We developed an alternative and highly sensitive detection method to study the myristoylation of proteins to replace the time consuming, expensive, hazardous, and laborious labeling of cells with radioactive fatty acids. This alternative method takes advantage of the Staudinger ligation’s unique chemoselective reactivity that can covalently link alkyl azides, such as azidomyristate, to a tagged triarylphosphine via
an amide bond, thereby allowing specific probing of azidomyristoylated proteins within the cell (15
). Indeed, the fact that neither alkyl azides nor phosphines are found in the biological milieu and that they do not react with cellular nucleophiles at ambient temperatures makes them ideal for in vivo
labeling and subsequent tagging of post-translationally modified proteins (15
). Important to the biological study of myristoylation, 12-azidododecanoate is an alkyl azide analog of myristate that, like most alkyl azides, has been shown previously to be nontoxic to cells and animals (29
). Furthermore, 12-azidododecanoate also is efficiently converted to its azidomyristoyl-CoA derivative by fatty acyl CoA synthetase (29
), which is a prerequisite to its use by NMT.
Herein, we demonstrate that the isosteric analog of myristate (29
), azidomyristate (12-azidododecanoate) can be specifically incorporated in a co- or post-translational fashion via
an alkali-resistant amide bond at the N-terminal glycine of exogenously or endogenously expressed proteins and readily detected with the phosphine-biotin using neutravidin/ECL. Recently, Hang et al
) also demonstrated that a variety of azido-fatty acid analogs can be used as chemical probes to monitor protein fatty acylation using the Staudinger ligation, therefore establishing the proof-of-principle for such a methodology. Of relevance to our study, they also showed that 12-azidododecanoate (azidomyristate) is preferentially incorporated into proteins via
an amide bond (30
). In our study, we established the proof-of-principle in greater detail and exploited this new technique to demonstrate the existence of several post-translationally myristoylated proteins in Jurkat T cells undergoing apoptosis. Furthermore, we designed a strategy that allowed us to successfully identify new post-translationally myristoylated proteins.
Our strategy is based on several studies that showed that the first 10–15 N-terminal amino acids of a known myristoylated protein are sufficient to confer myristoylation when appended to reporter proteins such as tumor necrosis factor or GFP (2
). Since we showed that the myristoylation of short chimeric WT-ctPAK2-N15
-EGFP and WT-Yes-N11
-GFP can readily be detected using azidomyristate cell labeling and reaction with phosphine-biotin (), we first identified internal myristoylation sites adjacent to caspase cleavage sites using computational prediction analysis (23
) and, second, incorporated these predicted NMT substrate sequences at the N terminus of EGFP at the cDNA level (Supplemental Table 2
). Finally, using our nonradioactive azidomyristate labeling/phosphine-biotin-based detection method, we assessed the myristoylation status of the chimeric EGFP proteins transiently expressed in COS-7 cells. In doing so, we identified ctBap31-, ctCD-IC2-, ctGCLC-, ctMST3-, and ctPKCε-N11
-EGFP as potential new substrates for NMT and reconfirmed the myristoylation of ctPAK2-N11
-EGFP. In our assay, ctPKCζ-N11
-EGFP was not a substrate for NMT, confirming the results of Utsumi et al
) using tumor necrosis factor as a reporter protein and [3
H]myristate as the label.
-EGFP and ctPKCε-N11
-EGFP provided the strongest signal via
Western blot analysis and were almost completely excluded from the nucleus like ctPAK2-N11
-EGFP as visualized by confocal microscopy () and, therefore, are likely very efficiently myristoylated and represent strong candidates for post-translational myristoylation of their respective full-length proteins in apoptotic cells. In contrast, despite the significant incorporation of azidomyristate into ctBap31-, ctGCLC-, and ctMST3-N11
-EGFP as assessed by Western blot analysis, these chimeras did not show significant nuclear exclusion, suggesting that they are only partially myristoylated, although this may still be of significance inside cells. It is possible that their endogenous counterparts may act as more desirable substrates for NMT or that there may be some unknown interferences in our system because we were using reporter constructs, which required cotranslational myristoylation of chimeric reporters as a means to assess the myristoylation status of naturally post-translationally myristoylated proteins. Furthermore, there are two NMT isoforms (NMT-1 and NMT-2) expressed in mammalian cells, which differ primarily at their N termini. While the NMT-1 N terminus is thought to be responsible for ribosome interactions required for cotranslational myristoylation, NMT-2 is more cytosolic (5
). The two isoforms have both overlapping and nonover-lapping substrate specificities (33
). Presently, we do not know which isoform is required for post-translational myristoylation, but based on localization studies and recent evidence that NMT-2 interacts with caspase 3 and Bcl-2, we hypothesize that NMT-2 may be the primary enzyme involved in post-translational myristoylation (32
Like other previously identified post-translationally myristoylated proteins, the proteins we identified are kinases, proapoptotic proteins, or regulators of the cytoskeleton structure (9
). Like ctPAK2, caspase cleavage of PKCε results in the loss of the N-terminal regulatory domain to generate a constitutively active C-terminal kinase domain (35
). PKCε contains two caspase cleavage sites at Asp383 and Asp451. The former appears to be the primary site of cleavage and is located within the hinge domain, between the regulatory and kinase domains, whereas cleavage at the second site is delayed and is found within the catalytic domain (35
). PKCε is a calcium-independent and diacylglycerol (DAG)-dependent kinase and post-translational myristoylation of ctPKCε at the primary caspase cleavage site (Asp383) may substitute the DAG-binding domain (found in the N terminus) to relocate the kinase domain to a new site within the cell where it could phosphorylate and regulate the activity of specific proteins during apoptosis.
