Role of a PDZ1 domain of NHERF1 in the binding of airway epithelial RACK1 to NHERF1

In past studies, we demonstrated regulation of CFTR Cl channel function by protein kinase C (PKC)- through the binding of PKC- to RACK1 (a receptor for activated C-kinase) and of RACK1 to human Na⁺/H⁺ exchanger regulatory factor (NHERF1). In this study, we investigated the site of RACK1 binding on NHERF1 using solid-phase and solution binding assays and pulldown, immunoprecipitation, and ³⁶Cl efflux experiments. Recombinant RACK1 binding to glutathione S-transferase (GST)-tagged PDZ1 domain of NHERF1 was 10-fold higher than its binding to GST-tagged PDZ2 domain of NHERF1. PDZ1 binds to RACK1 in a dose-dependent manner and vice versa, with similar binding constants of 1.67 and 1.26 µg, respectively. Interaction of the PDZ1 domain with RACK1 was not blocked by binding of activated PKC- to RACK1. A GST-tagged PDZ1 domain pulled down endogenous RACK1 from Calu-3 cell lysate. An internal 11-amino acid motif embedding the GYGF carboxylate binding loop of PDZ1 binds to RACK1, inhibits binding of recombinant NHERF1 and RACK1, pulls down endogenous RACK1 from Calu-3 cell lysate, and blocks coimmunoprecipitation of endogenous RACK1 with endogenous NHERF1 but does not affect cAMP-dependent activation of CFTR. A similar amino acid sequence in the PDZ2 domain did not bind RACK1. Our results indicate binding of Calu-3 RACK1 predominantly to the PDZ1 domain of NHERF1 at a site encompassing the GYGF loop of the PDZ1 domain and a site on RACK1 distinct from a PKC- binding site. CFTR activation by cAMP-generating agent is not affected by loss of RACK1-NHERF1 interaction. cystic fibrosis; cystic fibrosis transmembrane conductance regulator; protein-protein interaction; slot blot assay; pulldown; PDZ domain; chloride efflux; immunoprecipitation     

Protein kinase A gating of a pseudopodial-located RhoA/ROCK/p38/NHE1 signal module regulates invasion in breast cancer cell lines

Metastasis results from a sequence of selective events often involving interactions with elements of the tumor-specific physiological microenvironment. The low-serum component of this microenvironment confers increased motility and invasion in breast cancer cells by activating the Na+/H+ exchanger isoform 1 (NHE1). The present study was undertaken to characterize the signal transduction mechanisms underlying this serum deprivation-dependent activation of both the NHE1 and the concomitant invasive characteristics such as leading edge pseudopodia development and penetration of matrigel in breast cancer cell lines representing different stages of metastatic progression. Using pharmacological and genetic manipulation together with transport and kinase activity assays, we observe that the activation of the NHE1 and subsequent invasion by serum deprivation in metastatic human breast cells is coordinated by a sequential RhoA/p160ROCK/p38MAPK signaling pathway gated by direct protein kinase A phosphorylation and inhibition of RhoA. Fluorescence resonance energy transfer imaging of RhoA activity and immunofluorescence analysis of phospho-RhoA and NHE1 show that serum deprivation dynamically remodels the cell, forming long, leading edge pseudopodia and that this signal module is preferentially compartmentalized in these leading edge pseudopodia, suggesting a tight topographic relation of the signaling module to an invasion-specific cell structure.     

Canonical and Noncanonical Sites Determine NPT2A Binding Selectivity to NHERF1 PDZ1

Na⁺/H⁺ Exchanger Regulatory Factor-1 (NHERF1) is a scaffolding protein containing 2 PDZ domains that coordinates the assembly and trafficking of transmembrane receptors and ion channels. Most target proteins harboring a C-terminus recognition motif bind more-or-less equivalently to the either PDZ domain, which contain identical core-binding motifs. However some substrates such as the type II sodium-dependent phosphate co-transporter (NPT2A), uniquely bind only one PDZ domain. We sought to define the structural determinants responsible for the specificity of interaction between NHERF1 PDZ domains and NPT2A. By performing all-atom/explicit-solvent molecular dynamics (MD) simulations in combination with biological mutagenesis, fluorescent polarization (FP) binding assays, and isothermal titration calorimetry (ITC), we found that in addition to canonical interactions of residues at 0 and -2 positions, Arg at the -1 position of NPT2A plays a critical role in association with Glu43 and His27 of PDZ1 that are absent in PDZ2. Experimentally introduced mutation in PDZ1 (Glu43Asp and His27Asn) decreased binding to NPT2A. Conversely, introduction of Asp183Glu and Asn167His mutations in PDZ2 promoted the formation of favorable interactions yielding micromolar K _Ds. The results describe novel determinants within both the PDZ domain and outside the canonical PDZ-recognition motif that are responsible for discrimination of NPT2A between two PDZ domains. The results challenge general paradigms for PDZ recognition and suggest new targets for drug development.     

