Reversing chemoresistance in cisplatin-resistant human ovarian cancer cells: a role of c-Jun NH2-terminal kinase 1

To investigate the role of activation of c-Jun NH2-terminal kinase 1 (JNK1) in mediating cisplatin-induced apoptosis and the possibility of induction of JNK activity in triggering relation to DNA damage and drug resistance. We investigated the difference of cisplatin-induced activation of JNK pathway and H2O2 alteration between cisplatin-sensitive human ovarian carcinoma cell line A2780 and its resistant variant A2780/DDP. JNK, p-JNK protein, and extracellular H2O2 levels were determined in both A2780 and A2780/DDP cells which were transfected with dominant negative allele of JNK and recombinant JNK1 separately. Both A2780 and A2780/DDP were treated with CDDP, the JNK pathway was activated and a prolonged JNK activation was maintained for at least 12 h in A2780, and only a transient activation (3 h) was detected in A2780/DDP in response to cisplatin treatment. Inhibition of JNK activity by transfection with a dominant negative allele of JNK blocked CDDP-induced apoptosis significantly in A2780 cells. Selective stimulation of the JNK pathway by lipofectamine-mediated delivery of recombinant JNK1 led to activation of c-Jun and decrease of extracellular H2O2, as well as apoptosis sensitization to CDDP in A2780/DDP cells. We concluded that JNK pathway might play an important role in mediating cisplatin-induced apoptosis in A2780 cells, and the duration of JNK activation might be critical in determining whether cells survive or undergo apoptosis. The resistance to CDDP can be reversed through activating c-Jun and decreasing extracellular generation of H2O2 by pcDNA3(FLAG)-JNK1-wt transfection in A2780/DDP cells.     

Dominant negative Rac1 attenuates paclitaxel-induced apoptosis in human melanoma cells through upregulation of heat shock protein 27: a functional proteomic analysis

GTPase ras-related C3 botulinum toxin substrate 1 (Rac1) plays a role in various cellular processes pertinent to cancer development. In the present study, we investigated the molecular mechanisms underlying apoptosis regulation by Rac1 through functional proteomic analysis of three human melanoma M14 cell lines stably transfected with constitutively active Rac1V12, dominant negative Rac1N17, and empty vector (pIRES), respectively. We found that paclitaxel evoked apoptosis in the melanoma cell lines through the intrinsic (mitochondria) pathway in a caspsae-3-dependent manner. Compared to the Rac1pIRES and Rac1V12 cells, Rac1N17 cells were more resistant to paclitaxel-triggered caspase-3 activation and apoptosis. Protein composition comparisons amongst the three cell lines identified two peptide spots of interest. One was Hsp27, which was upregulated in Rac1N17 cells as assessed in our gel image interpretation, PMF and Western blot analysis. The other was identified as SR-25 protein (also known as the ADP-ribosylation factor-like factor 6-interacting protein 4; ARL6IP4) using PMF, which was separated only from the Rac1N17 cells under the experimental conditions. Moreover, knockdown of the protein level of Hsp27 using small interfering RNA in Rac1N17 cells significantly increased the paclitaxel-elicited caspase-3 activation and apoptosis. In conclusion, our results implicate that Hsp27 and SR-25 are mediators in Rac1 signaling pathway(s). It appears that the dominant negative Rac1N17 reduces the apoptosis sensitivity toward paclitaxel in the melanoma cells through upregulation of Hsp27, which inhibits its down stream drug-elicited caspase-3 activation.     

Hydrogen peroxide mediates arsenite activation of p70(s6k) and extracellular signal-regulated kinase