Interestingly and unlike PAK2 and PKCε, the C-terminal cleavage product of MST3 contains the regulatory domain of the protein (36
). Therefore, myristoylation of ctMST3 could relocalize its regulatory domain away from the catalytic domain, resulting in a constitutively active enzyme that could diffuse in the cytosol and translocate to the nucleus via
its nuclear localization sequence (36
), unlike constitutively active myr-ctPAK2, which was found primarily at the plasma membrane and at the surface of endosomes (9
). It is also possible that the two fragments remain associated, like cleaved Bid, and that the myristate provides a novel localization site for cleaved MST3 (10
). Similar to ctPAK2, MST3 regulates cell morphology but also plays a role in cell cycle progression (37
). In addition, overexpression of full-length MST3 or its N-terminal cleavage product is proapoptotic (36
). Although the myristoylation of ct-MST3-N11
-EGFP appeared to be partial, it may still be relevant and of significance inside cells.
CD-IC2 is required for the interaction between cytoplasmic dynein and dynactin through binding of p150Glued
its N terminus. Dynein and dynactin regulate the secretory and endocytic pathways and microtubule organization at interphase, and the ER serves as cargo for cytoplasmic dynein in Xenopus
egg extracts. During apoptosis, CD-IC2 is cleaved within the p150Glued
binding domain but remains associated with the heavy and intermediate light chains of dynein, probably via
its overlapping WD-40 repeats (38
). Very interestingly, light chain 8 of dynein associates with the proapoptotic protein Bcl-2 interacting mediator of cell death in normal cells, and during apoptosis this complex translocates to membranes of various organelles, including mitochondria, where it binds and inhibits BCL-2 or functional homologs (39
). It is possible that myr-ctCD-IC2 may be involved in this complex or required for a similar pathway to direct the progression of apoptosis.
Bap31 is an integral membrane protein of the ER and is thought to regulate the export of cellubrevin from the ER. During apoptosis, Bap31 forms a complex with caspase-8, BCL-2 or BCL-XL
, and what is thought to be a CED-4 homologue (40
). The ~20 kDa N-terminal fragment is a potent activator of apoptosis when overexpressed in cells. Interestingly, the C-terminal cleavage product, which has not been studied to the extent of the N-terminal fragment, contains overlapping leucine zippers and a weak homology death-effector domain that has been shown to interact with exogenously expressed CED-4 in mammalian cells, which, in turn, interacts with caspase-8 (41
). Again, these protein-binding domains may be required for complete and proper localization of the myr-ctBap31 protein. Like myr-ctBid and myr-ctPAK2, myristoylation of ctBap31 may ameliorate the proapoptotic effect of Bap31 by translocating caspase-8 to new substrates.
GCLC is the catalytic subunit of the heterodimeric enzyme glutamate-l
-cysteine ligase, which regulates the rate-limiting step in the synthesis of the antioxidant glutathione. A common feature during apoptosis is the depletion of glutathione in the cell. Although overex-pression of full-length GCLC is antiapoptotic, the effect of the caspase cleavage products of GCLC and the subsequent myristoylation of ctGCLC remains unclear at this time (42
Using this alternative detection methodology, we also demonstrate the existence of at least 15 proteins that undergo post-translational myristoylation in apoptotic Jurkat T cells (). This result suggests that post-translational myristoylation of caspase-cleaved proteins represents a mechanism widely used to regulate cell death. While the majority of cotranslationally myristoylated proteins were found in the membrane fraction () as assessed by our methodology, the majority of the post-translationally myristoylated proteins were found in the cytosolic fraction, perhaps suggesting alternative roles for myristoylation other than membrane tethering.
Because the azide moiety bound to myristate can be ligated with high chemoselectivity with a tagged phosphine via a highly stable amide bond, one of the most exciting future applications of this methodology resides in the proteomic identification of the complete set of myristoylated proteins in living and dying cells via affinity chromatography and identification by mass spectrometry. Although this methodology is designed as a chemical tool to label, discover, and identify myristoylated proteins, it is important to note that the biophysical properties of azidomyristate likely differ from that of myristate, and this dissimilarity obviously potentially restricts the use of azidomyristate in subcellular localization studies.
The ease, high sensitivity, and power of our new methodology to detect and identify new myristoylated proteins is undeniable and will greatly facilitate the study of the biology of co- and post-translational myristoylation of proteins in cells and eventually animals with the potential of unraveling new roles for this type of post-translational protein modification.