title: Tandem reporter assay for myristoylated proteins post-translationally (TRAMPP) identifies novel substrates for post-translational myristoylation: PKCε, a case study.
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.
authors.affiliation: Department of Cell Biology, University of Alberta, Edmonton, AB T6G 2H7, Canada. luc.berthiaume@ualberta.ca
authors.forename: Dale D O
authors.initials: DD
authors.lastname: Martin
authors.forename: Chrisselle Y
authors.initials: CY
authors.lastname: Ahpin
authors.forename: Ryan J
authors.initials: RJ
authors.lastname: Heit
authors.forename: Maneka A
authors.initials: MA
authors.lastname: Perinpanayagam
authors.forename: Megan C
authors.initials: MC
authors.lastname: Yap
authors.forename: Richard A
authors.initials: RA
authors.lastname: Veldhoen
authors.forename: Ing Swie
authors.initials: IS
authors.lastname: Goping
authors.forename: Luc G
authors.initials: LG
authors.lastname: Berthiaume
mesh_terms: D000818:Animals; D017209:Apoptosis / Q000502:physiology; D019556:COS Cells; D020169:Caspases / Q000378:metabolism; D002522:Chlorocebus aethiops; D003001:Cloning, Molecular / Q000379:methods*; D017930:Genes, Reporter / Q000235:genetics; D005822:Genetic Vectors / Q000235:genetics; D049452:Green Fluorescent Proteins / Q000235:genetics*; D006367:HeLa Cells; D006801:Humans; D019814:Myristic Acid / Q000378:metabolism*; D010957:Plasmids / Q000235:genetics*; D051744:Protein Kinase C-epsilon / Q000378:metabolism*; D011499:Protein Processing, Post-Translational / Q000235:genetics / Q000502:physiology*; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D015398:Signal Transduction / Q000502:physiology; D014162:Transfection / Q000379:methods; D054462:p21-Activated Kinases / Q000378:metabolism
chemical_list: D011994:Recombinant Proteins; C485184:enhanced green fluorescent protein; D019814:Myristic Acid; D049452:Green Fluorescent Proteins; C515907:PAK2 protein, human; D054462:p21-Activated Kinases; D051744:Protein Kinase C-epsilon; D020169:Caspases