Abstract
SummaryThe 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.
Highlights
The human kinome comprises more than 500 protein kinases, around 15% of which are predicted to be regulated by calcium/ calmodulin (Ca2+/CaM) binding (Manning et al, 2002)
We used the calcium/calmodulindependent members of the death-associated protein kinase (DAPK) family to investigate the role of a basic DAPK signature loop near the kinase active site
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
Summary
The human kinome comprises more than 500 protein kinases, around 15% of which are predicted to be regulated by calcium/ calmodulin (Ca2+/CaM) binding (Manning et al, 2002). Numerous structural studies of kinase ARD peptide-Ca2+/CaM complexes illustrate how both CaM lobes wrap an extended helical ARD segment that is structurally autonomous of the neighboring CD (Dagher et al, 2011; Kuczera and Kursula, 2012). This conclusion was confirmed by a structure of the prototypic calcium/calmodulin-dependent protein kinase II (CaMKII)Ca2+/CaM complex, in which the ARD-Ca2+/CaM-binding module is entirely separate from the CaMKII CD (PDB: 2WEL) (Rellos et al, 2010). The goal of this study was to investigate Ca2+/CaM binding to DAPKs to unravel the mechanism of activity regulation by Ca2+/ CaM binding
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