Identification and Characterization of PS-GAP as a Novel Regulator of Caspase-activated PAK-2

Abstract

p21-activated protein kinase (PAK)-2 is a member of the PAK family of serine/threonine kinases. PAKs are activated by the p21 G-proteins Rac and Cdc42 in response to a variety of extracellular signals and act in pathways controlling cell growth, shape, motility, survival, and death. PAK-2 is unique among the PAK family members because it is also activated through proteolytic cleavage by caspase-3 or similar proteases to generate the constitutively active PAK-2p34 fragment. Activation of fulllength PAK-2 by Rac or Cdc42 stimulates cell survival and protects cells from cell death, whereas caspase-activated PAK-2p34 induces a cell death response. Caspase-activated PAK-2p34 is rapidly degraded by the 26 S proteasome, but full-length PAK-2 is not. Stabilization of PAK2p34 by preventing its polyubiquitination and degradation results in a dramatic stimulation of cell death. Although many proteins have been shown to interact with and regulate full-length PAK-2, little is known about the regulation of caspase-activated PAK-2p34. Here, we identify PS-GAP as a regulator of caspase-activated PAK-2p34. PS-GAP is a GTPase-activating protein for Cdc42 and RhoA that was originally identified by its interaction with the tyrosine kinase PYK-2. PS-GAP interacts specifically with caspase-activated PAK-2p34, but not active or inactive full-length PAK-2, through a region between the GAP and SH3 domains. The interaction with PS-GAP inhibits the protein kinase activity of PAK-2p34 and changes the localization of PAK-2p34 from the nucleus to the perinuclear region. Furthermore, PS-GAP decreases the stimulation of cell death induced by stabilization of PAK-2p34.

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

“…In addition, it appears that the morphological changes seen in cells expressing myr-Ct-PAK2 undergoing programmed cell death do not activate the intrinsic apoptotic cell death pathway. These important observations are also supported by those of Lee et al (27), Jakobi et al (16), and Koeppel et al (24), who showed that expression of C-t-PAK2 led to adherent cell shrinking, rounding up, and nuclear condensation without exhibiting classical hallmarks of apoptosis (no phosphatidylserine externalization and absence of TUNEL reactivity).…”

“…Caspase-3-mediated cleavage of PAK2 has been observed during apoptosis (15,25,26), and overexpression of the Nterminally tagged C-terminal cleavage product C-t-PAK2 has been shown to induce cell death (16,24,27). However, all these previous studies assessed the apoptotic activity of the nonphysiologically relevant, nonmyristoylated form of C-t-PAK2.…”

“…To study the physiological form of C-t-PAK2 and assess the importance of posttranslational myristoylation of C-t-PAK2 in its ability to induce apoptosis, we compared the apoptotic properties of Gly-213-C-t-PAK2-myc and Ala-213-C by using an established EGFP cotransfection assay (16,24,27). Because the expression of C-t-PAK2 leads to nuclear condensation, adherent cell shrinking, and rounding up without exhibiting some of the classical hallmarks of apoptosis (no phosphatidylserine externalization and absence of TUNEL reactivity) (27,28), the percentage of cells undergoing apoptosis͞cell death at different times after cotransfection was calculated as the percentage of green cells presenting condensed͞fragmented nuclei and rounded͞retracted͞highly refractive morphology as described by Lee et al (27), Jakobi et al (16), and Koeppel et al (24). Expression of the myristoylatable Gly-213-C-t-PAK2-myc chimera resulted in a count of 52% apoptotic cells after 12 h, and this amount reached a maximal plateau at Ϸ68% within 20 h after transfection (Fig.…”

“…Therefore, our results explain why this artificial membrane tethering was required; like PAK2, xPAK1, which is the functional equivalent to PAK2 (28), contains a putative myristoylation signal downstream of its caspase-cleavage site (SATDGAESSVDKTKKKPK) followed by a short polybasic domain. Importantly, with the exception of the work by Rudel et al (26), all of the experiments in published articles were performed with nonphysiological forms of C-t-PAK2 bearing a variety of N-terminal epitope tags, therefore abolishing posttranslational myristoylation (16,24,27).…”

The ATDC (TRIM29) Protein Binds p53 and Antagonizes p53-Mediated Functions

“…For some experiments, apoptotic assays were performed as previously described (21,25) with minor modifications. For each assay, 10 5 cells were cotransfected with 0.1 g of pEGFP-C3 and either pcDNA3.1-HA vector or pcDNA3.1-HA-ATDC.…”