphorylation on caspase cleavage.
      One benefit of our experimental design was that it enabled us to monitor the effects of phosphorylation on cleavage irrespective of its distance from the scissile bond—an important consideration, as there is no way to predict how phosphorylation sites outside the caspase motif might affect the proteolysis of proteins. Importantly, our peptide cleavage assays revealed that phosphoserine within, or just proximal to, the canonical caspase consensus motif is primarily inhibitory. Taken together with the observation that MST3 proteolysis is promoted by phosphorylation, this supports a model by which substrate phosphorylation can promote caspase motif accessibility or structure when occurring outside of the context of the motif itself. One potential limitation of our approach relates to the high stoichiometry of caspase-site phosphorylation required in order for a difference in susceptibility to be measurable. In an effort to overcome this limitation, we treated cells with okadaic acid to preserve phosphosite occupancy. Similarly, the effect of phosphorylation on cleavage is not necessarily absolute, such as at P3′, for example. To avoid complete digestion of proteins with phosphorylation sites at these residues, we used two different caspase concentrations. Despite these caveats, we were able to uncover a number of modulated caspase substrates and validated Yap1, MST3, and Golgin-160 as caspase targets that experience altered degradation in response to lysate dephosphorylation.
      Yap1 and Golgin-160 have phosphoacceptor residues at positions that, when occupied with phosphate, were inhibitory toward the cleavage of peptide substrates. Although the phosphorylation status of P4 serine on Golgin-160 and P1′ threonine on Yap1 has not been confirmed in phospho-proteomics studies, their occupancy would be consistent with cleavage patterns we observed on peptide substrates and in lysate cleavage assays. At this point, we cannot rule out that remote phosphorylation sites may contribute to reduced susceptibility to proteolysis in a manner similar to how phosphorylation at S422 on Acinus-S reduces cleavage at D355 (
      • Hu Y.
      • Yao J.
      • Liu Z.
      • Liu X.
      • Fu H.
      • Ye K.
      Akt phosphorylates acinus and inhibits its proteolytic cleavage, preventing chromatin condensation.
      ). Interestingly, Golgin-160, like other members of the Golgi complex, is dismantled via caspase cleavage early in the apoptotic response (
      • Mukherjee S.
      • Chiu R.
      • Leung S.M.
      • Shields D.
      Fragmentation of the Golgi apparatus: an early apoptotic event independent of the cytoskeleton.
      ). Golgin-160 is cleaved at D59, D139, and D311—the site we identified—with purified caspases and in cells after treatment with diverse apoptotic stimuli (
      • Mancini M.
      • Machamer C.E.
      • Roy S.
      • Nicholson D.W.
      • Thornberry N.A.
      • Casciola-Rosen L.A.
      • Rosen A.
      Caspase-2 is localized at the Golgi complex and cleaves golgin-160 during apoptosis.
      ). In our validation studies, we inferred via differential migration of Golgin-160 cleavage products that two sites (D59 and D139) were cleaved in phosphorylated lysates but that proteolysis at SEVD311 was blocked by phosphorylation. Interestingly, in response to apoptotic signals initiating from death receptors or endoplasmic reticulum stress, noncleavable forms of Golgin-160 can actually block apoptotic progression (
      • Maag R.S.
      • Mancini M.
      • Rosen A.
      • Machamer C.E.
      Caspase-resistant Golgin-160 disrupts apoptosis induced by secretory pathway stress and ligation of death receptors.
      ), though the effect of blocking each caspase site in isolation remains unknown. Yap1 is another caspase target that we validated for which phosphorylation inhibits caspase cleavage. DEMD424 has not previously been identified as a caspase cleavage site, but ASTD111 is cleaved in Jurkat cells in response to apoptotic induction by TRAIL and staurosporine, perhaps explaining why we observed multiple cleavage products in our validation studies (
      • Crawford E.D.
      • Seaman J.E.
      • Agard N.
      • Hsu G.W.
      • Julien O.
      • Mahrus S.
      • Nguyen H.
      • Shimbo K.
      • Yoshihara H.A.
      • Zhuang M.
      • Chalkley R.J.
      • Wells J.A.
      The DegraBase: a database of proteolysis in healthy and apoptotic human cells.
      ). Furthermore, P3 and P2 of ASTD111 have previously been identified as phosphorylated in cells (
      • Gnad F.
      • Gunawardena J.
      • Mann M.
      PHOSIDA 2011: the posttranslational modification database.
