GTP binding to the ROC domain of DAP-kinase regulates its function through intramolecular signalling

Area of Science:

• Molecular Biology
• Biochemistry
• Cell Signaling

Background:

• Death-associated protein kinase (DAPk) is a serine/threonine kinase implicated in apoptosis.
• Recent sequence homology suggested DAPk belongs to the ROCO protein family, known for containing ROC and COR domains.
• The functional role of the ROC domain in DAPk regulation remained unclear.

Purpose of the Study:

• To investigate the functional role of the ROC domain in Death-associated protein kinase (DAPk).
• To elucidate the mechanism by which GTP binding regulates DAPk activity.
• To characterize the intramolecular signaling pathway involving the ROC domain.

Main Methods:

• GTP binding assays to confirm ROC domain functionality.
• Kinase activity assays to measure catalytic function.
• Site-directed mutagenesis to identify key regulatory residues.
• Analysis of autophosphorylation sites.

Main Results:

• The ROC domain of DAPk binds GTP via a conserved P-loop motif.
• GTP binding to the ROC domain leads to homo-oligomerization.
• GTP binding negatively regulates DAPk catalytic activity.
• GTP binding enhances autophosphorylation at a distal site, suppressing kinase activity.

Conclusions:

• DAPk's ROC domain is a functional GTP-binding module that acts as a negative regulator.
• GTP binding to the ROC domain initiates an intramolecular signal transduction cascade.
• This mechanism involves inhibitory autophosphorylation at a distal site, demonstrating a novel mode of kinase regulation.