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A protein kinase associated with apoptosis and tumor suppression: structure, activity, and discovery of peptide
A V Velentza1, A M Schumacher, C Weiss
1Drug Discovery Program and Department of Molecular Pharmacology and Biological Chemistry, Northwestern University, Chicago, Illinois 60611, USA.
Abstract:
Death-associated protein kinase (DAPK) has been implicated in apoptosis and tumor suppression, depending on cellular conditions, and associated with mechanisms of disease. However, DAPK has not been characterized as an enzyme due to the lack of protein or peptide substrates. Therefore, we determined the structure of DAPK catalytic domain, used a homology model of docked peptide substrate, and synthesized positional scanning substrate libraries in order to discover peptide substrates with K(m) values in the desired 10 microm range and to obtain knowledge about the preferences of DAPK for phosphorylation site sequences. Mutagenesis of DAPK catalytic domain at amino acids conserved among protein kinases or unique to DAPK provided a link between structure and activity. An enzyme assay for DAPK was developed and used to measure activity in adult brain and monitor protein purification based on the physical and chemical properties of the open reading frame of the DAPK cDNA. The results allow insight into substrate preferences and regulation of DAPK, provide a foundation for proteomic investigations and inhibitor discovery, and demonstrate the utility of the experimental approach, which can be extended potentially to kinase open reading frames identified by genome sequencing projects or functional genetics screens and lacking a known substrate.
Insights
Researchers identified peptide substrates for Death-associated protein kinase (DAPK), a key enzyme in apoptosis and tumor suppression. This discovery advances understanding of DAPK
Area of Science:
- Biochemistry
- Molecular Biology
- Enzymology
Background:
- Death-associated protein kinase (DAPK) is linked to apoptosis and tumor suppression but lacks characterized enzyme activity due to unknown substrates.
- Understanding DAPK's enzymatic function is crucial for elucidating its role in disease mechanisms.
Purpose of the Study:
- To identify specific peptide substrates for DAPK.
- To determine DAPK's substrate specificity and preferences for phosphorylation sites.
- To develop an enzyme assay for DAPK activity.
Main Methods:
- Determined the structure of the DAPK catalytic domain.
- Utilized homology modeling for docked peptide substrates.
- Synthesized positional scanning substrate libraries to discover substrates.
- Performed site-directed mutagenesis on the DAPK catalytic domain.
- Developed and applied an enzyme assay for DAPK activity.
Main Results:
- Discovered peptide substrates with K(m) values around 10 micromolar for DAPK.
- Elucidated DAPK's preferences for specific phosphorylation site sequences.
- Established a structure-activity relationship through mutagenesis.
- Developed a functional enzyme assay for DAPK, enabling activity measurement in adult brain tissue and monitoring protein purification.
Conclusions:
- The study provides the first characterization of DAPK as an enzyme by identifying its peptide substrates.
- Findings offer insights into DAPK substrate preferences and regulation, laying groundwork for proteomic studies and inhibitor development.
- The experimental approach is applicable to other kinases lacking known substrates.