Functional PAK-2 knockout and replacement with a caspase cleavage-deficient mutant in mice reveals differential requirements of full-length PAK-2 and caspase-activated PAK-2p34.

p21-Activated protein kinase 2 (PAK-2) has both anti- and pro-apoptotic functions depending on its mechanism of activation. Activation of full-length PAK-2 by the monomeric GTPases Cdc42 or Rac stimulates cell survival, whereas caspase activation of PAK-2 to the PAK-2p34 fragment is involved in the apoptotic response. In this study we use functional knockout of PAK-2 and gene replacement with the caspase cleavage-deficient PAK-2D212N mutant to differentiate the biological functions of full-length PAK-2 and caspase-activated PAK-2p34. Knockout of PAK-2 results in embryonic lethality at early stages before organ development, whereas replacement with the caspase cleavage-deficient PAK-2D212N results in viable and healthy mice, indicating that early embryonic lethality is caused by deficiency of full-length PAK-2 rather than lack of caspase activation to the PAK-2p34 fragment. However, deficiency of caspase activation of PAK-2 decreased spontaneous cell death of primary mouse embryonic fibroblasts and increased cell growth at high cell density. In contrast, stress-induced cell death by treatment with the anti-cancer drug cisplatin was not reduced by deficiency of caspase activation of PAK-2, but switched from an apoptotic to a nonapoptotic, caspase-independent mechanism. Homozygous PAK-2D212N primary mouse embryonic fibroblasts that lack the ability to generate the proapoptotic PAK-2p34 show less activation of the effector caspase 3, 6, and 7, indicating that caspase activation of PAK-2 amplifies the apoptotic response through a positive feedback loop resulting in more activation of effector caspases.

Targeting RhoA/Rho kinase and p21-activated kinase signaling to prevent cancer development and progression.

Elevated RhoA/Rho kinase and p21-activated kinase signaling have been shown to promote cancer development and metastasis and have drawn much attention as potential targets of anti-cancer therapy. Elevated RhoA and Rho kinase activity promote cancer cell invasion and eventually lead to metastasis by disrupting E-cadherin-mediated adherens junctions and degradation of the extracellular matrix. Elevated p21-activated kinase activity promotes invasion by stimulating cell motility but also promotes cancer cell survival and growth. In this review we describe normal functions of RhoA/Rho kinase and p21-activated kinase signaling, mechanisms that lead to constitutive activation of RhoA/Rho kinase and p21-activated kinase pathways, and processes by which constitutive RhoA/Rho kinase and p21-activated kinase activity promote cancer development and progression to more aggressive and metastatic phenotypes. In addition, we summarize relevant patents on RhoA/Rho kinase and p21-activated kinase as targets of anti-cancer therapy and discuss the clinical potential of different approaches to modulate RhoA/Rho kinase and p21-activated kinase signaling.

Elevated p21-activated kinase 2 activity results in anchorage-independent growth and resistance to anticancer drug-induced cell death.

p21-activated kinase 2 (PAK-2) seems to be a regulatory switch between cell survival and cell death signaling. We have shown previously that activation of full-length PAK-2 by Rac or Cdc42 stimulates cell survival, whereas caspase activation of PAK-2 to the proapoptotic PAK-2p34 fragment is involved in the cell death response. In this study, we present a role of elevated activity of full-length PAK-2 in anchorage-independent growth and resistance to anticancer drug-induced apoptosis of cancer cells. Hs578T human breast cancer cells that have low levels of PAK-2 activity were more sensitive to anticancer drug-induced apoptosis and showed higher levels of caspase activation of PAK-2 than MDA-MB435 and MCF-7 human breast cancer cells that have high levels of PAK-2 activity. To examine the role of elevated PAK-2 activity in breast cancer, we have introduced a conditionally active PAK-2 into Hs578T human breast cells. Conditional activation of PAK-2 causes loss of contact inhibition and anchorage-independent growth of Hs578T cells. Furthermore, conditional activation of PAK-2 suppresses activation of caspase 3, caspase activation of PAK-2, and apoptosis of Hs578T cells in response to the anticancer drug cisplatin. Our data suggest a novel mechanism by which full-length PAK-2 activity controls the apoptotic response by regulating levels of activated caspase 3 and thereby its own cleavage to the proapoptotic PAK-2p34 fragment. As a result, elevated PAK-2 activity interrupts the apoptotic response and thereby causes anchorage-independent survival and growth and resistance to anticancer drug-induced apoptosis.

RhoA mediates cyclooxygenase-2 signaling to disrupt the formation of adherens junctions and increase cell motility.