To define the mechanism of arsenite-induced tumor promotion, we examined the role of reactive oxygen species (ROS) in the signaling pathways of cells exposed to arsenite. Arsenite treatment resulted in the persistent activation of p70(s6k) and extracellular signal-regulated kinase 1/2 (ERK1/2) which was accompanied by an increase in intracellular ROS production. The predominant produced appeared to be H(2)O(2), because the arsenite-induced increase in dichlorofluorescein (DCF) fluorescence was completely abolished by pretreatment with catalase but not with heat-inactivated catalase. Elimination of H(2)O(2) by catalase or N-acetyl-L-cysteine inhibited the arsenite-induced activation of p70(s6k) and ERK1/2, indicating the possible role of H(2)O(2) in the arsenite activation of the p70(s6k) and the ERK1/2 signaling pathways. A specific inhibitor of p70(s6k), rapamycin, and calcium chelators significantly blocked the activation of p70(s6k) induced by arsenite. While the phosphatidylinositol 3-kinase (PI3K) inhibitors wortmannin and LY294002 completely abrogated arsenite activation of p70(s6k), ERK1/2 activation by arsenite was not affected by these inhibitors, indicating that H(2)O(2) might act as an upstream molecule of PI3K as well as ERK1/2. Consistent with these results, none of the inhibitors impaired H(2)O(2) production by arsenite. DNA binding activity of AP-1, downstream of ERK1/2, was also inhibited by catalase, N-acetyl-L-cysteine, and the MEK inhibitor PD98059, which significantly blocked arsenite activation of ERK1/2. Taken together, these studies provide insight into mechanisms of arsenite-induced tumor promotion and suggest that H(2)O(2) plays a critical role in tumor promotion by arsenite through activation of the ERK1/2 and p70(s6k) signaling pathways.     

Oncogenic H-Ras enhances DNA repair through the Ras/phosphatidylinositol 3-kinase/Rac1 pathway in NIH3T3 cells. Evidence for association with reactive oxygen species

This study investigated the role of oncogenic H-Ras in DNA repair capacity in NIH3T3 cells. Expression of dominant-positive H-Ras (V12-H-Ras) enhanced the host cell reactivation of luciferase activity from UV-irradiated and cisplatin-treated plasmids and also increased the unscheduled DNA synthesis following cisplatin or UV treatment of cells. This observed enhancement of DNA repair capacity was inhibited by transient transfection with dominant-negative H-Ras (N17-H-Ras) or Rac1 (N17-Rac1) plasmids. Moreover, stable transfection of dominant-positive Rac1 (V12-Rac1) further enhanced DNA repair capacity. Because reactive oxygen species (ROS) are known to be a downstream effector of oncogenic Ras, we examined the role of ROS in DNA repair capacity. We found that ROS production by V12-H-Ras expression was mediated by the Ras/phosphatidylinositol 3-kinase (PI3K)/Rac1/NADPH oxidase-dependent pathway and that pretreatment of V12-H-Ras-transformed cells with an antioxidant (N-acetylcysteine) and an NADPH oxidase inhibitor (diphenyleneiodonium) decreased DNA repair capacity. Similarly, treatment with PI3K inhibitors (wortmannin and LY294002) inhibited the ability of oncogenic H-Ras to enhance DNA repair capacity. Furthermore, inhibition of the Ras/PI3K/Rac1/NADPH oxidase pathway resulted in increased sensitivity to cisplatin and UV in V12-H-Ras-expressing NIH3T3 cells. Taken together, these results provide evidence that oncogenic H-Ras activates DNA repair capacity through the Ras/PI3K/Rac1/NADPH oxidase-dependent pathway and that increased ROS production via this signaling pathway is required for enhancement of the DNA repair capacity induced by oncogenic H-Ras.     

Redox regulation of human Rac1 stability by the proteasome in human aortic endothelial cells

Rac1 has been shown to activate a NADPH oxidase complex producing superoxide anions in a variety of mammalian cell types. We evaluated the impact of Rac1-induced reactive oxygen species production on the turnover of Rac1 itself in human aortic endothelial cells. The concentration of a constitutively active mutant of Rac1 (Rac1(V12)) was increased by treatment of the cells with diphenylene iodinium (DPI), an inhibitor of the NADPH oxidase. Such an effect was not observed for the dominant negative form of Rac1 (Rac1(N17)). We showed a decrease in proteolytic degradation of Rac1(V12) in the presence of DPI, and showed that short term treatment with H(2)O(2) reverses the effect of DPI. We found that proteasome inhibitors (lactacystin and MG132) increased Rac1(V12) protein level. In support of this finding, we have identified in the primary sequence of Rac1 a potential destruction box domain, which is known to be a signal for protein degradation mediated by the ubiquitin/proteasome system. We show that Rac1(V12) is ubiquitinated before degradation. By contrast Rac1(N17) induces an accumulation of the ubiquitinated form of Rac1. These results suggest that Rac1 activation of NADPH oxidase is necessary for the proteolytic degradation of Rac1 itself.     

