A PEF/Y Substrate Recognition and Signature Motif Plays a Critical Role in DAPK-Related Kinase Activity

• DAPK1 is inhibited by an interaction of the PEF/Y motif and autoregulatory domain
• PEF/Y mutations abolish DAPK1 kinetic activity in vitro and in cellular assays
• The PEF/Y motif is specific to DAPK1-related CaM kinases and distinct AGC kinases

SummaryKnowledge about protein kinase substrate preferences is biased toward residues immediately adjacent to the site of phosphorylation. By a combined structural, biochemical, and cellular approach, we have discovered an unexpected substrate recognition element with the consensus sequence PEF/Y in the tumor suppressor death-associated protein kinase 1. This motif can be effectively blocked by a specific pseudosubstrate-type interaction with an autoregulatory domain of this kinase. In this arrangement, the central PEF/Y glutamate interacts with a conserved arginine distant to the phosphorylation site in sequence and structure. We also demonstrate that the element is crucial for kinase activity regulation and substrate recognition. The PEF/Y motif distinguishes close death-associated protein kinase relatives from canonical calcium/calmodulin-dependent protein kinases. Insight into this signature and mode of action offers new opportunities to identify specific small molecule inhibitors in PEF/Y-containing protein kinases.

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