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This Article Full Text Full Text (PDF) Supplemental Data All Versions of this Article: fj.07-9198comv1 fj.07-9198comv2 22/3/797    most recent Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Google Scholar Articles by Martin, D. D. O. Articles by Berthiaume, L. G. PubMed PubMed Citation Articles by Martin, D. D. O. Articles by Berthiaume, L. G.

Rapid detection, discovery, and identification of post-translationally myristoylated proteins during apoptosis using a bio-orthogonal azidomyristate analog

Dale D. O. Martin^*, Gonzalo L. Vilas^*, Jennifer A. Prescher, Gurram Rajaiah, John R. Falck, Carolyn R. Bertozzi and Luc G. Berthiaume^*,1

^* Department of Cell Biology, University of Alberta, Edmonton, Alberta, Canada;

Department of Chemistry, Howard Hughes Medical Institute (HHMI), University of California, Berkeley, California, USA; and

Department of Biochemistry, University of Texas Southwestern Medical Center, Dallas, Texas, USA

¹Correspondence: Medical Sciences Bldg., University of Alberta, Faculty of Medicine and Dentistry, Department of Cell Biology, Edmonton, AB T6G 2H7, Canada. E-mail: luc.berthiaume{at}ualberta.ca

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, 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, D. D. O., Vilas, G. L., Prescher, J. A., Rajaiah, G., Falck, J. R., Bertozzi, C. R., Berthiaume, L. G. Rapid detection, discovery, and identification of post-translationally myristoylated proteins during apoptosis using a bio-orthogonal azidomyristate analog.

Key Words: fatty acylation • myristoylation • Staudinger ligation • cell death • triaryphosphine