Rapid detection, discovery, and identification of post-translationally myristoylated proteins during apoptosis using a bio-orthogonal azidomyristate analog.
Myristoylation is the attachment of the 14-carbon fatty acid myristate to the N-terminal glycine residue of proteins. Typically a co-translational modification, myristoylation of proapoptotic cysteinyl-aspartyl proteases (caspase)-cleaved Bid and PAK2 was also shown to occur post-translationally and is essential for their proper localization and proapoptotic function. Progress in the identification and characterization of myristoylated proteins has been impeded by the long exposure times required to monitor incorporation of radioactive myristate into proteins (typically 1-3 months). Consequently, we developed a nonradioactive detection methodology in which a bio-orthogonal azidomyristate analog is specifically incorporated co- or post-translationally into proteins at N-terminal glycines, chemoselectively ligated to tagged triarylphosphines and detected by Western blotting with short exposure times (seconds to minutes). This represents over a million-fold signal amplification in comparison to using radioactive labeling methods. Using rational prediction analysis to recognize putative internal myristoylation sites in caspase-cleaved proteins combined with our nonradioactive chemical detection method, we identify 5 new post-translationally myristoylatable proteins (PKC epsilon, CD-IC2, Bap31, MST3, and the catalytic subunit of glutamate cysteine ligase). We also demonstrate that 15 proteins undergo post-translational myristoylation in apoptotic Jurkat T cells. This suggests that post-translational myristoylation of caspase-cleaved proteins represents a novel mechanism widely used to regulate cell death.
Martin
Dale D O
DD
University of Alberta, Department of Cell Biology, Edmonton, AB T6G 2H7, Canada.
Vilas
Gonzalo L
GL
Prescher
Jennifer A
JA
Rajaiah
Gurram
G
Falck
John R
JR
Bertozzi
Carolyn R
CR
Berthiaume
Luc G
LG
D000818:Animals; D017209:Apoptosis; D001386:Azides / Q000737:chemistry*; D015153:Blotting, Western; D019556:COS Cells; D002460:Cell Line; D002623:Chemistry Techniques, Analytical / Q000379:methods; D002522:Chlorocebus aethiops; D005998:Glycine / Q000737:chemistry / Q000378:metabolism; D006801:Humans; D019169:Jurkat Cells; D009227:Myristic Acids / Q000737:chemistry / Q000378:metabolism*; D011499:Protein Processing, Post-Translational; D011506:Proteins / Q000032:analysis / Q000737:chemistry / Q000378:metabolism*; D013997:Time Factors
D001386:Azides; D009227:Myristic Acids; D011506:Proteins; D005998:Glycine