Roles of phosphatidylinositol 3-kinase and Rac in the nuclear signaling by tumor necrosis factor-alpha in rat-2 fibroblasts.

We investigated the extent to which phosphatidylinositol 3-kinase (PI 3-kinase) and Rac, a member of the Rho family of small GTPases, are involved in the signaling cascade triggered by tumor necrosis factor (TNF)-alpha leading to activation of c-fos serum response element (SRE) and c-Jun amino-terminal kinase (JNK) in Rat-2 fibroblasts. Inhibition of PI 3-kinase by LY294002 or wortmannin, two specific PI 3-kinase antagonists, or co-transfection with a dominant negative mutant of PI 3-kinase dose-dependently blocked stimulation of c-fos SRE by TNF-alpha. Similarly, LY294002 significantly diminished TNF-alpha-induced activation of JNK, suggesting that nuclear signaling triggered by TNF-alpha is dependent on PI 3-kinase-mediated activation of both c-fos SRE and JNK. We also found nuclear signaling by TNF-alpha to be Rac-dependent, as demonstrated by the inhibitory effect of transient co-transfection with a dominant negative Rac mutant, RacN17. Our findings suggest that Rac is situated downstream of PI 3-kinase in the TNF-alpha signaling pathway to the nucleus, and we conclude that PI 3-kinase and Rac each plays a pivotal role in the nuclear signaling cascade triggered by TNF-alpha.     

Leukotriene B(4) stimulates Rac-ERK cascade to generate reactive oxygen species that mediates chemotaxis.

Leukotriene B(4) is a potent chemoattractant known to be involved mainly in inflammation, immune responses, and host defense against infection, although the exact signaling mechanisms by which it exerts its effects are not well understood. Here we show that exogenous leukotriene B(4) induces reactive oxygen species (ROS) generation via a Rac-dependent pathway, and that stable expression of Rac(N17), a dominant negative Rac1 mutant, completely blocks leukotriene B(4)-induced ROS generation. In addition, leukotriene B(4)-induced ROS generation is selectively blocked by inhibition of ERK or cytosolic phospholipase A(2), but not p38 kinase, which is indicative of its dependence on ERK activation and synthesis of arachidonic acid. Consistent with those findings, leukotriene B(4) Rac-dependently stimulates ERK and cytosolic phospholipase A(2) activity, and transient transfection with plasmid expressing Rac(V12), a constitutively activated Rac1 mutant, also dose-dependently stimulates ERK activity. Our findings suggest that ERK and cytosolic phospholipase A(2) are situated downstream of Rac, and we conclude that Rac, ERK, and cytosolic phospholipase A(2) all play pivotal roles in mediating the ROS generation that appears to be a prerequisite for leukotriene B(4)-induced chemotaxis and cell proliferation.     

Wave2 activates serum response element via its VCA region and functions downstream of Rac

WAVE2 is a member of the WASP/WAVE family of protein effectors of actin reorganization and cell movement. In this report, we demonstrate that WAVE2 overexpression induces serum response element (SRE) activation through serum response factor. A WAVE2 mutant lacking the VCA region did not induce SRE activation and actin polymerization. WAVE2-induced SRE activation was blocked by exposure of cells to Latrunculin A, or overexpression of actin mutant R62D. The DeltaVCA mutant inhibited Rac V12-induced SRE activation, suggesting that WAVE2 lies downstream of Rac. Similar deletion of the VCA domain of WASP attenuated Cdc42 V12-mediated SRE activation, suggesting that WAVE2 acts in relation to Rac as WASP acts in relation to Cdc42. WAVE2 overexpression did not activate NF-kappaB.     

Roles of Rac and cytosolic phospholipase A2 in the intracellular signalling in response to titanium particles

