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 kinases (PAKs) are a family of serine/threonine protein kinases that are activated by binding of the p21 G proteins Cdc42 or Rac. The ubiquitous PAK-2 (␥-PAK) is unique among the PAK isoforms because it is also activated through proteolytic cleavage by caspases or caspase-like proteases. In response to stress stimulants such as tumor necrosis factor ␣ or growth factor withdrawal, PAK-2 is activated as a fulllength enzyme and as a proteolytic PAK-2p34 fragment. Activation of full-length PAK-2 stimulates cell survival, whereas proteolytic activation of PAK-2p34 is involved in programmed cell death. Here we provide evidence that the proapoptotic effect of PAK-2p34 is regulated by subcellular targeting and degradation by the proteasome. Full-length PAK-2 is localized in the cytoplasm, whereas the proteolytic PAK-2p34 fragment translocates to the nucleus. Subcellular localization of PAK-2 is regulated by nuclear localization and nuclear export signal motifs. A nuclear export signal motif within the regulatory domain prevents nuclear localization of fulllength PAK-2. Proteolytic activation removes most of the regulatory domain and disrupts the nuclear export signal. The activated PAK-2p34 fragment contains a nuclear localization signal and translocates to the nucleus. However, levels of activated PAK-2p34 are tightly regulated through ubiquitination and degradation by the proteasome. Inhibition of degradation by blocking polyubiquitination results in significantly increased levels of PAK-2p34 and as a consequence, in stimulation of programmed cell death. Therefore, nuclear targeting and inhibition of degradation appear to be critical for stimulation of the cell death response by PAK-2p34.

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.

The amplification from the crude lysates of the recombinant P. pastoris strains KM71 (pPIC9-MYO)-Mut S and GS115 (pPIC9-MYO)-Mut + , which contained the integration of the myostatin gene to chromosomal DNA, was completed. When Taq DNA polymerase was used for amplification, we observed either no or nonspecific amplification products. The positive control sample yielded the 831-bp specific fragment. When the HOTStar Taq DNA polymerase was used for the amplification, the 831-bp specific amplification product was visible. The wild-type gene AOX1 2.2-kb fragment was co-amplified in GS115-derived strains. The fragment is not visible in KM71-derived strains, because the gene is deleted from the parent strain. The highest yield was attained with 10 min of boiling at 80°C (Figure 1). Changing the volume of amplification mixture did not influence the result. No changes were found when the annealing temperature and number of amplification cycles were changed. Similar results were observed when the recombinant yeast P. pastoris strain KM71 (pPIC9-LEP)-Mut S with cloned gene for leptin was amplified using the same procedure. The results are demonstrated in Figure 2. In this experiment, a specific 912-bp product was observed as a positive result. No product from the 2.2-kb wild-type gene fragment was visible in these KM71-derived strains. From the presented results we can deduce that crude yeast lysates contain many PCR inhibitors, which decreases the specificity of the amplification and the amount of amplification product. In contrast to the amplification from the crude lysates of some bacterial cells, only some robust Taq DNA polymerases can overcome these inhibitors. However, fundamentally, the amplification from crude yeast lysates is possible, and the described method can be used as an inexpensive and quick alternative to isolation of chromosomal DNA from yeast cells.

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.