Phosphorylation Regulates Caspases Using Diverse Mechanisms

Caspases and kinases are caught in a web of complex interplay, and the battle between them tips the scale toward cell death or survival. Each caspase is phosphorylated in different locations by different kinases, which leads to differential regulation for each caspase. For example, caspase‐9, an initiator of apoptosis, is extensively phosphorylated on all its domains by multiple kinases. In contrast, a single phosphorylation site has been identified in caspase‐6. Our work seeks to understand the molecular details and mechanisms of inhibition or activation by phosphorylation of various caspases. We have interrogated the reported phosphorylation sites in caspase‐3, ‐6, ‐7 and ‐9 using phosphomimetic mutagenesis to pinpoint individual residues that result in altered caspase activity when phosphorylated. For example, in caspase‐9, S183E behaves dramatically differently from the wild‐type enzyme and is catalytically incompetent even when expressed in its fully processed form. In vitro phosphorylation of caspase‐9 by Protein Kinase A (PKA) revealed that the phosphorylation of S183 is the predominant inhibitory event upon the action of this kinase, and functions by blocking substrate binding. Similarly, caspase‐6 is inhibited by Ark‐5 mediated phosphorylation at S257. Our x‐ray structure of S257D suggests that phosphorylation leads to alterations in the loops that form the active site bundle. To date, our findings suggest two major classes of effects of phosphorylation on caspase function: 1) inhibition by directly impacting substrate binding and 2) disrupting CARD domain:core domain interactions.