DAP-kinase-mediated morphological changes are localization dependent and involve myosin-II phosphorylation

Area of Science:

• Cell Biology
• Molecular Biology
• Biochemistry

Background:

• Death-associated protein kinase (DAPk) is a Ser/Thr kinase involved in programmed cell death.
• Cytoskeletal dynamics play crucial roles in regulating cell morphology and apoptosis.

Purpose of the Study:

• To investigate the role of DAPk intracellular localization in mediating cytoskeletal rearrangements and cell morphology changes.
• To determine how different DAPk domains influence its interaction with cytoskeletal components and substrates.

Main Methods:

• Expression of GFP-tagged wild-type and mutant DAP-kinase constructs in cells.
• Analysis of cell morphology, membrane protrusions, and blebbing.
• Assessment of myosin-II distribution and regulatory light chain (RLC) phosphorylation.
• Investigation of DAPk interaction with actin stress fibers and focal contacts.

Main Results:

• Wild-type DAPk partially localized to actin stress fibers, inducing extensive membrane protrusions and increased phospho-RLC.
• A DAPk mutant lacking the cytoskeletal-interacting region showed diffuse localization, peripheral blebbing, and RLC phosphorylation.
• DAPk lacking ankyrin repeats mislocalized to focal contacts, causing no morphological changes.
• RLC phosphorylation by DAPk was independent of ROCK, but stress fiber-associated phospho-RLC increase required wild-type localization.

Conclusions:

• The intracellular localization of DAPk is critical for its function in regulating cell morphology.
• Specific DAPk localization determines its access to substrates like RLC, leading to distinct cellular responses.
• DAPk's interaction with the actin cytoskeleton mediates significant morphologic alterations crucial for cellular processes.