Posttranslational myristoylation of caspase-activated p21-activated protein kinase 2 (PAK2) potentiates late apoptotic events

Abstract

p21-activated protein kinase (PAK) 2 is a small GTPase-activated serine͞threonine kinase regulating various cytoskeletal functions and is cleaved by caspase-3 during apoptosis. We demonstrate that the caspase-cleaved PAK2 C-terminal kinase fragment (C-t-PAK2) is posttranslationally myristoylated, although myristoylation is typically a cotranslational process. Myristoylation and an adjacent polybasic domain of C-t-PAK2 are sufficient to redirect EGFP from the cytosol to membrane ruffles and internal membranes. Membrane localization and the ability of C-t-PAK2 to induce cell death are significantly reduced when myristoylation is abolished. In addition, the proper myristoylation-dependent membrane localization of C-t-PAK2 significantly increased signaling through the stress-activated c-Jun N-terminal kinase signaling pathway, which often regulates apoptosis. Interestingly, C-t-PAK2 promoted cell death without compromising mitochondrial integrity. Posttranslational myristoylation of caspase-cleaved proteins involved in cytoskeletal dynamics (e.g., PAK2, actin, and gelsolin) might be part of a unique series of mechanisms involved in the regulation of the later events of apoptosis.apoptosis ͉ cytoskeleton ͉ mitochondria ͉ membrane ͉ acylation

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

“…The ctPAK2-N 15 -EGFP and Yes-N 11 -EGFP constructs were from previous work (9). COS-7 cells were transiently transfected with the pEGFP-N1 (Invitrogen) vectors expressing the various chimeric enhanced green fluorescent proteins (EGFPs) using Fugene 6 (Roche, Indianapolis, IN, USA) in Dulbecco modified Eagle medium in the absence of FBS or antibiotics but supplemented with 1% fatty acid-free BSA (Sigma-Aldrich).…”

“…EGFP was detected on membranes using rabbit anti-GFP serum. (9). Approximately 1.0 × 10 7 Jurkat cells were incubated for 1 h at 37°C in the presence or absence of 1 mM 2-hydroxymyristic acid (HMA), an NMT inhibitor that does not inhibit protein palmitoylation (17)(18)(19), in RPMI without FBS or antibiotics but supplemented with 1.0% fatty acid-free BSA.…”

“…Following extensive washing with 0.1% SDS-RIPA, the immune complexes were boiled for 5 min in SDS-sample buffer, separated by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and electrotransferred to polyvinylidene difluo-ride (PVDF) membranes. Where indicated, the membranes were treated with either 0.2 M Tris-HCl pH 7.0 or 0.2 M potassium hydroxide (KOH) as described before (9), and biotinylated azidomyristoylated proteins were detected using neutravidin-HRP or the appropriate anti-FLAG antibodies followed by electrochemiluminescence (ECL) detection (Amersham). EGFP was detected on membranes using rabbit anti-GFP serum.…”

“…Post-translational myristoylation of caspase-cleaved proteins is rapidly emerging as a novel means to regulate programmed cell death (9)(10)(11). The programmed cleavage of hundreds of proteins during apoptosis warrants the search for additional post-translationally myristoylated proteins and the characterization of their involvement in the regulated demise of the cell.…”

“…In cotranslational myristoylation, the N-terminal glycine residue is exposed on removal of the initiator methionine by methionine aminopeptidase (6,8). However, during apoptosis, 4 proteins (Bid, p21-activated protein kinase [PAK2], actin, and gelsolin) were shown to undergo post-translational myristoylation on cysteinyl-aspartyl proteases (caspase) cleavage and the resulting exposure of an internal Gly residue within a myristoylation consensus sequence (9)(10)(11).…”