The potential for crosstalk between phosphorylation and proteolytic pathways in apoptosis and other cell biological processes has long been recognized (Kurokawa and Kornbluth, 2009
; Lopez-Otin and Hunter, 2010
); however, investigating such network interactions at a global level has proven technically challenging due to the lack of proteomic technologies that can coordinately profile protein phosphorylation and proteolysis in cells. qP-PROTOMAP addresses this problem by quantifying phosphorylation events in proteomes and incorporating these modifications into the topographical maps of proteins such that their relationship to proteolytic processing can be directly inferred. Using this approach, we have uncovered several ways that phosphorylation and proteolytic pathways intersect in apoptotic cells. This crosstalk is evident on a global level by the enrichment of phosphorylation events on proteolyzed proteins at locations that are in close proximity to caspase cleavage sites. From a functional perspective, we show that caspase cleavage can unveil new sites for phosphorylation on proteins and, surprisingly, apoptosis-specific phosphorylation events at the P3 position of caspase recognition sites can directly promote the cleavage of proteins (). To our knowledge, neither of these forms of crosstalk between caspases and kinases has been reported previously. Caspase cleavage can also activate kinases, like DNA-PK, that contribute to the creation of a network of phosphorylation events that are specific to apoptotic cells (). This network is enriched in phosphorylation events that lack literature precedent, further supporting their potentially special relationship to the cell death process.
Mechanisms of crosstalk between phosphorylation and proteolytic pathways in apoptosis
While we do not yet understand precisely how caspase cleavage promotes the phosphorylation of proteins, it is possible that the kinases responsible for these phosphorylation events cannot gain access to their substrates due to steric hindrance. Caspase cleavage at a proximal location along the protein backbone could then relieve this steric blockade to expose sites for phosphorylation (). Alternatively, there may be kinases that selectively phosphorylate proteins near their N- or C-termini, although we are not aware of any specific kinases that have been reported to show this substrate preference. Finally, it is possible that cleavage promotes the redistribution of kinases like DNA-PK to distinct subcellular compartments where they phosphorylate new sets of substrates.
Phosphorylation events that promote proteolysis were found to occur at the P3 position relative to caspase cleavage sites, where they dramatically enhanced substrate hydrolysis by caspase-8. This finding is unexpected and important because phosphorylation events within caspase consensus motifs (P4-P1’ residues) have, in the past, been exclusively found to hinder caspase cleavage (Kurokawa and Kornbluth, 2009
). Our results are, however, consistent with previous structural work on caspases, which have shown that caspase-8, as well as caspase-9, possess a unique arginine residue not found in other caspases that enhances binding to substrates with acidic residues in the P3 position (Blanchard et al., 2000
; Chereau et al., 2003
; Fuentes-Prior and Salvesen, 2004
). This feature has historically been interpreted to explain the preference that caspase-8 displays for substrates with a P3 glutamic acid residue (Fuentes-Prior and Salvesen, 2004
), but our data suggest another level of biological significance, namely, that caspase-8 may have evolved to recognize a set of substrates selectively in their phosphorylated state. We should mention, however, that so far, we have only assessed the impact of P3-phosphorylation on a handful of caspase substrates, and it is therefore not yet clear whether P3-phosphorylation will serve as a general or substrate-selective mechanism to enhance proteolysis by caspase-8.
The intricate, systems-level interactions between kinase and caspase networks uncovered by qP-PROTOMAP analysis of apoptotic cells sets the stage for several important lines of future research. First, we have only examined one cell line (Jurkat T-cells) and its response to a single apoptotic stimulus (STS). While the rapid and near-complete apoptotic progression observed in STS-treated Jurkat cells has made it a preferred model for cell biological and proteomic investigations of programmed cell death (Dix et al., 2008
; Mahrus et al., 2008
; Short et al., 2007
), and a recent study has shown that different apoptotic stimuli (STS versus TRAIL) cause similar overall patterns of protein cleavage in cells (Agard et al., 2012
), assessing the broader significance of our findings would certainly benefit from qP-PROTOMAP studies of additional cell types and with distinct apoptotic stimuli. Second, we do not yet fully understand which kinases are responsible for the phosphorylation events observed specifically in apoptotic cells. While our results indicate that DNA-PK makes a substantial contribution to this apoptosis-specific phosphorylation network, many of its constituent phosphorylation events do not conform to the [S/T]-Q motif preferred by DNA-PK (Figure S2
), pointing to the potential activation of other kinases (or inactivation of phosphatases) during apoptosis. Our functional proteomic data suggest candidates like AKT1 and 2, MAPK14, and BRAF for future investigation (). Disrupting such kinases could reveal the functional contribution that they (and their cognate substrates) make to apoptosis, as has been shown previously for DNA-PK (Bharti et al., 1998
; Chakravarthy et al., 1999
; Chen et al., 2005a
; Wang et al., 2000
). Finally, there are other potential forms of crosstalk between phosphorylation and proteolytic pathways that may have eluded detection in our study. Phosphorylation events that, for instance, block caspase cleavage would not have been easily identified because the resulting phosphoprotein would not be detected as a cleaved product. Future studies that compare the apoptotic process under different cellular conditions may reveal context-dependent changes in protein cleavage that are due to such “protective” phosphorylation events. In fact, others have speculated, for instance, that cancer cells displaying resistance to apoptosis may possess specific kinase networks that mark proteins with phosphorylation events that protect against caspase cleavage (Ahmed et al., 2002
). The qP-PROTOMAP method described herein represents a versatile proteomic platform for addressing such questions through its ability to generate global, quantitative, and integrated profiles of phosphorylation and proteolytic pathways in biological systems.