Tandem reporter assay for myristoylated proteins post‐translationally (TRAMPP) identifies novel substrates for post‐translational myristoylation: PKC∊, 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.

Post-translational myristoylation at the cross roads of cell death, autophagy and neurodegeneration

“…Of note, blocking caspase cleavage at D586 completely reverses the disease phenotype in the YAC128 HD mouse model [24]. In addition, we have shown that HTT is PTMyr at G553 following caspase cleavage at D552 [6,25] (Figure 2A). …”

“…It has also become apparent that caspases are required for multiple processes including cell survival, proliferation and differentiation [16,17], suggesting that PTMyr is not restricted to just apoptosis, but may be a constitutive process that provides a dual function and an additional layer of regulation for proteins following caspase cleavage during various cell pathways. This is supported by the fact that the overexpression of myristoylated C-terminal-truncated (myr-ct) gelsolin and myr-ctPKCε are implicated in cell survival [6,18] compared with myr-ctBID and myr-ctPAK2 [1,7], which promote cell death. This is supported by the fact that the overexpression of myristoylated C-terminal-truncated (myr-ct) gelsolin and myr-ctPKCε are implicated in cell survival [6,18] compared with myr-ctBID and myr-ctPAK2 [1,7], which promote cell death.…”

“…In fact, since the discovery of BID as a substrate of PTMyr many more proteins have been identified using in silico prediction analysis combined with overexpression systems [4][5][6]. Until recently, only BID [1] and p21activated kinase 2 (PAK2) [7] had been shown to be PTMyr in vivo.…”

A single gene connects stiffness in glaucoma and the vascular system

“…3). Because the half-life/photostability of the reporter proteins is 24–48 h for lacZ and 1 h for tdTomato (Martin et al, 2012; Smith et al, 1995), the protein marker expression observed in these studies represents the true transcription of the gene.…”

Regulation of co‐ and post‐translational myristoylation of proteins during apoptosis: interplay ofN‐myristoyltransferases and caspases

“…For many years, myristoylation was thought to occur only cotranslationally, until the C‐terminal fragment of caspase‐truncated Bid (ctBid) was demonstrated to be post‐translationally myristoylated during apoptosis (10). Since then, work done by the laboratories of Utsumi (11, 12) and ours (13, 14) have identified the caspase‐cleaved C‐terminal products of actin (11), gelsolin (12), p21‐activated kinase 2 (PAK2) (ref. 13), and PKCε (14) to be post‐translationally myristoylated.…”