Site-directed mutagenesis of the glycine-rich loop of death associated protein kinase (DAPK) identifies it as a key structure for catalytic activity.

Death associated protein kinase (DAPK) is a calmodulin (CaM)-regulated protein kinase that is a therapeutic target for central nervous system (CNS) disorders. We report here the results of studies that test the hypothesis of McNamara et al. (2009) that conformational selection in DAPK's glycine-rich region is key for catalytic activity. The hypothesis was tested by site-directed mutagenesis of glutamine-23 (Q23) in the middle of this loop. The glycine-rich loop exhibits localized differences in structure among DAPK conformations that correlate with different stages of the catalytic cycle. Changing the Q23 to a Valine (V23), found at the corresponding position in another CaM regulated protein kinase, results in a reduced catalytic efficiency. High resolution X-ray crystal structures of various conformations of the Q23V mutant DAPK and their superimposition with the corresponding conformations from wild type catalytic domain reveal localized changes in the glycine-rich region. The effect of the mutation on DAPK catalytic activity and the finding of only localized changes in the DAPK structure provide experimental evidence implicating conformational selection in this domain with activity. This article is part of a Special Issue entitled: 11th European Symposium on Calcium.

Homodimerization of the death-associated protein kinase catalytic domain: development of a new small molecule fluorescent reporter.

BACKGROUND: Death-Associated Protein Kinase (DAPK) is a member of the Ca2+/calmodulin regulated serine/threonine protein kinases. Its biological function has been associated with induced cell death, and in vivo use of selective small molecule inhibitors of DAPK catalytic activity has demonstrated that it is a potential therapeutic target for treatment of brain injuries and neurodegenerative diseases. METHODOLOGY/PRINCIPAL FINDINGS: In the in vitro study presented here, we describe the homodimerization of DAPK catalytic domain and the crucial role played by its basic loop structure that is part of the molecular fingerprint of death protein kinases. Nanoelectrospray ionization mass spectrometry of DAPK catalytic domain and a basic loop mutant DAPK protein performed under a variety of conditions was used to detect the monomer-dimer interchange. A chemical biological approach was used to find a fluorescent probe that allowed us to follow the oligomerization state of the protein in solution. CONCLUSIONS/SIGNIFICANCE: The use of this combined biophysical and chemical biology approach facilitated the elucidation of a monomer-dimer equilibrium in which the basic loop plays a key role, as well as an apparent allosteric conformational change reported by the fluorescent probe that is independent of the basic loop structure.

Structural insight into nucleotide recognition by human death-associated protein kinase.

Death-associated protein kinase (DAPK) is a member of the Ca(2+)/calmodulin-regulated family of serine/threonine protein kinases. The role of the kinase activity of DAPK in eukaryotic cell apoptosis and the ability of bioavailable DAPK inhibitors to rescue neuronal death after brain injury have made it a drug-discovery target for neurodegenerative disorders. In order to understand the recognition of nucleotides by DAPK and to gain insight into DAPK catalysis, the crystal structure of human DAPK was solved in complex with ADP and Mg(2+) at 1.85 A resolution. ADP is a product of the kinase reaction and product release is considered to be the rate-limiting step of protein kinase catalytic cycles. The structure of DAPK-ADP-Mg(2+) was compared with a newly determined DAPK-AMP-PNP-Mg(2+) structure and the previously determined apo DAPK structure (PDB code 1jks). The comparison shows that nucleotide-induced changes are localized to the glycine-rich loop region of DAPK.

A novel p38 alpha MAPK inhibitor suppresses brain proinflammatory cytokine up-regulation and attenuates synaptic dysfunction and behavioral deficits in an Alzheimer's disease mouse model.

BACKGROUND: An accumulating body of evidence is consistent with the hypothesis that excessive or prolonged increases in proinflammatory cytokine production by activated glia is a contributor to the progression of pathophysiology that is causally linked to synaptic dysfunction and hippocampal behavior deficits in neurodegenerative diseases such as Alzheimer's disease (AD). This raises the opportunity for the development of new classes of potentially disease-modifying therapeutics. A logical candidate CNS target is p38 alpha MAPK, a well-established drug discovery molecular target for altering proinflammatory cytokine cascades in peripheral tissue disorders. Activated p38 MAPK is seen in human AD brain tissue and in AD-relevant animal models, and cell culture studies strongly implicate p38 MAPK in the increased production of proinflammatory cytokines by glia activated with human amyloid-beta (A beta) and other disease-relevant stressors. However, the vast majority of small molecule drugs do not have sufficient penetrance of the blood-brain barrier to allow their use as in vivo research tools or as therapeutics for neurodegenerative disorders. The goal of this study was to test the hypothesis that brain p38 alpha MAPK is a potential in vivo target for orally bioavailable, small molecules capable of suppressing excessive cytokine production by activated glia back towards homeostasis, allowing an improvement in neurologic outcomes. METHODS: A novel synthetic small molecule based on a molecular scaffold used previously was designed, synthesized, and subjected to analyses to demonstrate its potential in vivo bioavailability, metabolic stability, safety and brain uptake. Testing for in vivo efficacy used an AD-relevant mouse model. RESULTS: A novel, CNS-penetrant, non-toxic, orally bioavailable, small molecule inhibitor of p38 alpha MAPK (MW01-2-069A-SRM) was developed. Oral administration of the compound at a low dose (2.5 mg/kg) resulted in attenuation of excessive proinflammatory cytokine production in the hippocampus back towards normal in the animal model. Animals with attenuated cytokine production had reductions in synaptic dysfunction and hippocampus-dependent behavioral deficits. CONCLUSION: The p38 alpha MAPK pathway is quantitatively important in the A beta-induced production of proinflammatory cytokines in hippocampus, and brain p38 alpha MAPK is a viable molecular target for future development of potential disease-modifying therapeutics in AD and related neurodegenerative disorders.