Titanium (Ti) particle is one of the prosthetic materials commonly used in implantation and has frequently been implicated in pathogenesis such as periprosthetic osteolysis. In the present study, we undertook to understand the intracellular signalling pathway stimulated by exogenous Ti at Rat-2 fibroblasts. By reporter gene analysis following transient transfections, exogenous Ti was shown to stimulate c-fos serum response element (SRE)-dependent luciferase activities in a dose-dependent manner. In addition, Ti-induced SRE activation was shown to be dramatically repressed by RacN17, a dominant negative mutant of Rac1, suggesting that Rac GTPase is essential for the signalling of Ti to c-fos SRE. Furthermore, pretreatment with MAFP, an inhibitor of cytosolic phospholipase A(2) (cPLA(2)), MK886, an inhibitor of 5-lipoxygenase (5-LO), or indomethacin, a general inhibitor of cyclooxygenase (COX), also significantly repressed Ti-induced SRE activation, suggesting mediatory roles of cPLA(2) and subsequent arachidonic acid (AA) metabolisms to leukotrienes (LTs) and prostaglandins (PGs) in the Ti signalling to c-fos SRE. Consistent with these results, intracellular levels of leukotriene B(4) (LTB(4)) and prostaglandin E(2) (PGE(2)) were Rac-dependently elevated in cells exposed to Ti particles.     

Induction of the nuclear proto-oncogene c-fos by the phorbol ester TPA and v-H-Ras

TPA is known to cooperate with an activated Ras oncogene in the transformation of rodent fibroblasts, but the biochemical mechanisms responsible for this effect have not been established. In the present study we used c-fos promoter-luciferase constructs as reporters, in transient transfection assays, in NIH3T3 cells to assess the mechanism of this cooperation. We found a marked synergistic interaction between TPA and a transfected v-Ha-ras oncogene in the activation of c-fos promoter and SRE. SRE has binding sites for TCF and SRF. A dominant-negative Ras (ras-N17) inhibited the TPA-Ras synergy by blocking the PKC-MAPK-TCF pathway. Dominant-negative RhoA and Rac1 (but not Cdc42Hs) inhibited the TPA-Ras synergy by blocking the Ras-Rho-SRF signaling pathway. Constitutively active PKCalpha and PKCepsilon showed synergy with v-Ras. These results suggest that the activation of two distinct pathways such as Ras-Raf-ERK-TCF pathway and Rho-SRF pathway are responsible for the induction of c-fos by TPA and Ras in mitogenic signaling pathways.     

Role of cytosolic phospholipase A(2) as a downstream mediator of Rac in the signaling pathway to JNK stimulation

Rac is an important regulatory molecule implicated in c-jun N-terminal kinase (JNK) activation in response to stress and cytokines. However, the signaling events that mediate the activation of JNK by Rac are not yet well characterized. To broaden our understanding of downstream mediators that link Rac signals to the JNK pathway, we investigated whether cytosolic phospholipase A(2) (cPLA(2)) is involved in Rac activation of JNK. In this report we demonstrate that either co-transfection with antisense cPLA(2) oligonucleotide or pretreatment with arachidonyltrifluoromethyl ketone (AACOCF3), a potent and specific inhibitor of cPLA(2), inhibits Rac-mediated JNK activation, implying a potential role of cPLA(2) in Rac-signaling to JNK activation. In accordance with this observation, we demonstrate that the addition of exogenous arachidonic acid (AA), a principal product of Rac-activated cPLA(2), or leukotrienes, products of 5-lipoxygenase (5-LO) of AA, caused a specific stimulation of JNK. Together, our findings suggest that cPLA(2) mediates, at least partly, the signaling cascade by which Rac stimulates JNK.     

Rac and protein kinase C-delta regulate ERKs and cytosolic phospholipase A2 in FcepsilonRI signaling to cysteinyl leukotriene synthesis in mast cells

Although cysteinyl leukotrienes (cysLTs) are known to be principal inflammatory lipid mediators released from IgE-stimulated mast cells, the signaling mechanisms involved in the synthesis of cysLTs remain largely unknown. In the present study, therefore, we investigated the signaling pathway by which IgE induces cysLTs synthesis after binding to its high affinity receptor (FcepsilonRI) in RBL-2H3 mast cells. We found that IgE-induced cysLT synthesis is completely abolished in RBL-2H3(Rac-N17) cells, a stable cell line expressing Rac(N17), a dominant negative Rac1 mutant; conversely, synthesis was enhanced in cells expressing Rac(V12), a constitutively active Rac1 mutant, suggesting that Rac1 is a key mediator of IgE signaling to cysLT synthesis. Further analysis aimed at identifying mediators downstream of Rac1 revealed that pretreating cells with a protein kinase C-delta (PKC-delta) inhibitor or infection with an adenoviral vector harboring a dominant negative PKC-delta mutant significantly attenuates IgE-induced ERKs phosphorylation, cytosolic phospholipase A(2) phosphorylation/translocation, and cysLT synthesis. In addition, the expression of Rac(N17) blocked PKC-delta translocation and impaired the phosphorylation of ERKs and cytosolic phospholipase A(2) otherwise elicited by IgE stimulation. Taken together these results suggest that PKC-delta also plays a critical mediatory role in the IgE signaling pathway leading to cysLT synthesis, acting downstream of Rac1. Finally, the physiological significance of PKC-delta in the IgE signaling pathway was demonstrated in an Ag (OVA)-challenged in vivo mouse model, in which induced levels of cysLTs and airway responsiveness in lung airways were significantly diminished by prior i.p. injection of a PKC-delta inhibitor.     

