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 serine/threonine 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.

Fig. 1.

C-t-PAK2 is posttranslationally myristoylated on Gly-213 by NMT during apoptosis in Jurkat T cells. (A) Jurkat T cells were metabolically labeled with [9,10(n)-³H]myristate and incubated for 5 h in the absence or presence of 2.5 μM STS and 5 μg/ml cycloheximide (STS) to induce apoptosis. Cells were lysed, and PAK2 was immunoprecipitated. Immunocomplexes were analyzed by Western blotting (W.B.) and fluorography (FL). (B) Jurkat T cells were metabolically labeled and induced to undergo apoptosis as in A in the absence or presence of 1 mM HMA. Samples were analyzed as in A. (C) COS-7 cells were transfected with plasmids allowing expression of Gly-213-C-t-PAK2-myc, the nonmyristoylatable Ala-213-C-t-PAK2-myc chimeras, vector alone, a palmitoylatable (nonmyristoylated) chimeric GAP-43-GFP, or GFP. Transfected cells were metabolically labeled with [9,10(n)-³H]myristate or [¹²⁵I]iodopalmitate for 4 h and lysed. Expressed proteins were immunoprecipitated and analyzed by Western blotting. PVDF membranes were then soaked for 24 h in either 0.2 M KOH or 1 M Tris·HCl (pH 7.0) and subjected to imaging with PhosphorImager screens (AR.). (D) Subcellular distribution of endogenous C-t-PAK2 in Jurkat T cells treated as in B were lysed and fractionated into total (T), cytosolic (S), and membrane (P) fractions. The presence of C-t-PAK2 was analyzed by Western blotting/enhanced chemiluminescence. (E) COS-7 cells were transfected with plasmids expressing Gly-213-C-t-PAK2-myc, the nonmyristoylatable Ala-213-C-t-PAK2-myc chimeras, or vector alone and were analyzed 12–14 h after transfection. Cells were subjected to subcellular fractionation and C-t-PAK2 detection as in D.

Fig. 2.

Myristoylation is required for proper intracellular localization of C-t-PAK2-myc. Indirect immunofluorescence confocal micrographs of COS-7 cells transfected with plasmids allowing expression of Gly-213-C-t-PAK2-myc, the nonmyristoylatable Ala-213-C-t-PAK2-myc chimeras, or vector alone at 12–14 h after transfection. (A) Gly-213-C-t-PAK2-myc localizes to the plasma membrane and membrane ruffles, whereas the Ala-213-C-t-PAK2-myc chimera remains cytosolic. Shown are C-t-PAK2-myc chimeras (anti-myc, green) and nuclei (Hoechst dye 33258 staining, blue). (B) Confirmation of Gly-213-C-t-PAK2-myc localization to plasma membrane ruffles as indicated by colocalization with actin (red). The merged images are presented in Right. Yellow indicates apparent colocalization of the green and red signals. (C) The N-terminal 14 amino acids of C-t-PAK2 encompassing the myristoylation signal and a short polybasic domain are sufficient to localize EGFP to membranes. COS-7 cells were transfected with plasmids expressing chimeric EGFPs in which the first 14 amino acids of C-t-PAK2 (Gly-213-N15-EGFP or the corresponding nonmyristoylatable mutant Ala-213-N15-EGFP) were inserted after the initiator methionine or vector alone. Images were obtained 12–14 h after transfection. EGFP was detected either by live cell fluorescence microscopy (Live) or indirect immunofluorescence (Fixed) by confocal microscopy. (Scale bars, 10 μm.)

Fig. 3.

Myristoylation enhances the morphological apoptotic effects of C-t-PAK2 and signaling through JNK. (A) COS-7 cells were cotransfected with plasmids expressing EGFP and plasmids expressing Gly-213-C-t-PAK2-myc (hatched bars), Ala-213-C-t-PAK2-myc chimeras (gray bars), or vector alone (open bars). At the indicated times, the percentage of cotransfected apoptotic cells for each time point was calculated as described in Materials and Methods and plotted. Data are means ± SE from three independent experiments. (B) Expression of C-t-PAK2 induces JNK phosphorylation. COS-7 cells were transfected with Gly-213-C-t-PAK2-myc, Ala-213-C-t-PAK2-myc chimeras, or vector alone; 14–16 h after transfection, cell lysates were made and total JNK, phospho-JNK, and myc were electroblotted and immunodetected with the proper antibodies. The intensity of the bands was determined by densitometry. Data in the histogram are presented as fold phosphorylation over vector normalized by the corresponding total JNK and C-t-PAK2-myc expression. Data represent the means ± SE of three individual experiments. ∗, P ≤ 0.05.

Fig. 4.

C-t-PAK2 expression induces mitochondrial-independent cell death. COS-7 cells were mock-transfected or transfected with either vector expressing Gly-213-C-t-PAK2-myc or vector alone. At 16 h after transfection, cells were treated with 1 μM STS for 4 h or 5 μM para-trifluoromethoxy carbonyl cyanide phenylhydrazone for 15 min or left untreated. Cells were then incubated in the presence of the potentiometric dye MitoTracker red CM-H₂XRos for 30 min. After incubation, cells were processed for confocal immunofluorescence microscopy to detect C-t-PAK2-myc (green), cytochrome c (light blue), nuclei (blue), and mitochondrial membrane potential (red). The merged images are presented in the far-right column. (Scale bars, 10 μm.)