      ), perhaps clarifying results from our Western blots that indicated multiple cleavage products were differentially regulated between the phosphorylated and phosphatase-treated lysates. In terms of functionality, Yap1 is a transcriptional effector of the MST1 and MST2 kinases (
      • Hong W.
      • Guan K.L.
      The YAP and TAZ transcription co-activators: key downstream effectors of the mammalian Hippo pathway.
      ), which are also targeted by caspases and function in apoptotic progression (
      • Graves J.D.
      • Gotoh Y.
      • Draves K.E.
      • Ambrose D.
      • Han D.K.
      • Wright M.
      • Chernoff J.
      • Clark E.A.
      • Krebs E.G.
      Caspase-mediated activation and induction of apoptosis by the mammalian Ste20-like kinase Mst1.
      ,
      • Lee K.K.
      • Ohyama T.
      • Yajima N.
      • Tsubuki S.
      • Yonehara S.
      MST, a physiological caspase substrate, highly sensitizes apoptosis both upstream and downstream of caspase activation.
      ). Interestingly, MST1 and MST2 contain previously identified phosphorylation sites at P1′ and P3′, suggesting that caspase cleavage of multiple constituents of the same pathway is hierarchically regulated by phosphorylation (
      • Turowec J.P.
      • Duncan J.S.
      • Gloor G.B.
      • Litchfield D.W.
      Regulation of caspase pathways by protein kinase CK2: identification of proteins with overlapping CK2 and caspase consensus motifs.
      ). As well, MST1 phosphorylation by Akt at T387, a site distant from the scissile bond, blocks cleavage (
      • Jang S.W.
      • Yang S.J.
      • Srinivasan S.
      • Ye K.
      Akt phosphorylates MstI and prevents its proteolytic activation, blocking FOXO3 phosphorylation and nuclear translocation.
      ), further implicating the control of proteolytic processing of this pathway as an important signaling event.
      In the case of MST3, treating cells with the phosphatase inhibitor calyculin A promotes phosphorylation, activation, and structural reordering that alters its interaction partners (
      • Fuller S.J.
      • McGuffin L.J.
      • Marshall A.K.
      • Giraldo A.
      • Pikkarainen S.
      • Clerk A.
      • Sugden P.H.
      A novel non-canonical mechanism of regulation of MST3 (mammalian Sterile20-related kinase 3).
      ). One interpretation of our data supports a model whereby MST3 is preferentially cleaved in its active form because our N-terminomic strategies used lysates from cells treated with okadaic acid, a phosphatase inhibitor that, like calyculin A, inhibits PP1 and PP2 (
      • Fuller S.J.
      • McGuffin L.J.
      • Marshall A.K.
      • Giraldo A.
      • Pikkarainen S.
      • Clerk A.
      • Sugden P.H.
      A novel non-canonical mechanism of regulation of MST3 (mammalian Sterile20-related kinase 3).
      ). As well, MST3 is a pro-apoptotic kinase that exhibits increases in activity after the removal of its C terminus by caspase cleavage (
      • Huang C.Y.
      • Wu Y.M.
      • Hsu C.Y.
      • Lee W.S.
      • Lai M.D.
      • Lu T.J.
      • Huang C.L.
      • Leu T.H.
      • Shih H.M.
      • Fang H.I.
      • Robinson D.R.
      • Kung H.J.
      • Yuan C.J.
      Caspase activation of mammalian sterile 20-like kinase 3 (Mst3). Nuclear translocation and induction of apoptosis.
      ). A function of this hypothetical hierarchical layer of regulation is that phosphorylation and cleavage could provide graded levels of MST3 activity.
      As a logical extension of the unbiased investigation of the effect of phosphorylation on caspase-mediated degradation in the proteome, we were also interested in systematically evaluating the determinant properties of phosphorylated residues in the context of model substrate peptides. Given the size and charge of phosphoryl residues, we speculated that phosphorylation might have effects outside of the canonical P4–P1′ caspase motif. Along these lines, even unmodified amino acids have exhibited determinant properties at P6, P5, P2′, and P3′, and Dix et al. (
      • Dix M.M.
      • Simon G.M.
      • Wang C.
      • Okerberg E.
      • Patricelli M.P.
      • Cravatt B.F.
      Functional interplay between caspase cleavage and phosphorylation sculpts the apoptotic proteome.
      ) recently catalogued a large number of caspase sites with phosphorylated residues in an extended motif, though precise effects of phosphorylated residues at many positions remain unknown (
      • Demon D.
      • Van Damme P.
      • Vanden Berghe T.
      • Deceuninck A.
      • Van Durme J.
      • Verspurten J.
      • Helsens K.
      • Impens F.