Ras-Raf-Arf signaling critically depends on the Dmp1 transcription factor

Dmp1 prevents tumor formation by activating the Arf-p53 pathway. In cultured primary cells, the Dmp1 promoter was efficiently activated by oncogenic Ha-Ras(V12), but not by overexpressed c-Myc or E2F-1. Dmp1 promoter activation by Ras(V12) depended on Raf-MEK-ERK signaling. Induction of p19(Arf) and p21(Cip1) by oncogenic Raf was compromised in Dmp1-null cells, which were resistant to Raf-mediated premature senescence. A Ras(V12)-responsive element was mapped to the 5' leader sequence of the murine Dmp1 promoter, where endogenous Fos and Jun family proteins bind. Dmp1 promoter activation by Ras(V12) was strikingly impaired in c-Jun as well as in JunB knock-down cells, suggesting the critical role of Jun proteins in the activation of the Dmp1 promoter. A Ras(V12)-responsive element was mapped to the unique Dmp1/Ets site on the Arf promoter, where endogenous Dmp1 proteins bind upon oncogenic Raf activation. Therefore, activation of Arf by Ras/Raf signaling is indirectly mediated by Dmp1, explaining why Dmp1-null primary cells are highly susceptible to Ras-induced transformation. Our data indicate the presence of the novel Jun-Dmp1 pathway that directly links oncogenic Ras-Raf signaling and p19(Arf), independent of the classical cyclin D1/Cdk4-Rb-E2F pathway.     

G13alpha-mediated PYK2 activation. PYK2 is a mediator of G13alpha -induced serum response element-dependent transcription

G(12)alpha/G(13)alpha transduces signals from G-protein-coupled receptors to stimulate growth-promoting pathways and the early response gene c-fos. Within the c-fos promoter lies a key regulatory site, the serum response element (SRE). Here we show a critical role for the tyrosine kinase PYK2 in muscarinic receptor type 1 and G(12)alpha/G(13)alpha signaling to an SRE reporter gene. A kinase-inactivate form of PYK2 (PYK2 KD) inhibits muscarinic receptor type 1 signaling to the SRE and PYK2 itself triggers SRE reporter gene activation through a RhoA-dependent pathway. Placing PYK2 downstream of G-protein activation but upstream of RhoA, the expression of PYK2 KD blocks the activation of an SRE reporter gene by GTPase-deficient forms of G(12)alpha or G(13)alpha but not by RhoA. The GTPase-deficient form of G(13)alpha triggers PYK2 kinase activity and PYK2 tyrosine phosphorylation, and co-expression of the RGS domain of p115 RhoGEF inhibits both responses. Finally, we show that in vivo G(13)alpha, although not G(12)alpha, readily associates with PYK2. Thus, G-protein-coupled receptors via G(13)alpha activation can use PYK2 to link to SRE-dependent gene expression.     

Observations of rotation within the F(o)F(1)-ATP synthase: deciding between rotation of the F(o)c subunit ring and artifact