Important role for Rac1 in regulating reactive oxygen species generation and pulmonary arterial smooth muscle cell growth

Vascular NADPH oxidases have been shown to be a major source of reactive oxygen species (ROS). Recent studies have also implicated ROS in the proliferation of vascular smooth muscle cells. However, the components required for activation of the NADPH oxidase complex have not been clearly elucidated. Here we demonstrate that ROS generation in ovine pulmonary arterial smooth muscle cells (PASMCs) requires the activation of Rac1, implicating this protein as an important subunit of the NADPH oxidase complex. Our results, using a geranylgeranyl transferase inhibitor (GGTI-287), demonstrated a dose-dependent inhibition of Rac1 activity and ROS production. This was associated with an inhibition of PASMC proliferation with an arrest at G(2)/M. The inhibition of Rac1 by GGTI-287 led us to more specifically target Rac1 to investigate its role in the generation of ROS and cellular proliferation. To accomplish this, we utilized a dominant negative Rac1 (N17Rac1) and a constitutively active Rac1 (V12Rac1). These two forms of Rac1 were transiently expressed in PASMCs using adenovirus-mediated gene transfer. N17Rac1 expression resulted in decreased cellular Rac1 activity, whereas V12Rac1 infection showed increased activity. Compared with controls, the V12Rac1-expressing cells had higher levels of ROS production and increased proliferation, whereas the N17Rac1-expressing cells had decreased ROS generation and proliferation and cell cycle arrest at G(2)/M. However, the inhibition of cell growth produced by N17Rac1 overexpression could be overcome if cells were co-incubated with the Cu,Zn superoxide dismutase inhibitor DETC. These results indicate the importance of Rac1 in ROS generation and proliferation of vascular smooth muscle cells.     

DEF6, a novel PH-DH-like domain protein, is an upstream activator of the Rho GTPases Rac1, Cdc42, and RhoA

In this paper, we describe the characterization of DEF6, a novel PH-DH-like protein related to SWAP-70 that functions as an upstream activator of Rho GTPases. In NIH 3T3 cells, stimulation of the PI 3-kinase signaling pathway with either H2O2 or platelet-derived growth factor (PDGF) resulted in the translocation of an overexpressed DEF6-GFP fusion protein to the cell membrane and induced the formation of filopodia and lamellipodia. In contrast to full-length DEF6, expression of the DH-like (DHL) domain as a GFP fusion protein potently induced actin polymerization, including stress fiber formation in COS-7 cells, in the absence of PI 3-kinase signaling, indicating that it was constitutively active. The GTP-loading of Cdc42 was strongly enhanced in NIH 3T3 cells expressing the DH domain while filopodia formation, membrane ruffling, and stress fiber formation could be inhibited by the co-expression of the DH domain with dominant negative mutants of either N17Rac1, N17Cdc42, or N19RhoA, respectively. This indicated that DEF6 acts upstream of the Rho GTPases resulting in the activation of the Cdc42, Rac1, and RhoA signaling pathways. In vitro, DEF6 specifically interacted with Rac1, Rac2, Cdc42, and RhoA, suggesting a direct role for DEF6 in the activation of Rho GTPases. The ability of DEF6 to both stimulate actin polymerization and bind to filamentous actin suggests a role for DEF6 in regulating cell shape, polarity, and movement.     