cGMP inhibition of Na+/H+ antiporter 3 (NHE3) requires PDZ domain adapter NHERF2, a broad specificity protein kinase G-anchoring protein

Electroneutral NaCl absorption mediated by Na+/H+ exchanger 3 (NHE3) is important in intestinal and renal functions related to water/Na+ homeostasis. cGMP inhibits NHE3 in intact epithelia. However, unexpectedly it failed to inhibit NHE3 stably transfected in PS120 cells, even upon co-expression of cGMP-dependent protein kinase type II (cGKII). Additional co-expression of NHERF2, the tandem PDZ domain adapter protein involved in cAMP inhibition of NHE3, restored cGMP as well as cAMP inhibition, whereas NHERF1 solely restored cAMP inhibition. In vitro conditions were identified in which NHERF2 but not NHERF1 bound cGKII. The NHERF2 PDZ2 C terminus, which binds NHE3, also bound cGKII. A non-myristoylated mutant of cGKII did not support cGMP inhibition of NHE3. Although cGKI also bound NHERF2 in vitro, it did not evoke inhibition of NHE3 unless a myristoylation site was added. These results show that NHERF2, acting as a novel protein kinase G-anchoring protein, is required for cGMP inhibition of NHE3 and that cGKII must be bound both to the plasma membrane by its myristoyl anchor and to NHERF2 to inhibit NHE3.     

NHERF1 and NHERF2 are necessary for multiple but usually separate aspects of basal and acute regulation of NHE3 activity

Na(+)/H(+) exchanger 3 (NHE3) is expressed in the brush border (BB) of intestinal epithelial cells and accounts for the majority of neutral NaCl absorption. It has been shown that the Na(+)/H(+) exchanger regulatory factor (NHERF) family members of multi-PDZ domain-containing scaffold proteins bind to the NHE3 COOH terminus and play necessary roles in NHE3 regulation in intestinal epithelial cells. Most studies of NHE3 regulation have been in cell models in which NHERF1 and/or NHERF2 were overexpressed. We have now developed an intestinal Na(+) absorptive cell model in Caco-2/bbe cells by expressing hemagglutinin (HA)-tagged NHE3 with an adenoviral infection system. Roles of NHERF1 and NHERF2 in NHE3 regulation were determined, including inhibition by cAMP, cGMP, and Ca(2+) and stimulation by EGF, with knockdown (KD) approaches with lentivirus (Lenti)-short hairpin RNA (shRNA) and/or adenovirus (Adeno)-small interfering RNA (siRNA). Stable infection of Caco-2/bbe cells by NHERF1 or NHERF2 Lenti-shRNA significantly and specifically reduced NHERF protein expression by >80%. NHERF1 KD reduced basal NHE3 activity, while NHERF2 KD stimulated NHE3 activity. siRNA-mediated (transient) and Lenti-shRNA-mediated (stable) gene silencing of NHERF2 (but not of NHERF1) abolished cGMP- and Ca(2+)-dependent inhibition of NHE3. KD of NHERF1 or NHERF2 alone had no effect on cAMP inhibition of NHE3, but KD of both simultaneously abolished the effect of cAMP. The stimulatory effect of EGF on NHE3 was eliminated in NHERF1-KD but occurred normally in NHERF2-KD cells. These findings show that both NHERF2 and NHERF1 are involved in setting NHE3 activity. NHERF2 is necessary for cGMP-dependent protein kinase (cGK) II- and Ca(2+)-dependent inhibition of NHE3. cAMP-dependent inhibition of NHE3 activity requires either NHERF1 or NHERF2. Stimulation of NHE3 activity by EGF is NHERF1 dependent.     