Annexin-I (ANX-I) is a 37-kDa protein with a calcium-dependent phospholipid-binding property. Previously we have observed the inhibition of cytosolic phospholipase A2 (cPLA2) by ANX-I in the studies using purified recombinant ANX-I, and proposed a specific interaction model for the mechanism of cPLA2 inhibition by ANX-I [Kim et al. (1994) FEBS Lett. 343, 251-255]. Here we have studied the role of ANX-I in the cPLA2 signaling pathway by transient transfection assay. The stimulation of Rat2 fibroblast cells with phorbol 12-myristate 13-acetate (PMA) induced the c-fos serum response element (SRE). The SRE stimulation by PMA was dramatically reduced by (1) pretreatment with a cPLA2-specific inhibitor, arachidonyltrifluoromethyl ketone, or (2) co-transfection with antisense cPLA2 oligonucleotide, indicating that the SRE activation was through cPLA2 activation. Co-transfection with an ANX-I expression vector also reduced the SRE stimulation by PMA, suggesting the inhibition of cPLA2 by ANX-I. The active domain of ANX-I was mapped using various deletion mutants. ANX-I(1-113) and ANX-I(34-346) were fully active, whereas ANX-I(114-346) abolished the activity. Therefore the activity was in the amino acid 34 to 113 region, which corresponds to the conserved domain I of ANX-I.     

Oncoprotein-mediated signalling cascade stimulates c-Jun activity by phosphorylation of serines 63 and 73.

In resting cells, c-Jun is phosphorylated on five sites. Three of these sites reside next to its DNA binding domain and negatively regulate DNA binding. In response to expression of oncogenic Ha-Ras, phosphorylation of these sites decreases, while phosphorylation of two other sites within c-Jun's activation domain is greatly enhanced. Phosphorylation of these residues, serines 63 and 73, stimulates the transactivation function of c-Jun and is required for oncogenic cooperation with Ha-Ras. We now show that the same changes in c-Jun phosphorylation are elicited by a variety of transforming oncoproteins with distinct biochemical activities. These oncoproteins, v-Sis, v-Src, Ha-Ras, and Raf-1, participate in a signal transduction pathway that leads to increased phosphorylation of serines 63 and 73 on c-Jun. While oncogenic Ha-Ras is a constitutive stimulator of c-Jun activity and phosphorylation, the normal c-Ha-Ras protein is a serum-dependent modulator of c-Jun's activity. c-Jun is therefore a downstream target for a phosphorylation cascade involved in cell proliferation and transformation.     

Signaling through CD5 Activates a Pathway Involving Phosphatidylinositol 3-Kinase, Vav, and Rac1 in Human Mature T Lymphocytes

CD5 acts as a coreceptor on T lymphocytes and plays an important role in T-cell signaling and T-cell–B-cell interactions. Costimulation of T lymphocytes with anti-CD5 antibodies results in an increase of the intracellular Ca²⁺ levels, and subsequently in the activation of Ca²⁺/calmodulin-dependent (CaM) kinase type IV. In the present study, we have characterized the initial signaling pathway induced by anti-CD5 costimulation. The activation of phosphatidylinositol (PI) 3-kinase through tyrosine phosphorylation of its p85 subunit is a proximal event in the CD5-signaling pathway and leads to the activation of the lipid kinase activity of the p110 subunit. The PI 3-kinase inhibitors wortmannin and LY294002 inhibit the CD5-induced response as assessed in interleukin-2 (IL-2) secretion experiments. The expression of an inactivated Rac1 mutant (Rac1 · N17) in T lymphocytes transfected with an IL-2 promoter-driven reporter construct also abrogates the response to CD5 costimulation, while the expression of a constitutively active Rac1 mutant (Rac1-V12) completely replaces the CD5 costimulatory signal. The Rac1-specific guanine nucleotide exchange factor Vav is heavily phosphorylated on tyrosine residues upon CD5 costimulation, which is a prerequisite for its activation. A role for Vav in the CD5-induced signaling pathway is further supported by the findings that the expression of a dominant negative Vav mutant (Vav-C) completely abolishes the response to CD5 costimulation while the expression of a constitutively active Vav mutant [Vav(Δ1–65)] makes the CD5 costimulation signal superfluous. Wortmannin is unable to block the Vav(Δ1–65)- or Rac1 · V12-induced signals, indicating that both Vav and Rac1 function downstream from PI 3-kinase. Vav and Rac1 both act upstream from the CD5-induced activation of CaM kinase IV, since KN-62, an inhibitor of CaM kinases, and a dominant negative CaM kinase IV mutant block the Vav(Δ1–65)-and Rac1 · V12-mediated signals. We propose a model for the CD5-induced signaling pathway in which the PI 3-kinase lipid products, together with tyrosine phosphorylation, activate Vav, resulting in the activation of Rac1 by the Vav-mediated exchange of GDP for GTP.     