Rac1-induced endocytosis is associated with intracellular proteolysis during migration through a three-dimensional matrix

Transfection of Rat1 fibroblasts with an activated form of rac1 (V12rac1) stimulated cell migration in vitro compared to transfection of Rat1 fibroblasts with vector only or with dominant negative rac1 (N17rac1). To investigate the involvement of proteases in this migration, we used a novel confocal assay to evaluate the ability of the Rat1 transfectants to degrade a quenched fluorescent protein substrate (DQ-green bovine serum albumin) embedded in a three-dimensional gelatin matrix. Cleavage of the substrate results in fluorescence, thus enabling one to image extracellular and intracellular proteolysis by living cells. The Rat1 transfectants accumulated degraded substrate intracellularly. V12rac1 increased accumulation of the fluorescent product in vesicles that also labeled with the lysosomal marker LysoTracker. Treatment of the V12rac1-transfected cells with membrane-permeable inhibitors of lysosomal cysteine proteases and a membrane-permeable selective inhibitor of the cysteine protease cathepsin B significantly reduced intracellular accumulation of degraded substrate, indicating that degradation occurred intracellularly. V12rac1 stimulated uptake of dextran 70 (a marker of macropinocytosis) and polystyrene beads (markers of phagocytosis) into vesicles that also labeled for cathepsin B. Thus, stimulation of the endocytic pathways of macropinocytosis and phagocytosis by activated Rac1 may be responsible for the increased internalization and subsequent degradation of extracellular proteins.     

Rac1蛋白活化加速缺氧诱导的人血管内皮细胞衰老

为阐明Rac1蛋白在人脐静脉内皮细胞（human umbilical vein endothelial cells,HUVECs）衰老中的作用及分子机制,我们采用持续缺氧的方法诱导内皮细胞衰老,检测缺氧前后内皮细胞衰老标志基因SA-β-Gal和PAI-1的表达、细胞周期分布和细胞增殖情况,同时分析缺氧前后细胞内Rac1蛋白的表达.结果显示,持续缺氧96 h后,HUVECs体积变大,细胞浆内颗粒和空泡增多,SA-β-Gal活性明显增加,PAI-1基因表达升高,细胞发生G1期阻滞,细胞增殖受抑,活化型Rac1蛋白表达上调,提示持续缺氧诱导的内皮细胞衰老可能与Rac1蛋白的活化有关.为进一步明确内皮细胞衰老与Rac1蛋白的关系,应用逆转录病毒将持续活化型Rac1（V12Rac1）和主导抑制型Rac1（N17Rac1）基因分别瞬时感染HUVECs,比较三种HUVECs（HUVECs,V12Rac1-HUVECs,N17Rac1-HUVECs）缺氧后的衰老变化,并分析其下游调控分子--血清反应因子（serum response factor,SRF）的表达和定位变化.研究发现,缺氧培养V12Rac1-HUVECs 48 h即可引起细胞衰老,表现为SA-β-Gal活性明显增加,PAI-1基因表达升高,细胞出现明显的G1期阻滞并且细胞增殖受抑,其改变与缺氧96 h的HUVECs相似;而N17Rac1明显抑制缺氧引起的内皮细胞衰老发生.上述结果说明,Rac1蛋白活化可以加速缺氧诱导的内皮细胞衰老,而抑制Rac1蛋白的活性则可抑制缺氧诱导的内皮细胞衰老.为进一步研究Rac1蛋白引起内皮细胞衰老的机制,通过免疫荧光染色及Western blot分析检测三种细胞缺氧处理后SRF的表达,发现：与HUVECs细胞比较,V12Rac1引起缺氧48 h HUVECs核蛋白中SRF的表达明显下降,SRF入核转位受到明显抑制;而N17Rac1感染后,缺氧HUVECs细胞核蛋白中SRF表达明显增多.上述结果提示：缺氧状态下Rac1蛋白活化能够明显加速HUVECs衰老,而抑制Rac1蛋白活性则明显抑制缺氧诱导的HUVECs衰老,SRF蛋白的核转位活化参与了Rac1蛋白调控HUVECs衰老的发生.     