      • Wejda M.
      • Schymkowitz J.
      • Rousseau F.
      • Madder A.
      • Vandekerckhove J.
      • Declercq W.
      • Gevaert K.
      • Vandenabeele P.
      Proteome-wide substrate analysis indicates substrate exclusion as a mechanism to generate caspase-7 versus caspase-3 specificity.
      ). In systematically analyzing the positional effect of phosphoserine on caspase cleavage, we found that phosphorylated residues outside the canonical caspase motif inhibited caspase-mediated cleavage. Prime side (C-terminal of the scissile bond) determinants inhibited cleavage as far away as four residues from the scissile bond, and the inhibitory capacity was inversely proportional to the distance from the catalytic machinery. This finding may be partly explained by previous observations made by Stennicke et al. (
      • Stennicke H.R.
      • Renatus M.
      • Meldal M.
      • Salvesen G.S.
      Internally quenched fluorescent peptide substrates disclose the subsite preferences of human caspases 1, 3, 6, 7 and 8.
      ), who showed that negative determinants at P1′ fail to stabilize the transition state, and therefore the bulky, highly charged phosphoserine might act similarly, but with diminishing effects when distanced from the catalytic machinery. There is also evidence of an S5 binding site (interacting with P5) on caspases in that P5 residues positively influence cleavage by caspase-3 (
      • Fu G.
      • Chumanevich A.A.
      • Agniswamy J.
      • Fang B.
      • Harrison R.W.
      • Weber I.T.
      Structural basis for executioner caspase recognition of P5 position in substrates.
      ) and direct selectivity between caspase-3 and caspase-7 (
      • Demon D.
      • Van Damme P.
      • Vanden Berghe T.
      • Deceuninck A.
      • Van Durme J.
      • Verspurten J.
      • Helsens K.
      • Impens F.
      • Wejda M.
      • Schymkowitz J.
      • Rousseau F.
      • Madder A.
      • Vandekerckhove J.
      • Declercq W.
      • Gevaert K.
      • Vandenabeele P.
      Proteome-wide substrate analysis indicates substrate exclusion as a mechanism to generate caspase-7 versus caspase-3 specificity.
      ). We investigated the effect of phosphorylation at this position, observing inhibitory effects consistent with a preference for small non-polar residues by caspase-3 (
      • Demon D.
      • Van Damme P.
      • Vanden Berghe T.
      • Deceuninck A.
      • Van Durme J.
      • Verspurten J.
      • Helsens K.
      • Impens F.
      • Wejda M.
      • Schymkowitz J.
      • Rousseau F.
      • Madder A.
      • Vandekerckhove J.
      • Declercq W.
      • Gevaert K.
      • Vandenabeele P.
      Proteome-wide substrate analysis indicates substrate exclusion as a mechanism to generate caspase-7 versus caspase-3 specificity.
      ). The determinant properties of P5 on caspase-7 are less obvious, making specific reasons for phospho-P5 difficult to reconcile (
      • Demon D.
      • Van Damme P.
      • Vanden Berghe T.
      • Deceuninck A.
      • Van Durme J.
      • Verspurten J.
      • Helsens K.
      • Impens F.
      • Wejda M.
      • Schymkowitz J.
      • Rousseau F.
      • Madder A.
      • Vandekerckhove J.
      • Declercq W.
      • Gevaert K.
      • Vandenabeele P.
      Proteome-wide substrate analysis indicates substrate exclusion as a mechanism to generate caspase-7 versus caspase-3 specificity.
      ). Interestingly, phosphoserine at P5 in the Golgin-160 series resulted in little change in cleavage, suggesting that the effects at this position might be context dependent.
      Inhibitory effects at P4, P2, and P1′ were consistent with the literature, but we did note a discrepancy at the P3 position. Dix et al. (
      • Dix M.M.
      • Simon G.M.
      • Wang C.
      • Okerberg E.
      • Patricelli M.P.
      • Cravatt B.F.
      Functional interplay between caspase cleavage and phosphorylation sculpts the apoptotic proteome.
      ) found that phosphorylated P3 on substrate peptides is strictly a positive factor for caspase-8 cleavage and either promotes or has no effect on caspase-3 mediated degradation, whereas, like Toszer et al. (
      • Tozser J.
      • Bagossi P.
      • Zahuczky G.
      • Specht S.I.
      • Majerova E.
      • Copeland T.D.
      Effect of caspase cleavage-site phosphorylation on proteolysis.
      ), we observed no effect at this site for both caspase-3 and caspase-8. We cannot definitively explain this discordance, but we can speculate that reaction conditions might have played a role. First, Dix et al. (
      • Dix M.M.