Cyclooxygenase-2 (COX-2) represents an important target for treatment and prevention of colorectal cancer. Although COX-2 signaling is implicated in promoting tumor cell growth and invasion, the molecular mechanisms that mediate these processes are largely unknown. In this study, we show that the RhoA pathway mediates COX-2 signaling to disrupt the formation of adherens junctions and increase cell motility. Disruption of adherens junctions promotes tumor cell invasion and metastasis and is often associated with tumor progression. We detected high levels of RhoA activity in HCA-7 colon carcinoma cells that constitutively express COX-2. Inhibition of COX-2 significantly reduced the levels of RhoA activity in HCA-7 cells, suggesting that constitutive expression of COX-2 stimulates RhoA activity. Interestingly, inhibition of COX-2 or silencing of COX-2 expression with small interfering RNA (siRNA) stimulated the formation of adherens junctions, concomitant with increased protein levels of E-cadherin and alpha-catenin. Furthermore, inhibition of RhoA or silencing of RhoA expression with siRNA increased the levels of E-cadherin and alpha-catenin. Inhibition of Rho kinases (ROCK), the RhoA effector proteins, also increased levels of E-cadherin and alpha-catenin and stimulated formation of adherens junctions. The motility of HCA-7 cells was significantly decreased when COX-2 or RhoA was inhibited. Therefore, our data reveal a novel molecular mechanism that links COX-2 signaling to disrupt the formation of adherens junctions; COX-2 stimulates the RhoA/ROCK pathway, which reduces levels of E-cadherin and alpha-catenin leading to disruption of adherens junction formation and increased motility. Understanding of COX-2 downstream signaling pathways that promote tumor progression is crucial for the development of novel therapeutic strategies.

Differential regulation of cyclooxygenase 2 expression by small GTPases Ras, Rac1, and RhoA.

Cyclooxygenase 2 (COX-2) is an immediate early gene induced by a variety of stimuli and its expression is stimulated by individual activation of Ras or Rho GTPases. Here we investigate the role of coordinate activation of Ras and Rho GTPases in the induction of COX-2. Individual expression of constitutively active Ras, RhoA, or Rac1 was capable of stimulating COX-2 expression in NIH3T3 cells, but co-expression of constitutively active RhoA with either constitutively active Ras or Rac1 was required for full stimulation of COX-2 expression. Serum growth factors differentially activated Ras, RhoA, and Rac1, which correlated with the activation of Raf-1, ERK, and c-Jun as well as with induction of COX-2. Inhibition of Ras significantly blocked the activation of Raf-1, ERK, and c-Jun and the stimulation of COX-2 expression in response to serum. In contrast, inhibition of Rho family GTPases partially blocked serum induction of ERK activation but had little effects on COX-2 expression. Both inhibitors of MEK (PD098059) and JNK (SP600125) inhibited serum induction of COX-2. PD98059 only inhibited constitutively active Ras-induced COX-2 expression, while SP600125 significantly inhibited both constitutively active Ras- and RhoA-induced COX-2 expression. Together, our data suggest that constitutively active oncogenic Ras and Rho coordinately stimulate COX-2 expression whereas transient activation of Ras but not RhoA or Rac1 mediates the induction of COX-2 in response to serum. Furthermore, ERK and JNK activation are both required for serum- and oncogenic Ras-mediated COX-2 expression whereas only JNK activation is required for oncogenic RhoA-mediated stimulation of COX-2 expression.

Identification and characterization of PS-GAP as a novel regulator of caspase-activated PAK-2.

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 full-length 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 PAK-2p34 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.

Subcellular targeting regulates the function of caspase-activated protein kinases in apoptosis.

Subcellular localization and targeting of proteins play important roles in signal transduction pathways that regulate cell survival and programmed cell death. The regulation of cell survival and cell death requires translocation of many anti- and pro-apoptotic signaling molecules from one subcellular compartment to another. In many cases translocation is triggered by caspase cleavage. Caspase cleavage removes the regulatory domains of the protein kinases MEKK1, Mst-1 and PAK-2 resulting in activation and in relocalization of the catalytic fragments. Caspase-activated MEKK1 translocates from a particulate compartment to the cytosol; caspase-activated Mst-1 and PAK-2 translocate from the cytoplasm to the nucleus. Caspase activation of these protein kinases induces a cell death response. Relocalization of the catalytic fragments to a pro-apoptotic location appears to be required to induce cell death. It is suggested that translocation to a pro-apoptotic location results in phosphorylation of pro-apoptotic substrates. Therefore, these protein kinases could represent novel targets for cancer therapy. Compounds that stimulate cleavage of MEKK1, Mst-1 and PAK-2 or compounds that cause translocation to a pro-apoptotic location could be used to induce cell death of cancer cells.

Caspase-activated PAK-2 is regulated by subcellular targeting and proteasomal degradation.

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 (gamma-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 alpha or growth factor withdrawal, PAK-2 is activated as a full-length 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 full-length 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.