Differential requirement for Rho family GTPases in an oncogenic insulin-like growth factor-I receptor-induced cell transformation

Insulin-like growth factor I receptor (IGFR) plays an important role in cell growth and transformation. We dissected the downstream signaling pathways of an oncogenic variant of IGFR, Gag-IGFR, called NM1. Loss of function mutants of NM1, Phe-1136 and dS2, that retain kinase activity but are attenuated in their transforming ability were used to identify signaling pathways that are important for transformation of NIH 3T3 cells. MAPK, phospholipase C gamma, and Stat3 were activated to the same extent by NM1 and its two mutants, suggesting that activation of these pathways, individually or in combination, was not sufficient for NM1-induced cell transformation. The mutant dS2 has decreased IRS-1 phosphorylation levels and IRS-1-associated phosphatidylinositol 3'-kinase activity, suggesting that this impairment may be in part responsible for the defectiveness of dS2. We show that Rho family members, RhoA, Rac1, and Cdc42 are activated by NM1, and this activation, particularly RhoA and Cdc42, is attenuated in both mutants of NM1. Dominant negative mutants of Rho, Rac, and Cdc42 inhibited NM1-induced cell transformation, as measured by focus and colony forming ability. Dominant negative Rho most potently inhibited the focus forming activity, whereas Cdc42 was most effective in inhibiting the colony forming ability of NM1-expressing cells. Conversely, constitutively activated (ca) Rho is more effective than ca Rac or ca Cdc42 in rescuing the focus forming ability of the mutants. By contrast, ca Cdc42 is most effective in rescuing the colony forming ability of both mutants.     

Tumor necrosis factor-alpha generates reactive oxygen species via a cytosolic phospholipase A2-linked cascade

Reactive oxygen species (ROS) are important regulatory molecules implicated in the signaling cascade triggered by tumor necrosis factor (TNF)-alpha, although the events through which TNF-alpha induces ROS generation are not yet well characterized. We therefore investigated selected candidates likely to mediate TNF-alpha-induced ROS generation. Consistent with the role of Rac in that process, stable expression of Rac(Asn-17), a dominant negative Rac1 mutant, completely blocked TNF-alpha-induced ROS generation. To understand better the mediators downstream of Rac, we investigated the involvement of cytosolic phospholipase A(2) (cPLA(2)) activation and metabolism of the resultant arachidonic acid (AA) by 5-lipoxygenase (5-LO). TNF-alpha-induced ROS generation was blocked by inhibition of cPLA(2) or 5-LO, but not cyclooxygenase, suggesting that TNF-alpha-induced ROS generation is dependent on synthesis of AA and its subsequent metabolism to leukotrienes. Consistent with that hypothesis, TNF-alpha Rac-dependently stimulated endogenous production of leukotriene B(4) (LTB(4)), while exogenous application of LTB(4) increased levels of ROS. In contrast, application of leukotrienes C(4), D(4), and E(4) or prostaglandin E(2) had little effect. Our findings suggest that LTB(4) production by 5-LO is situated downstream of the Rac-cPLA(2) cascade, and we conclude that Rac, cPLA(2), and LTB(4) play pivotal roles in the ROS-generating cascade triggered by TNF-alpha.     

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.     

Differential activation of the Rac pathway by Ha-Ras and K-Ras

Ras proteins are key regulators of cell growth and differentiation. Mammalian cells express three closely related Ras proteins: Ha-Ras, K-Ras, and N-Ras. We have compared the abilities of the Ha-Ras and K-Ras isoforms to activate the Rac effector pathway, using three Rac-dependent readouts: induction of membrane ruffling and pinocytosis, stimulation of cell motility, and Pak binding. The total surface area of membrane ruffles induced by K-RasV12 was 2-fold greater than that induced by Ha-RasV12. Likewise, the number of K-RasV12-induced pinocytic vesicles per cell was approximately 2-fold greater than that induced by Ha-RasV12. In a wound healing assay, K-RasV12-injected cells migrated twice as fast as Ha-RasV12-injected cells. Moreover, the Pak binding activity of Rac, which is indicative of the amount of GTP-bound Rac, was higher in K-RasV12-expressing cells than Ha-RasV12-expressing cells. These results suggest that K-Ras activates Rac more efficiently than Ha-Ras. The preferential activation of Rac by K-Ras is dependent on the mode of membrane anchoring and impacts on the ability of K-Ras to regulate cell survival.     