      • Simon G.M.
      • Wang C.
      • Okerberg E.
      • Patricelli M.P.
      • Cravatt B.F.
      Functional interplay between caspase cleavage and phosphorylation sculpts the apoptotic proteome.
      ) used enzyme:substrate ratios 10-fold to 100-fold greater than our own, and reaction times that were 20 times longer, suggesting that their enzymes were less stable. For caspase-8, we used the chaotropic agent sodium citrate, which promotes caspase activity by stabilizing the dimeric form (
      • Boatright K.M.
      • Renatus M.
      • Scott F.L.
      • Sperandio S.
      • Shin H.
      • Pedersen I.M.
      • Ricci J.E.
      • Edris W.A.
      • Sutherlin D.P.
      • Green D.R.
      • Salvesen G.S.
      A unified model for apical caspase activation.
      ). Collectively, it is possible that determinant properties of phosphorylated P3 might depend on reaction conditions that affect enzyme stability. Another possibility is that the results were affected by the substrate peptides used; Dix and colleagues (
      • Dix M.M.
      • Simon G.M.
      • Wang C.
      • Okerberg E.
      • Patricelli M.P.
      • Cravatt B.F.
      Functional interplay between caspase cleavage and phosphorylation sculpts the apoptotic proteome.
      ) tended toward longer peptides and those with sequences that differed from our model, suggesting that lengthier peptides or pairwise primary amino acid interactions could affect cleavage. At minimum, our mutual findings are consistent in that phosphorylated P3 did not universally inhibit cleavage.
      In accordance with the literature, phospho-P2 and phospho-P1′ were also inhibitory toward proteolysis (
      • Tozser J.
      • Bagossi P.
      • Zahuczky G.
      • Specht S.I.
      • Majerova E.
      • Copeland T.D.
      Effect of caspase cleavage-site phosphorylation on proteolysis.
      ,
      • Duncan J.S.
      • Turowec J.P.
      • Duncan K.E.
      • Vilk G.
      • Wu C.
      • Luscher B.
      • Li S.S.
      • Gloor G.B.
      • Litchfield D.W.
      A peptide-based target screen implicates the protein kinase CK2 in the global regulation of caspase signaling.
      ). Of note was the measureable activity of caspase-8 against these peptides, whereas activity of caspase-3 and caspase-7 was not detected under the conditions used. Furthermore, baseline levels of cleavage by caspase-8 for peptides phosphorylated at P2′ and P4 were also above background, perhaps suggesting greater tolerance against inhibitory effects of caspase-8 site phosphorylation. Along these lines, the only known caspase-8 protein substrates negatively regulated by phosphorylation are phosphorylated at two sites within the cleavage motif; phosphorylation at P2 and P1′ of procaspase-3 blocks its cleavage by caspase-8 (
      • Duncan J.S.
      • Turowec J.P.
      • Duncan K.E.
      • Vilk G.
      • Wu C.
      • Luscher B.
      • Li S.S.
      • Gloor G.B.
      • Litchfield D.W.
      A peptide-based target screen implicates the protein kinase CK2 in the global regulation of caspase signaling.
      ), and phosphorylated P2 and P2′ prevents cleavage of Bid (
      • Desagher S.
      • Osen-Sand A.
      • Montessuit S.
      • Magnenat E.
      • Vilbois F.
      • Hochmann A.
      • Journot L.
      • Antonsson B.
      • Martinou J.C.
      Phosphorylation of bid by casein kinases I and II regulates its cleavage by caspase 8.
      ). In this way, phosphorylation at multiple, slightly tolerated positions might function to grade cleavage and modulate signaling output.
      In summary, we used two strategies to elucidate the interplay between caspase substrate phosphorylation and proteolysis. We systematically characterized the primary sequence determinant properties of phosphoserine within peptide substrates, and we also utilized an unbiased, N-terminomic approach to monitor caspase substrates that were regulated by phosphorylation in the context of the proteome. Overall, we anticipate that efforts such as ours that clarify the hierarchical regulation of caspase substrate phosphorylation and cleavage will improve our understanding of apoptotic signaling and the regulatory mechanisms that control cell survival.

      Acknowledgments

      We thank Dr. Sean Cregan for providing access to a fluorescent plate reader; Dr. Huadong Liu for help with peptide synthesis; and Nathan Cox, Dennis Goldfarb, Paula Pittock, Dr. Chris Hughes, and past and present members of the Litchfield laboratory for helpful discussions.

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