In response to stress stimulants, cells activate opposing signaling pathways for cell survival and programmed cell death. p21-activated protein kinase ␥-PAK is involved in both cell survival and cell death pathways. Many stress stimulants activate ␥-PAK as a full-length enzyme and as a proteolytic fragment. Caspase-mediated proteolytic activation parallels cell death and appears to be a pro-apoptotic factor in stress-induced cell death. Here, we show that activation of full-length ␥-PAK promotes cell survival and suppresses stress-induced cell death. Expression of constitutively active ␥-PAK-T402E, which mimics activated full-length ␥-PAK, stimulates cell survival of BALB3T3 fibroblasts in response to tumor necrosis factor ␣, growth factor withdrawal, and UVC light. This stimulation of cell survival is mainly due to protection of cells from cell death rather than by stimulation of proliferation. Expression of ␥-PAK-T402E increases phosphorylation of the proapoptotic Bcl-2 family protein Bad and protects from cell death induced by ectopic expression of Bad. In response to tumor necrosis factor ␣, expression of ␥-PAK-T402E increases the early but reduces the late activation of ERK, JNK, and p38. Our results indicate that the ubiquitous ␥-PAK may have a crucial function in cell survival by regulating the pro-apoptotic activity of Bad and the stress-induced activation of ERK, JNK, and p38 pathways.

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.

Figure 2. Expression of FLAG-and EGFP-tagged fusion proteins and EGFP-tagged antisense RNA. cDNAs for p21-activated protein kinase (PAK) and the p21 G-protein Cdc42 were subcloned into pKoz/M-Flag, pKoz/EGFP or pKoz/EGFP-anti to generate FLAG-or EGFP-tagged cDNA constructs or EGFP-tagged antisense RNA constructs. Tagged constructs were subcloned into the inducible retroviral expression vector pRevTRE and transduced into BALB3T3 fibroblasts. (A) Expression of FLAG-tagged PAK before (-Dox) and after (+Dox) induction with doxycycline was detected in a western blot with anti-FLAG antibody in transduced cells and untransduced cells (control). (B) Expression of EGFP-tagged PAK and Cdc42 after induction with doxycycline was detected in a western blot with an anti-EGFP antibody. (C) Levels of EGFP and endogenous PAK before (-Dox) and after (+Dox) induction with doxycycline were detected in western blots with anti-EGFP and anti-PAK antibodies in cells transduced with EGFP-tagged PAK antisense RNA. (D) Expression of EGFP-tagged PAK or Cdc42 and EGFP-tagged PAK antisense RNA was detected by fluorescence microscopy.