PerspectiveCell Biology

Protein Kinases Curb Cell Death

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Sci. Signal.  10 May 2011:
Vol. 4, Issue 172, pp. pe26
DOI: 10.1126/scisignal.2001921

Abstract

Networks of aspartic acid–directed caspases play a major role in the execution of programmed cell death. Studies have provided evidence that caspases or their substrates are subjected to phosphorylation, which suggests a potential convergence of protein kinase and caspase signaling pathways. Various caspase substrates, but also several procaspases, are protected from cleavage when they are phosphorylated at sites adjacent to caspase cleavage sites. Whereas many distinct protein kinases could potentially protect substrates from caspase-mediated cleavage, a study has identified protein kinase CK2 as the most prominent kinase that exerts a global inhibition of caspase signaling pathways. CK2 is a component of protein kinase networks that are involved in tumors derived from various tissues. Its dysregulation in many cancers, together with its dual function in promoting cell growth and in suppressing apoptosis, is particularly relevant to its oncogenic potential. Thus, this study suggests that the ability of CK2 to contribute to tumorigenesis resides, at least in part, in its ability to phosphorylate caspases or their targets.

In every organ, the number of cells and their functional quality must be strictly controlled through a balance of cell-fate decisions (self-renewal, differentiation, migration, and death), which are governed by the complex interplay between extracellular and intracellular signals.

In 1972, the term “apoptosis” was introduced to describe a stereotypical set of cellular changes that are observed during cell death in various situations, ranging from development to stress responses (1). Since this pioneering observation, it has been established that a family of cysteine-dependent aspartic acid–directed proteases called caspases plays a major role in this cell death pathway through the proteolytic cleavage of various cellular proteins that are required for cell survival. These enzymes, which are synthesized as proenzymes, are organized into cascades that define two pathways involved in the execution of apoptosis. The first, the extrinsic pathway, is triggered upon ligation of the death receptors, such as CD95 (also known as Apo-1 and Fas) and the tumor necrosis factor receptor (2, 3), which subsequently stimulate initiator procaspases that interact with adaptor molecules to form the death-inducing signaling complex. The second pathway, the intrinsic pathway, requires the critical contribution of mitochondrial-derived death amplification factors of the Bcl-2 family, such as the proapoptotic proteins Bax, Bak, and Bid, which modify the mitochondrial outer membrane potential, thus enabling the cytosolic translocation of cytochrome C. Finally, effector caspases are activated by being cleaved by upstream initiator caspases, which, in turn, are able to cleave a number of cellular proteins.

In most cancers, perturbations of the delicate balance of cell-fate decisions are the result of positive and negative external influences that lead to the activation of oncogenic signals and the repression of death-signaling pathways. Failure to undergo apoptotic cell death in response to appropriate signals may result in cancer and resistance to chemotherapy. Accordingly, caspase-regulated events have emerged as attractive prospects for therapeutic intervention (4). In addition to caspases, cells also contain networks of protein kinases that play a major role in the regulation of cell survival through their involvement in the control of the stability and localization of key signaling proteins. Furthermore, the realization that alterations to protein kinases and protein kinase–mediated signal transduction events are linked to several forms of cancers has also propelled these enzymes onto the stage as promising therapeutic targets (5). Thus, it is of paramount importance that two crucial processes such as proteolysis (an irreversible process) and protein phosphorylation (a reversible covalent modification) act in a concerted way (611). In this respect, the emerging picture shows that a number of proteins experience changes in their susceptibility to cleavage by caspases when they undergo phosphorylation at a site proximal to the caspase-recognition sequence. Consequently, a convergence between multiple protein kinases and caspase signaling networks is of particular importance in the context in which abnormally active, cancer-associated kinases promote the enhanced survival of tumor cells (5).

Studies investigating the sequence requirements for the recognition by caspases of their substrates identified a stringent specificity for an aspartic acid (Asp) residue within the cleavage site (12). In addition, the presence of bulky or charged residues adjacent to the Asp residue of position P1′ is poorly tolerated in caspase catalysis. In a landmark study, Tözsér et al. showed that the phosphorylation of caspase substrates by protein kinases at or near this cleavage site inhibits their ability to be cleaved by caspases. In this study, phosphorylation of peptides representing known caspase-cleavage sites inhibited their cleavage by effector caspases (7). Later, several groups provided additional evidence that protein kinase–dependent phosphorylation of caspases, their substrates, or both may inhibit caspase-mediated cleavage, highlighting the convergence of multiple protein kinases in the regulation of caspase signaling pathways (6, 8, 10, 1315).

