Myristoylation, the addition of a 14-carbon
fatty acid to the N-terminal
glycine of a
protein, is key to
protein-membrane and
protein-
protein interactions. Typically, myristoylation occurs cotranslationally; however, post-translational myristoylation of
caspase-cleaved
proteins is now emerging as a well-established
protein modification and as a novel regulator of apoptosis. To identify additional post-translationally myristoylated
proteins, we engineered a plasmid vector encoding for a
caspase-cleavable reporter
protein named tandem reporter assay for myristoylation of
proteins post-translationally (TRAMPP). pTRAMPP consists of
tdTomato-DEVD-"test myristoylation sequence"-
enhanced green fluorescent protein (EGFP). After induction of apoptosis, the reporter
protein is cleaved by
caspases, which frees a new N-terminal
glycine residue attached to EGFP that can be myristoylated. We used pTRAMPP in appropriately transfected cells to identify 7 post-translationally myristoylated
proteins. First, we confirmed the post-translational myristoylation of two previously identified putative substrates,
cytoplasmic dynein intermediate chain 2A and PKCε (ctPKCε), and identified 5 more
caspase-cleaved potential substrates for myristoylation that include the antiapoptotic regulator of apoptosis, Mcl-1, and the causative agent of
Huntington's disease,
huntingtin protein. Further investigation revealed that post-translationally myristoylated ctPKCε localized to membranes and increased Erk signaling and degradation of the proapoptotic
protein Bim, which prevented a significant loss of mitochondrial potential of 17% over nonmyristoylated ctPKCε in HeLa cells in the presence of apoptotic stimuli. Taken together, these findings suggest a possible antiapoptotic role for post-translationally myristoylated
caspase-cleaved ctPKCε.