Activation of Rac1, RhoA, and mitogen-activated protein kinases is required for Ras transformation.

Although substantial evidence supports a critical role for the activation of Raf-1 and mitogen-activated protein kinases (MAPKs) in oncogenic Ras-mediated transformation, recent evidence suggests that Ras may activate a second signaling pathway which involves the Ras-related proteins Rac1 and RhoA. Consequently, we used three complementary approaches to determine the contribution of Rac1 and RhoA function to oncogenic Ras-mediated transformation. First, whereas constitutively activated mutants of Rac1 and RhoA showed very weak transforming activity when transfected alone, their coexpression with a weakly transforming Raf-1 mutant caused a greater than 35-fold enhancement of transforming activity. Second, we observed that coexpression of dominant negative mutants of Rac1 and RhoA reduced oncogenic Ras transforming activity. Third, activated Rac1 and RhoA further enhanced oncogenic Ras-triggered morphologic transformation, as well as growth in soft agar and cell motility. Finally, we also observed that kinase-deficient MAPKs inhibited Ras transformation. Taken together, these data support the possibility that oncogenic Ras activation of Rac1 and RhoA, coupled with activation of the Raf/MAPK pathway, is required to trigger the full morphogenic and mitogenic consequences of oncogenic Ras transformation.     

Protein kinase Ciota is required for Ras transformation and colon carcinogenesis in vivo

Protein kinase C iota (PKCiota) has been implicated in Ras signaling, however, a role for PKCiota in oncogenic Ras-mediated transformation has not been established. Here, we show that PKCiota is a critical downstream effector of oncogenic Ras in the colonic epithelium. Transgenic mice expressing constitutively active PKCiota in the colon are highly susceptible to carcinogen-induced colon carcinogenesis, whereas mice expressing kinase-deficient PKCiota (kdPKCiota) are resistant to both carcinogen- and oncogenic Ras-mediated carcinogenesis. Expression of kdPKCiota in Ras-transformed rat intestinal epithelial cells blocks oncogenic Ras-mediated activation of Rac1, cellular invasion, and anchorage-independent growth. Constitutively active Rac1 (RacV12) restores invasiveness and anchorage-independent growth in Ras-transformed rat intestinal epithelial cells expressing kdPKCiota. Our data demonstrate that PKCiota is required for oncogenic Ras- and carcinogen-mediated colon carcinogenesis in vivo and define a procarcinogenic signaling axis consisting of Ras, PKCiota, and Rac1.     

K-Ras mediated murine epidermal tumorigenesis is dependent upon and associated with elevated Rac1 activity

A common goal for potential cancer therapies is the identification of differences in protein expression or activity that would allow for the selective targeting of tumor vs. normal cells. The Ras proto-oncogene family (K-Ras, H-Ras and N-Ras) are amongst the most frequently mutated genes in human cancers. As a result, there has been substantial effort dedicated to determining which pathways are activated by Ras signaling and, more importantly, which of these contribute to cancer. Although the most widely studied Ras-regulated signaling pathway is the Raf/mitogen-activated protein kinase cascade, previous research in model systems has revealed that the Rac1 GTP-binding protein is also required for Ras-induced biological responses. However, what have been lacking are rigorous in vivo Rac1 target validation data and a clear demonstration that in Ras-driven hyperplastic lesions, Rac1 activity is increased. Using a combination of genetically-modified mouse models that allow for the tissue-selective activation or deletion of signaling molecules and an activation-state sensitive Rac1 antibody that detects GTP-bound Rac1, we found that Rac1 contributes to K-Ras induced epidermal papilloma initiation and growth and that Rac1 activity is elevated by oncogenic K-Ras in vivo. Previously, it was not practical to assess Rac1 activation status in the most commonly used format for clinical tumor specimens, formalin-fixed paraffin embedded (FFPE) tissues samples. However, this study clearly demonstrates that Rac1 is essential for K-Ras driven epithelial cell hyperproliferation and that Rac1 activity is elevated in tissues expressing mutant oncogenic K-Ras, while also characterizing the activation-state specific Rac1-GTP antibody as a probe to examine Rac1 activation status in FFPE samples. Our findings will facilitate further research on the status of Rac1 activity in human tumors and will help to define the tumor types of the patient population that could potentially benefit from therapies targeting Rac activation or downstream effector signaling pathways.