DAP-kinase-mediated morphological changes are localization dependent and involve myosin-II phosphorylation.
DAP-kinase (DAPk) is a Ser/Thr kinase that regulates cytoplasmic changes associated with programmed cell death. It is shown here that a GFP-DAPk fusion, which partially localized to actin stress fibers, induced extensive membrane protrusions. This phenotype correlated with changes in myosin-II distribution and with increased phosphorylation of the myosin-II regulatory light chain (RLC). A mutant lacking the cytoskeletal-interacting region (GFP-DAPkDeltaCyto) displayed diffuse cytoplasmic localization, and induced peripheral membrane blebbing, instead of the extensive protrusions. In contrast, deletion of the ankyrin repeats led to mislocalization of the kinase to focal contacts, where it failed to elicit any changes in cell morphology. While both wild-type DAPk and DAPkDeltaCyto induced RLC phosphorylation independently of the Rho-activated kinase ROCK, only the wild type led to increases in stress-fiber associated phospho-RLC. Thus, the precise intracellular localization of DAPk is critical for exposure to its substrates, including the RLC, which mediate varying morphologic changes.
Bialik
S
S
Department of Molecular Genetics, Weizmann Institute of Science, Rehovot, Israel.
Bresnick
A R
AR
Kimchi
A
A
D000199:Actins / Q000378:metabolism; D051017:Apoptosis Regulatory Proteins; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000235:genetics / Q000378:metabolism*; D003599:Cytoskeleton / Q000378:metabolism; D064550:Death-Associated Protein Kinases; D017930:Genes, Reporter; D006367:HeLa Cells; D006801:Humans; D024462:Myosin Type II / Q000378:metabolism*; D010766:Phosphorylation; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D017384:Sequence Deletion
D000199:Actins; D051017:Apoptosis Regulatory Proteins; D011993:Recombinant Fusion Proteins; D064550:Death-Associated Protein Kinases; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D024462:Myosin Type II