Dual Site Phosphorylation of Caspase-7 by PAK2 Blocks Apoptotic Activity by Two Distinct Mechanisms

Eron, Scott J.; Raghupathi, Kishore; Hardy, James L.

doi:10.1016/j.str.2016.11.001

Cited by 35 publications

(40 citation statements)

References 64 publications

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“…There has been some suggestion in the literature that in vitro phosphorylation of kinase substrates sometimes differs from cellular phosphorylation (21,30,31). We have not previously observed irregular phosphorylation of caspase substrates by any of the kinases we have studied (19,32,60). Once again in this work, we found that in vitro phosphorylation by c-Abl accurately reflected the intracellular phosphorylation specificity we observed.…”

Section: Discussionsupporting

confidence: 80%

“…This fidelity between in vitro and cell-based observations is probably due to casp-9 being a direct substrate of c-Abl. Our data from multiple kinase-caspase pairs suggest that when the appropriate kinase is studied, in vitro and cellular phosphorylation patterns are conserved (19,32,60).…”

Section: Discussionmentioning

confidence: 84%

“…Phosphorylation of residues adjacent to or within the cleavage site(s) in the intersubunit linker has been shown to block linker cleavage, as observed in casp-3 (54), -8 (55,56), and -9 (10). Recently, phosphorylation of casp-7 at a prodomainadjacent Ser-30 was observed to block interaction with casp-9, leading to failure of casp-7 cleavage and activation (32). Phosphorylation also impacts the catalytic activity of mature caspases.…”

Section: Caspase-9 Phosphorylation By C-ablmentioning

confidence: 99%

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Active site–adjacent phosphorylation at Tyr-397 by c-Abl kinase inactivates caspase-9

Serrano¹,

Szydlo

Alfandari

et al. 2017

Journal of Biological Chemistry

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Caspase-9 (casp-9) is an initiator caspase and plays a central role in activating apoptotic cell death. Control of all caspases is tightly regulated by a series of phosphorylation events enacted by several different kinases. Caspase-9 is the most heavily phosphorylated of all caspases, with phosphorylation of at least 11 distinct residues in all three caspase-9 domains by nine kinases. Caspase-9 phosphorylation by the non-receptor tyrosine kinase c-Abl at Tyr-153 reportedly leads to caspase-9 activation. All other phosphorylation events on caspases have been shown to block proteolytic function by a number of mechanisms, so we sought to unravel the molecular mechanism of the putative caspase-9 activation by phosphorylation. Surprisingly, we observed no evidence for Tyr-153 phosphorylation of caspase-9 or in cells, suggesting that Tyr-153 is not phosphorylated by c-Abl. Instead, we identified a new site for c-Abl-mediated phosphorylation, Tyr-397. This residue is adjacent to the caspase-9 active site but, as a member of the second shell, not a residue that directly contacts substrate. Our results further indicate that Tyr-397 is the dominant site of c-Abl phosphorylation both and upon c-Abl activation in cells. Of note, phosphorylation at this site inhibits caspase-9 activity, and the bulk of the added phosphate moiety appeared to directly block substrate binding. c-Abl plays both proapoptotic and prosurvival roles, and our findings suggest that c-Abl's effects on caspase-9 activity promote the prosurvival mode.

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Section: Discussionsupporting

confidence: 80%

Section: Discussionmentioning

confidence: 84%

Section: Caspase-9 Phosphorylation By C-ablmentioning

confidence: 99%

See 1 more Smart Citation

Active site–adjacent phosphorylation at Tyr-397 by c-Abl kinase inactivates caspase-9

Serrano¹,

Szydlo

Alfandari

et al. 2017

Journal of Biological Chemistry

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“…Caspases have numerous substrates and often rely on exosites to maintain specificity. 41,42,55 It is possible that a zinc-binding site on caspase-3 could disrupt critical protein-protein interactions and limit substrate accessibility or even redirect hydrolysis towards particular proteins of interest.…”

