C-terminal 15 kDa fragment of cytoskeletal actin is posttranslationally N-myristoylated upon caspase-mediated cleavage and targeted to mitochondria

• Published in: FEBS letters Vol. 539; no. 1; pp. 37 - 44
• Main Authors: Utsumi, Toshihiko, Sakurai, Nagisa, Nakano, Kengo, Ishisaka, Rumi
• Format: Journal Article
• Language: English
• Published: England Elsevier B.V 27.03.2003
• Subjects: Actin; Actins - genetics; Actins - metabolism; Amino Acid Sequence; Animals; Apoptosis; BH3 Interacting Domain Death Agonist Protein; Carrier Proteins - metabolism; Caspase substrate; Caspases - metabolism; Cercopithecus aethiops; COS Cells; DPBS, Dulbecco's phosphate-buffered saline; Gelsolin - metabolism; Humans; Mitochondria - metabolism; Molecular Sequence Data; Myristic Acid - metabolism; NMT, N-myristoyltransferase; PAGE, polyacrylamide gel electrophoresis; PCR, polymerase chain reaction; Peptide Fragments; Plasmids; Posttranslational modification; Posttranslational N-myristoylation; Protein acylation; Protein N-myristoylation; Protein Processing, Post-Translational; Protein Transport; Recombinant Fusion Proteins - metabolism; SDS, sodium dodecyl sulfate; tActin, truncated actin; tBID, truncated BID; TNF, tumor necrosis factor; tActin, truncated actin; TNF, tumor necrosis factor; Protein N-myristoylation; NMT, N-myristoyltransferase; SDS, sodium dodecyl sulfate; DPBS, Dulbecco’s phosphate-buffered saline; Posttranslational modification; Caspase substrate; Actin; PAGE, polyacrylamide gel electrophoresis; Posttranslational N-myristoylation; Protein acylation; PCR, polymerase chain reaction; tBID, truncated BID
• ISSN: 0014-5793; 1873-3468
• DOI: 10.1016/S0014-5793(03)00180-7

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 [ 3H]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 [ 3H]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.