Sphingosine 1-phosphate inhibits migration and RANTES production in human bronchial smooth muscle cells

Sphingosine 1-phosphate (S1P), a bioactive lipid mediator, has been shown to be increased in bronchoalveolar lavage fluid after allergen challenge in asthmatic patients. Here, we examined S1P actions and their intracellular signalings in cultured human bronchial smooth muscle cells (BSMCs). Expression of mRNAs of three subtypes of S1P receptors, including S1P(1), S1P(2), and S1P(3), was detected in BSMCs, and exposure of the cells to S1P inhibited platelet-derived growth factor (PDGF)-induced migration and tumor necrosis factor-alpha-induced RANTES production. S1P also inhibited PDGF-induced Rac1 activation, and dominant negative Rac1 inhibited PDGF-induced migration. On the other hand, dominant negative Galpha(q) attenuated the S1P-induced inhibition of RANTES production. Finally, an S1P(2)-selective antagonist, JTE-013, suppressed the S1P-induced inhibition of migration response and RANTES production. These results suggest that S1P attenuates cell migration by inhibiting a Rac1-dependent signaling pathway and decreases RANTES production by stimulating a Galpha(q)-dependent mechanism both possibly through the S1P(2) receptors.     

Activated Rac1 Selectively Up-regulates the Expression of Integrin α6β4 and Induces Cell Adhesion and Membrane Ruffles of Nonadherent Colon Cancer Colo201 Cells

Functions of small GTPases in integrin expression were investigated when the interaction of nonadherent human colon carcinoma 201 cells with the extracellular matrix (ECM) was examined. By transfection of the constitutively active form of a small GTPase Rac1, Rac V12, adhesion of cells to the ECM increased with concomitant cell spreading and formation of membrane ruffles. Activated Cdc42 and Cdc42 V12, but not wild-type Rac1, Cdc42, or RhoA, also induced the adhesion and spreading of Colo201 cells. This adhesion is integrin β4 dependent since an antibody for integrin β4 inhibited the RacV12-dependent cell adhesion and numbers of adhesive cells on laminin-coated plates exceeded those on collagen- and fibronectin-coated plates. By immunofluorescence, in addition to clustering of integrin molecules, expression of integrin α6β4 on the cell surface of Rac V12- and Cdc42 V12-expressing cells was selectively up-regulated without an increase in biosynthesis of α6β4 integrin. Treatment of Rac V12-expressing cells with wortmannin or LY294002, specific inhibitors of phosphoinositide 3-OH kinase, decreased the up-regulated α6β4 and cell adhesion. In light of this evidence, we propose that the regulation of integrin α6β4 expression induced by Rac1 and Cdc42 may play an important role in cell adhesion and tumorigenesis of colon carcinoma cells.     

A novel function of peroxiredoxin 1 (Prx-1) in apoptosis signal-regulating kinase 1 (ASK1)-mediated signaling pathway

We report a novel function of peroxiredoxin-1 (Prx-1) in the ASK1-mediated signaling pathway. Prx-1 interacts with ASK1 via the thioredoxin-binding domain of ASK1 and this interaction is highly inducible by H2O2. However, catalytic mutants of Prx1, C52A, C173A, and C52A/C173A, could not undergo H2O2 inducible interactions, indicating that the redox-sensitive catalytic activity of Prx-1 is required for the interaction with ASK1. Prx-1 overexpression inhibited the activation of ASK1, and resulted in the inhibition of downstream signaling cascades such as the MKK3/6 and p38 pathway. In Prx-1 knockdown cells, ASK1, p38, and JNK were quickly activated, leading to apoptosis in response to H2O2. These findings suggest a negative role of Prx-1 in ASK1-induced apoptosis.     

Nerve growth factor-induced neuronal differentiation requires generation of Rac1-regulated reactive oxygen species

Nerve growth factor (NGF) stimulation of pheochromocytoma PC12 cells transiently increased the intracellular concentration of reactive oxygen species (ROS). This increase was blocked by the chemical antioxidant N-acetylcysteine and a flavoprotein inhibitor, diphenylene iodonium. NGF responses of PC12 cells, including neurite outgrowth, tyrosine phosphorylation, and AP-1 activation, was inhibited when ROS production was prevented by N-acetylcysteine and diphenylene iodonium. The expression of dominant negative Rac1N17 blocked induction of both ROS generation and morphological differentiation by NGF. The ROS produced appears to be H(2)O(2), because the introduction of catalase into the cells abolished NGF-induced neurite outgrowth, ROS production, and tyrosine phosphorylation. These results suggest that the ROS, perhaps H(2)O(2), acts as an intracellular signal mediator for NGF-induced neuronal differentiation and that NGF-stimulated ROS production is regulated by Rac1 and a flavoprotein-binding protein similar to the phagocytic NADPH oxidase.     

