C-terminal 15 kDa fragment of cytoskeletal actin is posttranslationally N-myristoylated upon caspase-mediated cleavage and targeted to mitochondria.
To detect the posttranslational N-myristoylation of caspase substrates, the susceptibility of the newly exposed N-terminus of known caspase substrates to protein N-myristoylation was evaluated by in vivo metabolic labeling with [(3)H]myristic acid in transfected cells using a fusion protein in which the query sequence was fused to a model protein. As a result, it was found that the N-terminal nine residues of the newly exposed N-terminus of the caspase-cleavage product of cytoskeletal actin efficiently direct the protein N-myristoylation. Metabolic labeling of COS-1 cells transiently transfected with cDNA coding for full-length truncated actin (tActin) revealed the efficient incorporation of [(3)H]myristic acid into this molecule. When COS-1 cells transiently transfected with cDNA coding for full-length actin were treated with staurosporine, an apoptosis-inducing agent, an N-myristoylated tActin was generated. Immunofluorescence staining coupled with MitoTracker or fluorescence tagged-phalloidin staining revealed that exogenously expressed tActin colocalized with mitochondria without affecting cellular and actin morphology. Taken together, these results demonstrate that the C-terminal 15 kDa fragment of cytoskeletal actin is posttranslationally N-myristoylated upon caspase-mediated cleavage during apoptosis and targeted to mitochondria.
Utsumi
Toshihiko
T
Department of Biological Chemistry, Faculty of Agriculture, Yamaguchi University, Yamaguchi 753-8515, Japan. utsumi@agr.yamaguchi-u.ac.jp
Sakurai
Nagisa
N
Nakano
Kengo
K
Ishisaka
Rumi
R
D000199:Actins / Q000235:genetics / Q000378:metabolism*; D000595:Amino Acid Sequence; D000818:Animals; D017209:Apoptosis; D051023:BH3 Interacting Domain Death Agonist Protein; D019556:COS Cells; D002352:Carrier Proteins / Q000378:metabolism; D020169:Caspases / Q000378:metabolism; D002522:Chlorocebus aethiops; D018260:Gelsolin / Q000378:metabolism; D006801:Humans; D008928:Mitochondria / Q000378:metabolism*; D008969:Molecular Sequence Data; D019814:Myristic Acid / Q000378:metabolism*; D010446:Peptide Fragments; D010957:Plasmids; D011499:Protein Processing, Post-Translational; D021381:Protein Transport; D011993:Recombinant Fusion Proteins / Q000378:metabolism
D000199:Actins; D051023:BH3 Interacting Domain Death Agonist Protein; C494814:BID protein, human; D002352:Carrier Proteins; D018260:Gelsolin; D010446:Peptide Fragments; D011993:Recombinant Fusion Proteins; D019814:Myristic Acid; D020169:Caspases