CD44 interaction with Na+-H+ exchanger (NHE1) creates acidic microenvironments leading to hyaluronidase-2 and cathepsin B activation and breast tumor cell invasion

We have explored CD44 (a hyaluronan (HA) receptor) interaction with a Na(+)-H(+) exchanger (NHE1) and hyaluronidase-2 (Hyal-2) during HA-induced cellular signaling in human breast tumor cells (MDA-MB-231 cell line). Immunological analyses demonstrate that CD44s (standard form) and two signaling molecules (NHE1 and Hyal-2) are closely associated in a complex in MDA-MB-231 cells. These three proteins are also significantly enriched in cholesterol and ganglioside-containing lipid rafts, characterized as caveolin and flotillin-rich plasma membrane microdomains. The binding of HA to CD44 activates Na(+)-H(+) exchange activity which, in turn, promotes intracellular acidification and creates an acidic extracellular matrix environment. This leads to Hyal-2-mediated HA catabolism, HA modification, and cysteine proteinase (cathepsin B) activation resulting in breast tumor cell invasion. In addition, we have observed the following: (i) HA/CD44-activated Rho kinase (ROK) mediates NHE1 phosphorylation and activity, and (ii) inhibition of ROK or NHE1 activity (by treating cells with a ROK inhibitor, Y27632, or NHE1 blocker, S-(N-ethyl-N-isopropyl) amiloride, respectively) blocks NHE1 phosphorylation/Na(+)-H(+) exchange activity, reduces intracellular acidification, eliminates the acidic environment in the extracellular matrix, and suppresses breast tumor-specific behaviors (e.g. Hyal-2-mediated HA modification, cathepsin B activation, and tumor cell invasion). Finally, down-regulation of CD44 or Hyal-2 expression (by treating cells with CD44 or Hyal-2-specific small interfering RNAs) not only inhibits HA-mediated CD44 signaling (e.g. ROK-mediated Na(+)-H(+) exchanger reaction and cellular pH changes) but also impairs oncogenic events (e.g. Hyal-2 activity, hyaluronan modification, cathepsin B activation, and tumor cell invasion). Taken together, our results suggest that CD44 interaction with a ROK-activated NHE1 (a Na(+)-H(+) exchanger) in cholesterol/ganglioside-containing lipid rafts plays a pivotal role in promoting intracellular/extracellular acidification required for Hyal-2 and cysteine proteinase-mediated matrix degradation and breast cancer progression.     

Hsp90-mediated inhibition of FKBP38 regulates apoptosis in neuroblastoma cells

The FK506-binding protein 38 (FKBP38) is a pro-apoptotic regulator of Bcl-2 in neuroblastoma cells. Hsp90 inhibits the pro-apoptotic FKBP38/CaM/Ca(2+) complex and thus prevents interactions between FKBP38 and Bcl-2. Here we show that Hsp90 increases cell survival rates of neuroblastoma cells after apoptosis induction. Depletion of FKBP38 by short interference RNA significantly decreased the anti-apoptotic effect of Hsp90 expression. In addition, the influence of high cellular Hsp90 levels was only observed in post-stimulation apoptosis that is sensitive to selective FKBP38 active site inhibition. Similar anti-apoptotic effects in neuroblastoma cells were observed after stimulation of endogenous Hsp90 expression. Hence, the inhibition of FKBP38 by Hsp90 participates in programmed cell death control of neuroblastoma cells.     

Association of plasminogen with dipeptidyl peptidase IV and Na+/H+ exchanger isoform NHE3 regulates invasion of human 1-LN prostate tumor cells

Binding of plasminogen type II (Pg 2) to dipeptidyl peptidase IV (DPP IV) on the surface of the highly invasive 1-LN human prostate tumor cell line induces an intracellular Ca2+ ([Ca2+]i) signaling cascade accompanied by a rise in intracellular pH (pHi). In endothelial cells, Pg 2 regulates intracellular pH via Na+/H+ exchange (NHE) antiporters; however, this mechanism has not been demonstrated in any other cell type including prostate cancer cells. Because the Pg 2 receptor DPP IV is associated with NHE3 in kidney cell plasma membranes, we investigated a similar association in 1-LN human prostate cancer cells and a mechanistic explanation for changes in [Ca2+]i or pHi induced by Pg 2 in these cells. Our results suggest that the signaling cascade initiated by Pg 2 and its receptor proceeds via activation of phospholipase C, which promotes formation of inositol 3,4,5-trisphosphate, an inducer of Ca2+ release from endoplasmic reticulum stores. Furthermore, our results suggest that Pg 2 may regulate pHi via an association with NHE3 linked to DPP IV in these cells. These associations suggest that Pg has the potential to simultaneously regulate calcium signaling pathways and Na+/H+ exchanges necessary for tumor cell proliferation and invasiveness.     

