Posttranslational myristoylation of caspase-activated p21-activated protein kinase 2 (PAK2) potentiates late apoptotic events -- Vilas et al. 103 (17): 6542 Data Supplement - HTML Page - index.htslp -- Proceedings of the National Academy of Sciences

Vilas et al. 10.1073/pnas.0600824103.

Supporting Information

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Supporting Figure 5
Supporting Figure 6
Supporting Figure 7
Supporting Materials and Methods

Fig. 5. The N-terminal decapeptide of C-t-PAK2 is myristoylated in vitro. (A) A schematic representation of the interdomain region of PAK2. The diagram shows the cleavage site for caspase-3, the putative myristoylation sequence, and a polybasic stretch of amino acids required for stable membrane binding and targeting. (B) Decapeptides encompassing either the first 10 amino acids of C-t-PAK2 (Gly-213-N10-C-t-PAK2) or a nonmyristoylatable Ala-213-N10-C-t-PAK2 were incubated in the presence of NMT and myristoyl-CoA followed by MS analysis. Gly-213-N10-C-t-PAK2 and a chemically myristoylated Gly-213-N10-PAK2 (myr-N10-C-t-PAK2) were used as negative and positive myristoylation controls, respectively. Black arrows indicate the position of the nonmyristoylated Gly-213-N10-C-t-PAK2; red and black arrowheads indicate the position of myristoylated-Gly-213-N10-C-t-PAK2 and nonmyristoylated Ala-213-N10-C-t-PAK2, respectively. All peptides were synthesized at Service de Synthèse de Peptides de L’Est du Québec, Le Centre Hospitalier de L’Université Laval (Quebec).

Fig. 6. Myristoylation is required for localization of C-t-PAK2-myc to membrane ruffles and plasma membrane. Confocal image depicting a Z stack reconstruction of a COS-7 cell transfected with a plasmid allowing expression of Gly-213-C-t-PAK2-myc analyzed 12-14 h after transfection by indirect immunofluorescence.

Fig. 7. Myristoylation enhances the apoptotic effects of C-t-PAK2. COS-7 cells were cotransfected with plasmids expressing EGFP and plasmids expressing Gly-213-C-t-PAK2-myc, Ala-213-C-t-PAK2-myc chimeras, or vector alone. Green cells presenting condensed/fragmented nuclei and rounded/retracted/highly refractive morphology as described by Lee et al. (1), Jakobi et al. (2), and Koeppel et al. (3) were scored as "apoptotic" or undergoing programmed cell death. Representative green fluorescence, phase-contrast, and Hoechst fluorescence images of EGFP cotransfected cells taken 24 h posttransfection are shown. Merged images are presented in the bottom row. Arrows indicate cells undergoing apoptotic cell death.

1. Lee, N., MacDonald, H., Reinhard, C., Halenbeck, R., Roulston, A., Shi, T. & Williams, L. T. (1997) Proc. Natl. Acad. Sci. USA 94, 13642-13647.

2. Jakobi, R., McCarthy, C. C., Koeppel, M. A. & Stringer, D. K. (2003) J. Biol. Chem. 278, 38675-38685.

3. Koeppel, M. A., McCarthy, C. C., Moertl, E. & Jakobi, R. (2004) J. Biol. Chem. 279, 53653-53664.

Materials and Methods
Cell Lines, Antibodies, and Reagents.
Jurkat T cells and COS-7 cells were grown, respectively, in RPMI medium 1640 and DMEM media (Invitrogen) supplemented with 10% FBS/100 units/ml penicillin G (sodium salt)/100 mg/ml streptomycin sulfate at 37°C in a humidified incubator with a 7% CO₂ atmosphere.

Goat anti-gPAK (C-19) antibody that reacts with an epitope mapping to the C terminus of human PAK2, donkey anti-goat-horseradish peroxidase (HRP), rabbit anti-JNK (C-17), and goat anti-p-JNK (Thr 183/Tyr 185) were from Santa Cruz Biotechnology. Rabbit and goat anti-GFP antibodies were from Eusera (Edmonton, AB, Canada). Anti-myc tag clone 4A6 was from Upstate Biotechnology (Lake Placid, NY). Rabbit polyclonal to myc tag antibody was from Abcam, Inc. (Cambridge, MA). Alexa Fluor 488 chicken anti-rabbit, Alexa Fluor 488 goat anti-mouse, and Alexa Fluor 633 phalloidin were from Molecular Probes. Sheep anti-mouse-HRP was from Amersham Pharmacia Biosciences. Cycloheximide was purchased from ICN. 2-Hydroxy-myristic acid was from Sigma. Complete Protease Inhibitor Mixture Tablets were purchased from Roche Diagnostics. The BCA Protein Assay Reagent Kit was from Pierce. Electrophoresis reagents were from Bio-Rad. All other reagents used were purchased from Sigma.

Immunocytochemistry.
Appropriately transfected COS-7 cells grown on poly(L-lysine)-coated cover slips were washed in PBS, fixed in 4% paraformaldehyde in PBS (pH 7.4) for 20 min, quenched with 50 mM NH₄Cl for 10 min, and permeabilized with 0.1% Triton X-100 in PBS for 1 min at room temperature, followed by a 30-min block with 0.2% gelatin (Fisher Scientific) in PBS. All antibodies used were diluted in 0.2% gelatin in PBS. For colocalization of myc chimeras and various organelles and cellular structures, anti-myc tag clone 4A6 (1:500), Alexa Fluor 633 phalloidin (3 units per coverslip), and Hoechst 33258 (50 ng/ml) were used to detect the myc epitope, actin cytoskeleton, and nuclei, respectively. Alexa Fluor 488 goat anti-mouse (1:1,000) was used to detect anti-myc antibodies. Mitochondrial potential was detected with MitoTracker Red CM-H₂XRos (1 mM, Molecular Probes) as recommended by the supplier. Cytochrome c was detected by using sheep anti-cytochrome c antibody (1:1,000, Sigma).