Section: Discussionmentioning

confidence: 99%

Multiple Mechanisms of Zinc-Mediated Inhibition for the Apoptotic Caspases-3, -6, -7, and -8

et al. 2018

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Zinc is emerging as a widely used and important biological regulatory signal. Cellular zinc levels are tightly regulated by a complex array of zinc importer and exporters to control processes such as apoptotic cell death. While caspase inhibition by zinc has been reported previously, the reported inhibition constants were too weak to suggest a critical biological role for zinc-mediated inhibition. In this work we have adopted a method of assessing available zinc. This allowed assessment of the accurate inhibition constants for apoptotic caspases, caspase-3, -6, -7 and -8. Each of these caspases are inhibited by zinc at intracellular levels, however, with widely differing inhibition constants and different zinc binding stoichiometries. Caspase -3, -6 and -8 appear to be constitutively inhibited by typical zinc levels and this inhibition must be lifted to allow activation. The inhibition constant for caspase-7 (76 nM) is much weaker than for the other apoptotic caspases (2.6–6.9 nM) suggesting that caspase-7 is not inactivated by normal zinc concentrations but can be inhibited under conditions of zinc stress. Caspase-3, -7, and -8 were found to bind three, one, and two zincs respectively. In each of these caspases, zinc was present in the active site, in contrast to caspase-6, which binds one zinc allosterically. The most notable new mechanism to emerge from this work is for zinc-mediated inhibition of caspase-8. Zinc binds caspase-8 directly at the active site and at a second site. Zinc binding inhibits formation of the caspase-8 dimer, the activated form of the enzyme. Together these findings suggest that zinc plays a critical role in regulation of apoptosis by direct inactivation of caspases, in a manner that is unique for each caspase.

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“…The data reveal that, like caspase-3, the mutations at this conserved site in caspases-6 and -7 also had no significant effect on the activity of these enzymes (Table 1). We note that a recent study by Hardy and coworkers showed similar results for a comparable variant of caspase-7, that of T173E (34). In addition to introducing phospho-mimetic mutations, we removed the salt-bridge between E176 and R271' in caspase-7 by replacing E176 with glycine, either individually or in combination with the T173D phospho-mimetic.…”

Section: Evolutionary Conservation Of Helix-3 Cterminal Loopmentioning

confidence: 53%

Modifications to a common phosphorylation network provide individualized control in caspases

Thomas

Grinshpon

Swartz

et al. 2018

Journal of Biological Chemistry

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Caspase-3 activation and function has been well defined during programmed cell death, but caspase activity, at low levels, is also required for developmental processes such as lymphoid proliferation and erythroid differentiation.Post-translational modification of caspase-3 is one method used by cells to fine-tune activity below the threshold required for apoptosis, but the allosteric mechanism that reduces activity is unknown. Phosphorylation of caspase-3 at a conserved allosteric site by p38-MAPK promotes survival in human neutrophils, and the modification of the loop is thought to be a key regulator in many developmental processes. We utilized phylogenetic, structural, and biophysical studies to define the interaction networks that facilitate the allosteric mechanism in caspase-3. We show that, within the modified loop, S150 evolved with the apoptotic caspases, while T152 is a more recent evolutionary event in mammalian caspase-3. Substitutions at S150 result in a pH-dependent decrease in dimer stability, and localized changes in the modified loop propagate to the active site of the same protomer through a connecting surface helix. Likewise, a cluster of hydrophobic amino acids connects the conserved loop to the active site of the second protomer. The presence of T152 in the conserved loop introduces a "kill switch" in mammalian caspase-3 while the more ancient S150 reduces without abolishing enzyme activity. These data reveal how evolutionary changes in a conserved allosteric site result in both a common pathway for lowering activity during development as well as introducing a more recent cluster-specific switch to abolish activity.

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Dual Site Phosphorylation of Caspase-7 by PAK2 Blocks Apoptotic Activity by Two Distinct Mechanisms

Cited by 35 publications

References 64 publications

Active site–adjacent phosphorylation at Tyr-397 by c-Abl kinase inactivates caspase-9

Active site–adjacent phosphorylation at Tyr-397 by c-Abl kinase inactivates caspase-9

Multiple Mechanisms of Zinc-Mediated Inhibition for the Apoptotic Caspases-3, -6, -7, and -8

Modifications to a common phosphorylation network provide individualized control in caspases

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