Elevated calcium acutely regulates dynamic interactions of NHERF2 and NHE3 proteins in opossum kidney (OK) cell microvilli

The brush border (BB) Na(+)/H(+) exchanger NHE3 is rapidly activated or inhibited by changes in trafficking, which mimics renal and intestinal physiology. However, there is a paradox in that NHE3 has limited mobility in the BB due to its binding to the multi-PDZ domain containing the NHERF family. To allow increased endocytosis, as occurs with elevated intracellular Ca(2+), we hypothesized that NHE3 had to be, at least transiently, released from the BB cytoskeleton. Because NHERF1 and -2 are localized at the BB, where they bind NHE3 as well as the cytoskeleton, we tested whether either or both might dynamically interact with NHE3 as part of Ca(2+) signaling. We employed FRET to study close association of NHE3 and these NHERFs and fluorescence recovery after photobleaching to monitor NHE3 mobility in the apical domain in polarized opossum kidney cells. Under basal conditions, NHERF2 and NHE3 exhibited robust FRET signaling. Within 1 min of A23187 (0.5 μm) exposure, the NHERF2-NHE3 FRET signal was abolished, and BB NHE3 mobility was transiently increased. The dynamics in FRET signal and NHE3 mobility correlated well with a change in co-precipitation of NHE3 and NHERF2 but not NHERF1. We conclude the following. 1) Under basal conditions, NHE3 closely associates with NHERF2 in opossum kidney cell microvilli. 2) Within 1 min of elevated Ca(2+), the close association of NHE3-NHERF2 is abolished but is re-established in ～60 min. 3) The change in NHE3-NHERF2 association is accompanied by an increased BB mobile fraction of NHE3, which contributes to inhibition of NHE3 transport activity via increased endocytosis.     

The Nck-interacting kinase (NIK) phosphorylates the Na+-H+ exchanger NHE1 and regulates NHE1 activation by platelet-derived growth factor

NIK, a recently identified Nck-interacting kinase, acts upstream of the MEK kinase MEKK1 to activate the c-Jun N-terminal kinase JNK. We now show that NIK binds to and divergently activates the plasma membrane Na(+)-H(+) exchanger NHE1. In a genetic screen, NHE1 interacted with NIK at a site N-terminal (amino acids 407-502) to the Nck-binding domain, and this site is critical for its association with NHE1 in vivo. NIK also phosphorylates NHE1; however, the phosphorylation sites, which are distal to amino acid 638, are distinct from the NIK-binding site on NHE1 (amino acids 538-638). Expression of wild-type, but not a kinase-inactive, NIK in fibroblasts increased NHE1 phosphorylation and activity. The kinase domain of NIK, however, was not sufficient for this response in vivo. Full phosphorylation and activation of NHE1 required both the kinase and the NHE1-binding domains of NIK, suggesting that the NHE1-binding site functions as a targeting signal. The functional significance of an interaction between NIK and NHE1 was confirmed by the ability of a kinase-inactive NIK to selectively inhibit activation of NHE1 by platelet-derived growth factor but not by thrombin. Moreover, although NIK activates JNK through a mechanism dependent on MEKK1, it phosphorylated and activated NHE1 independently of MEKK1. These findings indicate that NIK acts downstream of platelet-derived growth factor receptors to phosphorylate and activate NHE1 divergently of its activation of JNK.     