A study by Duncan and colleagues now provides new insights into the functional interplay between caspase and protein kinase signaling pathways (16). Consensus sequences can serve as a guide for the prediction of substrates for individual caspases. Therefore, the authors searched for protein sequences in the human proteome that contained overlapping recognition motifs for kinases and caspases. To investigate whether phosphorylation of protein targets conferred protection from caspase-3–mediated cleavage, the authors selected 10 protein kinases implicated in survival and tumorigenesis. The consensus phosphorylation sites for each kinase and the corresponding overlapping caspase-3 target sequence were used in a peptide match program to screen the human proteome. Intriguingly, the protein kinase CK2 (also known as casein kinase 2) emerged from this analysis as a prevalent protein kinase that exhibited the most overlapping consensus phosphorylation sites within a cleavage recognition motif for caspase-3. Because acidic residues represent specificity determinants for CK2 and caspases have stringent specificity for an aspartic acid within the cleavage site, this finding could explain the tendency of CK2 to recognize caspase-3 recognition sites. Peptides corresponding to known caspase substrates were susceptible to caspase-mediated cleavage, and peptides containing a putative CK2 and caspase target consensus sequence were efficiently phos­phorylated by CK2 (16).

With a fluorescence-based assay, Duncan et al. investigated whether the candidate peptides phosphorylated by CK2 were also cleaved by caspases. Indeed, peptides phosphorylated at the P2 or the P1′ residues were protected from caspase-mediated cleavage. Importantly, this analysis also revealed that procaspase-3, which must be cleaved by caspase-8 to become activated, was a candidate substrate of CK2. Kinase assays showed that procaspase-3 was phosphoryl­ated on Thr174 and Ser176 by CK2 and that this phosphorylation resulted in the protection of procaspase-3 from caspase-8– and caspase-9–mediated cleavage in vitro (16). Furthermore, CK2 and procaspase-3 physically associated with each other in cells, providing evidence of a role for CK2 in the progression of apoptosis through caspase-3 signaling. Finally, the authors demonstrated that a reduction in the activity of CK2 in cells [by chemical inhibition or RNA interference (RNAi)–mediated knockdown] facilitated the generation of active caspase-3. This is consistent with a role for CK2 in the prevention of caspase-3 activation and the protection of targets of caspase-3 from cleavage. Overlap with the targets of CK2 was also observed for caspase-3, -7, -8, and -9, suggesting a global protective effect of CK2 for various caspase targets. Overall, this study by Duncan et al. provides solid evidence that CK2 could have an important role in regulating cell survival through its widespread ability to modulate the sensitivities of proteins to caspases (16).

Despite substantial advances in cancer management, the efficacy of therapeutic drugs is often compromised by the development of resistance to chemotherapy; however, a notable conceptual advance was achieved with the realization that the inefficient execution of signals regulating the cell death machinery is paramount to an effective response of cancer cells to drug-induced apoptosis. Increasing evidence indicates that CK2 is a component of protein kinase networks that control several aspects of transformation and cancer. Dysregulated CK2 can promote tumorigenesis, because its abundance and activity are enhanced in human tumors and transformed cell lines. Furthermore, enhanced CK2 abundance is an unfavorable prognostic marker in lung cancer (17), acute myeloid leukemia (18), and prostate adenocarcinoma (19), and it is correlated with the risk of metastasis in breast carcinoma (20). Whereas a precise understanding of the regulation of CK2 in cells remains incomplete, recent evidence suggests that CK2 also promotes cell survival. The emerging picture is that this kinase, by cooperating with other survival-promoting pathways, is central to many of the molecular switches that protect the cell against stress (21).

