Tandem reporter assay for myristoylated proteins post-translationally (TRAMPP) identifies novel substrates for post-translational myristoylation: PKCε, a case study.
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| Abstract |
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.
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| Authors | Dale D O Martin, Chrisselle Y Ahpin, Ryan J Heit, Maneka A Perinpanayagam, Megan C Yap, Richard A Veldhoen, Ing Swie Goping, Luc G Berthiaume |
| Journal | FASEB journal : official publication of the Federation of American Societies for Experimental Biology (FASEB J) Vol. 26 Issue 1 Pg. 13-28 (Jan 2012) ISSN: 1530-6860 [Electronic] United States |
| PMID | 21965604 (Publication Type: Journal Article, Research Support, Non-U.S. Gov't) |
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