PAK1 regulates RUFY3-mediated gastric cancer cell migration and invasion

Actin protrusion at the cell periphery is central to the formation of invadopodia during tumor cell migration and invasion. Although RUFY3 (RUN and FYVE domain containing 3)/SINGAR1 (single axon-related1)/RIPX (Rap2 interacting protein X) has an important role in neuronal development, its pathophysiologic role and relevance to cancer are still largely unknown. The purpose of this study was to elucidate the molecular mechanisms by which RUFY3 involves in gastric cancer cell migration and invasion. Here, our data show that overexpression of RUFY3 leads to the formation of F-actin-enriched protrusive structures at the cell periphery and induces gastric cancer cell migration. Furthermore, P21-activated kinase-1 (PAK1) interacts with RUFY3, and promotes RUFY3 expression and RUFY3-induced gastric cancer cell migration; inhibition of PAK1 attenuates RUFY3-induced SGC-7901 cell migration and invasion. Importantly, we found that the inhibitory effect of cell migration and invasion is significantly enhanced by knockdown of both PAK1 and RUFY3 compared with knockdown of RUFY3 alone or PAK1 alone. Strikingly, we found significant upregulation of RUFY3 in gastric cancer samples with invasive carcinoma at pathologic TNM III and TNM IV stages, compared with their non-tumor counterparts. Moreover, an obvious positive correlation was observed between the protein expression of RUFY3 and PAK1 in 40 pairs of gastric cancer samples. Therefore, these findings provide important evidence that PAK1 can positively regulate RUFY3 expression, which contribute to the metastatic potential of gastric cancer cells, maybe blocking PAK1-RUFY3 signaling would become a potential metastasis therapeutic strategy for gastric cancer.Gastric cancer is the second leading cause of cancer-related death worldwide, and the underlying molecular mechanisms responsible for gastric cancer metastasis are needed to be elucidated. Invasion of tumor cells is the key step in determining the aggressive phenotype of human cancers and compose the paramount causes of cancer deaths.¹ The motility and invasion of cancer cell participates in a complex and integrated series of events that are primarily controlled by the regulation and reorganization of the actin cytoskeleton.^{1, 2} Regulation of actin polymerization is responsible for the formation of protrusive structures that are essential for tumor cell movement and invasion, including filopodia, lamellipodia and invadopodia.³ To improve the survival rate of cancer patients, it is of practical significance to investigate the proteins governing metastasis and to identify novel prognostic markers and therapeutic targets.Human RUFY3 (RUN and FYVE domain containing 3), also known as RIPX (Rap2 interacting protein X) or Singar1 (single axon-related1), is a 469-amino-acid protein and is the highly expressed in brain tissue. The N-terminal region of RUFY3 and its homologs, including RPIP8⁴ and RPIP9,⁵ contains the RUN domain, which can interact with Rap2^{4, 5, 6} and Rab.^{7, 8} The crystal structures indicate that RUFY3 contains a RUN domain⁹ and two coiled-coil domains.¹⁰ Several proteins containing RUN domain have been shown to be involved in Ras-like GTPase signaling¹¹ and Rab-mediated membrane trafficking.^{12, 13, 14, 15, 16} RUFY3 is thought to localize in growth cones and have a role in neuronal development by suppressing the formation of surplus axons to maintain optimal neuronal polarity.^{17, 18} However, up to date, its pathophysiologic role and relevance to cancer metastasis are still unexplored.The human RUFY3 was identified by a yeast two-hybrid assay using P21-activated kinase-1 (PAK1) as a bait protein in our studies. The PAKs, a family of serine/threonine protein kinases, have pivotal roles in cytoskeletal reorganization,¹⁹ survival,²⁰ motility^{21, 22} and tumorigenesis.²³ There has been mounting evidence that PAK1 is tightly related to the progression and metastasis of cancer and may become a promising diagnostic and therapeutic target for cancer.^{24, 25} For example, elevated PAK1 expression is correlated with cancer progression and lymph node metastases in gastric cancer tissues.^{26, 27} Therefore, it is worthwhile to study the novel binding partners of PAK1.In this study, we report that RUFY3 localizes in F-actin-enriched invadopodia and induces the formation of protrusive structures. Importantly, we found that the overexpression of RUFY3 promotes gastric cancer cell migration and invasion. Furthermore, we showed that PAK1 can affect RUFY3-mediated gastric cancer cell migration and invasion by regulating its expression. In gastric cancer samples, we showed a positive relationship between PAK1 and RUFY3, and that increased expression of RUFY3 is positively correlated with clinical gastric cancer samples. This report is the first investigation focused on exploring the role of RUFY3 in cancer cells and the relationship between PAK1 and RUFY3.     