An important task for the future is to delineate the mechanism by which CK2 functions, especially how it relates to the delivery of survival signals that contribute to tumorigenesis. In this respect, inhibition of CK2 activity by RNAi or pharmacological inhibitors compromises cell survival, rendering cancer cells sensitive to various apoptotic stimuli, including radiotherapy, chemotherapy, and death receptor ligands (2224). Alternatively, overexpression of CK2 attenuates apoptotic cell death induced by chemotherapeutic drugs (25, 26). Collectively, these studies support the notion that CK2 mediates its cancer-promoting effect, at least in part, by inhibiting apoptotic signals. This idea has found support with the observation that the amounts of a number of key signaling proteins that are involved in apoptotic pathways increase in response to overexpression of CK2 or are subjected to CK2-mediated inactivation, resulting in the resistance of cancer cells to drug-induced apoptosis (25, 27, 28). Survivin, a protein inhibitor of apoptosis, is increased in abundance when CK2 is overexpressed (27). Upon phosphorylation by CK2, other proteins—such as Max, Bid, PTEN, and the connexin channel—are protected from caspase-mediated cleavage (Fig. 1A) (2831). The catalytic activity of caspases is under the control of caspase-inhibiting proteins such as apoptosis repressor with caspase recruitment domain (ARC), which binds to and inhibits the activation of caspase-8, thereby inhibiting apoptosis induced by various stimuli. ARC binds to caspase-8 only when ARC is localized to mitochondria, which occurs when it is phosphorylated by CK2 at Thr149 (32).

Fig. 1

Regulation of caspases and caspase substrates by the protein kinase CK2. (A) Caspases are synthesized as procaspases and organized into cascades. Procaspases must first be cleaved by upstream caspases to be activated. Phosphorylation of procaspases by CK2 on a serine or threonine residue adjacent to the aspartic acid residue of the caspase cleavage site inhibits their activation. (B) Caspase-mediated cleavage of key proteins leads to apoptosis; however, phosphorylation by CK2 of a serine or threonine residue adjacent to the aspartic acid of the cleavage site effectively blocks cleavage, enabling cell survival. α, catalytic subunit; β, regulatory subunit; P, phosphate group.

CREDIT: Y. HAMMOND/SCIENCE SIGNALING

In addition to regulating substrates or protein inhibitors of caspases, CK2 also directly regulates the activity of caspases themselves (Fig. 1B). Caspase-9 is protected from caspase-8–mediated cleavage by being phosphorylated by CK2 (15), whereas, by phosphorylating caspase-2, CK2 prevents its dimerization-dependent activation (33). Similarly, phosphorylation of procaspase-3 by CK2 protects it from cleavage by caspase-8 and -9 (16). Clearly, CK2 has a profound impact on the catalytic activity of caspases and on their substrates, thereby contributing to the resistance of cancer cells to drug-induced apoptosis. CK2 phosphoryl­ation consensus sites were also identified in other caspase family members (16). This raises several questions about the interplay between CK2 and caspases. First, is CK2 playing the role of a common inhibitor of the activation of every procaspase? If so, this would suggest a widespread protective effect of CK2 that could provide the cell with a very efficient brake on caspase signaling pathways. Observations showing that inhibition of CK2 activity is sufficient to restore the susceptibility of cells to apoptotic stimuli are consistent with this notion (34). However, the convergence between multiple protein kinase and caspase pathways in the regulation of apoptosis suggests that CK2 is not unique in its regulation of caspase signaling. With the emergence of global phosphoproteomic studies, additional proteins with phosphorylation sites that overlap with caspase cleavage sites will undoubtedly be discovered. Second, what are the upstream signals that target CK2 to specific components of the caspase cascade? As a signaling molecule, CK2 is a mobile enzyme that could be targeted to different cellular compartments in response to different stimuli (35). In this scenario, stress-induced signaling pathways could trigger a subpopulation of CK2 to colocalize with the apoptotic machinery. Third, the inefficient execution of signals stimulating the cell death machinery was observed in cells with abnormally high amounts of CK2. Thus, what is the threshold amount of CK2 protein or extent of activity that is necessary to curb caspase-mediated apoptosis? Although CK2 supports multiple key prosurvival signaling pathways, future experiments that address these questions will improve our understanding of the “supervision” of apoptotic pathways that is performed by CK2. Caspase-regulated events are attractive prospects for therapeutic intervention (4). This notion can now be extended to CK2, because this kinase is a potential target in cancer, and new drugs targeting CK2 are entering the clinic. This is illustrated by the recent launching of the first orally bioavailable CK2 inhibitor, which is currently in clinical trials for the treatment of cancer (36).

References and Notes

  1. Funding: Studies on CK2 in our laboratory are supported by the Institut National de la Santé et de la Recherche Médicale (INSERM), the Centre National pour la Recherche Scientifique (CNRS), the Commissariat à l’Energie atomique (CEA), the Institut Curie, the Ligue Nationale Contre le Cancer (équipe labellisée 2010), and the Institut National du Cancer (grant no. 57).
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