Pak regulates calpain-dependent degradation of E3b1

E3b1, a binding partner of Eps8, plays a critical role in receptor tyrosine kinase (RTK)-mediated Rac activation by facilitating the interaction of Eps8 with Sos-1 and the consequent activation of the Rac-specific guanine nucleotide exchange factor activity of Sos-1. Here we present evidence that E3b1 levels are regulated by the Ca(2+)-activated protease calpain, and also by Pak, a downstream target of Rac signaling. Serum starvation of Rat2 or COS7 cells resulted in rapid loss of E3b1 that was reversed by calpain inhibitors. Loss was also prevented by expressing the constitutively active Pak1 mutant, Pak1(H83,86L). Activation of endogenous Pak by platelet-derived growth factor or the constitutively active Rac1 mutant, Rac1(G12V), also inhibited degradation. In contrast, inhibition of endogenous Pak activity by expressing the Pak auto-inhibitory domain caused degradation of over-expressed E3b1 even in the presence of serum. Taken together, these findings indicate that E3b1 is down-regulated by calpain activation and stabilized by Pak activation. They also suggest that RTK-mediated Rac activation can be modulated by changes in the level of E3b1 in response to signals that affect the activity of calpain or Pak.     

Sphingosine 1-phosphate stimulation of NADPH oxidase activity: relationship with platelet-derived growth factor receptor and c-Src kinase

This study demonstrates for the first time that sphingosine 1-phosphate (S1P) increases H2O2 production in NIH3T3 fibroblasts through NADPH oxidase activation, confirming the involvement of phosphoinositide-3-kinase and protein kinase C in the activation of this enzyme in non-phagocyte mammalian cells. The results demonstrate also that both platelet-derived growth factor (PDGF) and S1P-mediated NADPH oxidase activation and H2O2 production by Gi-protein coupled receptors (GPCRs) and c-Src kinase. Moreover, both PDGF and S1P activate c-Src kinase through GPCRs, indicating that this kinase can constitute a connection factor between PDGF and S1P signaling, confirming the cross-talk previously found between their receptors. Thus, Gi-protein-mediated NADPH oxidase activation with the consequent H2O2 increase constitutes an early event in the PDGF and S1P pathways. However, a different time course of H2O2 production in S1P-stimulated cells compared to that obtained in PDGF-stimulated cells has been observed, and this seems to be related to the different activation behavior of c-Src kinase induced after S1P or PDGF stimulation. Finally, these data demonstrate that S1P-induced H2O2 production is necessary to maximize c-Src kinase activation, confirming that this is a redox regulated kinase. After which, c-Src plays an important role both upstream and downstream from NADPH oxidase activation.     

Rac-dependent anti-apoptotic signaling by the insulin receptor cytoplasmic domain.

Mutations in the cytoplasmic domain of the insulin receptor that block the ability of the receptor to stimulate glucose uptake do not block the receptor's ability to inhibit apoptosis (Boehm, J. E., Chaika, O. V., and Lewis, R. E. (1998) J. Biol. Chem. 273, 7169-7176). To characterize this survival pathway we used a chimeric receptor (CSF1R/IR) consisting of the ligand-binding domain of the colony-stimulating factor-1 receptor spliced to the cytoplasmic domain of the insulin receptor and a mutated version of the chimeric receptor containing a 12-amino acid deletion of the juxtamembrane domain (CSF1R/IRDelta960). In addition to the inhibition of apoptosis, activation of either the CSF1R/IR or the CSF1R/IRDelta960 rapidly induced membrane ruffling in Rat1 fibroblasts. The small GTPase Rac mediates membrane ruffling. Activated and dominant-inhibitory mutants of Rac and other small GTPases were expressed in Rat1 fibroblasts to examine a potential link between the intracellular pathways that induce membrane ruffling and promote cell survival. The anti-apoptotic action of the CSF1R/IRDelta960 was reversed by dominant-inhibitory Rac(N17), but not by Ras(N17) or Cdc42(N17). Activated Rac(V12), but not Ras(D12) or Cdc42(V12), promoted cell survival in the absence of insulin. These data implicate Rac as a mediator of an unique anti-apoptotic signaling pathway activated by the insulin receptor cytoplasmic domain.     