Overexpression, purification, and characterization of PDZ domain proteins NHERF and E3KARP in Escherichia coli

NHERF (Na(+)/H(+) exchanger regulatory factor) and E3KARP (NHE3 kinase A regulatory protein or NHERF2) are structurally related adapter proteins that contain two tandem PDZ (PSD-95/Dlg-1/ZO-1) domains. Recent studies suggest that these proteins play important roles in the membrane targeting, trafficking, and sorting of several ion channels, transmembrane receptors, and signaling proteins in many tissues. Both NHERF and E3KARP interact with NHE3 through their C-terminally extended second PDZ domain, and the last 30 amino acids of these PDZ domain proteins interact with ezrin. However, the structural bases of the full-length human NHERF and E3KARP, in their physiological roles on the regulation of NHE3 trafficking, are still unknown. To obtain pure and soluble proteins for crystallization and X-ray studies, NHERF and E3KARP were subcloned into pET-30b and pET-30a vectors, and overexpressed in Escherichia coli strains of BL21(DE3). The soluble NHERF and E3KARP proteins were purified using Ni-NTA, anion-exchange column and gel filtration chromatography. The purity, molecular mass, and the conformation of the proteins were determined by high-performance liquid chromatography, matrix-assisted laser desorption-ionization-time-of-flight mass spectroscopy and circular dichroism studies, respectively.     

Resveratrol inhibits heregulin-beta1-mediated matrix metalloproteinase-9 expression and cell invasion in human breast cancer cells

The growth factor heregulin-β1 (HRG-β1), which is expressed in breast cancer, activates the HER-2 signaling pathway through induction of heterodimeric complexes of HER-2 with HER-3 or HER-4. It has been shown in many studies that HRG-β1 induces the tumorigenicity and metastasis of breast cancer cells. Matrix metalloproteinase (MMP) 9 is a key enzyme in the degradation of extracellular matrices, and its expression may be dysregulated in breast cancer invasion and metastasis. Resveratrol, a major component in grape, exhibited potential anticarcinogenic activities in both in vitro and in vivo studies. However, the inhibitory effect of resveratrol on HER-2-mediated expression of MMP-9 has not been demonstrated yet.

In the present study, we investigated the anti-invasive mechanism of resveratrol in human breast cancer cells. Human breast cancer MCF-7 cells were exposed to resveratrol (2, 5 and 10 μM). The expression activity of MMP-9 was measured by zymogram analysis. Phosphorylated levels of HER-2 and mitogen-activated protein kinase (MAPK)/ERK were measured by Western blot analysis. Total actin was used as internal control for protein expression. HRG-β1 induced the phosphorylation of HER-2/neu receptor and MMP-9 expression in human breast cancer MCF-7 cells. Resveratrol significantly inhibited HRG-β1-mediated MMP-9 expression in human breast cancer cells. MEK inhibitor induced a marked reduction in MMP-9 expression, and it suggested that ERK1/2 cascade could play an important role in HRG-β1-mediated MMP-9 expression. Furthermore, resveratrol significantly suppressed HRG-β1-mediated phosphorylation of ERK1/2 and invasion of breast cancer cells. However, resveratrol had negligible effects on either HRG-β1-mediated phosphorylation of HER-2 receptor or expression of the tissue inhibitor of MMP, tissue inhibitor metalloproteinase protein 1.

Taken together, our results suggest that resveratrol inhibited MMP-9 expression in human breast cancer cells. The inhibitory effects of resveratrol on MMP-9 expression and invasion of breast cancer cells are, in part, associated with the down-regulation of the MAPK/ERK signaling pathway.     

The CRAL/TRIO and GOLD domain protein TAP-1 regulates RAF-1 activation

The activation of the protein kinase Raf at the cell membrane is a critical step in cell signaling during development, but the mechanisms that regulate Raf activity remain incompletely defined. We previously demonstrated that the C. elegans cgr-1 gene encodes a CRAL/TRIO domain-containing protein that is a critical modulator of Ras-dependent cell fate specification during C. elegans development. Here we identify the mammalian α-tocopherol associated protein-1 (TAP-1) as a functional ortholog of cgr-1. TAP-1 mRNA was expressed in many tissues, and TAP-1 protein colocalized with Ras and Raf at the cell membrane. Reducing TAP-1 expression by RNA interference increased Ras/ERK signaling in multiple cell types. These functional studies demonstrate that CRAL/TRIO domain proteins play a conserved role in regulating Ras signaling. Biochemical analyses indicated that TAP-1 operates at the level of Raf, since TAP-1 function negatively regulated the amount of Raf-1 recruited to GTP-bound Ras at the cell membrane. TAP-1 plays a significant physiological role in controlling cell division, since reducing TAP-1 expression increased the oncogenic capacity of Ras transformed human cancer cell lines. These studies identify TAP-1 as a critical modulator of Ras-mediated cellular signaling.