Death-Associated Protein Kinase Activity Is Regulated by Coupled Calcium/Calmodulin Binding to Two Distinct Sites

Summary The regulation of many protein kinases by binding to calcium/calmodulin connects two principal mechanisms in signaling processes: protein phosphorylation and responses to dose- and time-dependent calcium signals. We used the calcium/calmodulin-dependent members of the death-associated protein kinase (DAPK) family to investigate the role of a basic DAPK signature loop near the kinase active site. In DAPK2, this loop comprises a novel dimerization-regulated calcium/calmodulin-binding site, in addition to a well-established calcium/calmodulin site in the C-terminal autoregulatory domain. Unexpectedly, impairment of the basic loop interaction site completely abolishes calcium/calmodulin binding and DAPK2 activity is reduced to a residual level, indicative of coupled binding to the two sites. This contrasts with the generally accepted view that kinase calcium/calmodulin interactions are autonomous of the kinase catalytic domain. Our data establish an intricate model of multi-step kinase activation and expand our understanding of how calcium binding connects with other mechanisms involved in kinase activity regulation.
Graphical Abstract
Highlights • Members of the DAPK family share a specific basic-loop-mediated dimerization motif • DAPK2 contains a kinase-mediated and dimerization-regulated CaM-binding site • Autoregulatory segment CaM binding depends on kinase-mediated CaM binding • DAPK2 activity is regulated by coupled CaM binding to two different sites
Simon et al. show that human death-associated protein kinase activity is regulated by coupled binding of calcium/calmodulin to two distinct sites, a novel kinase-mediated site and an autoregulatory segment-mediated site, and thus establish a new link to the role of calcium in kinase signaling.