Basic fibroblast growth factor stimulates activation of Rac1 through a p85 betaPIX phosphorylation-dependent pathway

In a previous study (Shin, E. Y., Shin, K. S., Lee, C. S., Woo, K. N., Quan, S. H., Soung, N. K., Kim, Y. G., Cha, C. I., Kim, S. R., Park, D., Bokoch, G. M., and Kim, E. G. (2002) J. Biol. Chem. 277, 44417-44430) we reported that phosphorylation of p85 betaPIX, a guanine nucleotide exchange factor (GEF) for Rac1/Cdc42, is a signal for translocation of the PIX complex to neuronal growth cones and is associated with basic fibroblast growth factor (bFGF)-induced neurite outgrowth. However, the issue of whether p85 betaPIX phosphorylation affects GEF activity on Rac1/Cdc42 is yet to be explored. Here we show that Rac1 activation occurs in a p85 betaPIX phosphorylation-dependent manner. A GST-PBD binding assay reveals that Rac1 is activated in a dose- and time-dependent manner in PC12 cells in response to bFGF. Inhibition of ERK or PAK2, the kinases upstream of p85 betaPIX in the bFGF signaling, prevents Rac1 activation, suggesting that phosphorylation of p85 betaPIX functions upstream of Rac1 activation. To directly address this issue, transfection studies with wild-type and mutant p85 betaPIX (S525A/T526A, a non-phosphorylatable form) were performed. Expression of mutant PIX markedly inhibits both bFGF- and nerve growth factor (NGF)-induced activation of Rac1, indicating that phosphorylation of p85 betaPIX is responsible for activation of this G protein. Both wild-type and mutant p85 betaPIX displaying negative GEF activity (L238R/L239S) are similarly recruited to growth cones, suggesting that Rac1 activation is not essential for translocation of the PIX complex (PAK2-p85 betaPIX-Rac1). However, expression of mutant p85 betaPIX (L238R/L239S) results in retraction of the pre-existing neurites. Our results provide evidence that bFGF- and NGF-induced phosphorylation of p85 betaPIX mediates Rac1 activation, which in turn regulates cytoskeletal reorganization at growth cones, but not translocation of the PIX complex.     

Implication of a small GTPase Rac1 in the activation of c-Jun N-terminal kinase and heat shock factor in response to heat shock

Heat shock induces c-Jun N-terminal kinase (JNK) activation as well as heat shock protein (HSP) expression through activation of the heat shock factor (HSF), but its signal pathway is not clearly understood. Since a small GTPase Rac1 has been suggested to participate in the cellular response to stresses, we examined whether Rac1 is involved in the heat shock response. Here we show that moderate heat shock (39-41 degrees C) induces membrane translocation of Rac1 and membrane ruffling in a Rac1-dependent manner. In addition, Rac1N17, a dominant negative mutant of Rac1, significantly inhibited JNK activation by heat shock. Since Rac1V12 was able to activate JNK, it is suggested that heat shock may activate JNK via Rac1. Similar inhibition by Rac1N17 of HSF activation in response to heat shock was observed. However, inhibitory effects of Rac1N17 on heat shock-induced JNK and HSF activation were reduced as the heat shock temperature increased. Rac1N17 also inhibited HSF activation by l-azetidine-2-carboxylic acid, a proline analog, and heavy metals (CdCl)), suggesting that Rac1 may be linked to HSF activation by denaturation of polypeptides in response to various proteotoxic stresses. However, Rac1N17 did not prevent phosphorylation of HSF1 in response to these proteotoxic stresses. Interestingly, a constitutively active mutant Rac1V12 did not activate the HSF. Therefore, Rac1 activation may be necessary, but not sufficient, for heat shock-inducible HSF activation and HSP expression, or otherwise a signal pathway(s) involving Rac1 may be indirectly involved in the HSF activation. In sum, we suggest that Rac1 may play a critical role(s) in several aspects of the heat shock response.