Modification of plant Rac/Rop GTPase signalling using bacterial toxin transgenes

Bacterial protein toxins which modify Rho GTPase are useful for the analysis of Rho signalling in animal cells, but these toxins cannot be taken up by plant cells. We demonstrate in vitro deamidation of Arabidopsis Rop4 by Escherichia coli Cytotoxic Necrotizing Factor 1 (CNF1) and glucosylation by Clostridium difficile toxin B. Expression of the catalytic domain of CNF1 caused modification and activation of co‐expressed Arabidopsis Rop4 GTPase in tobacco leaves, resulting in hypersensitive‐like cell death. By contrast, the catalytic domain of toxin B modified and inactivated co‐expressed constitutively active Rop4, blocking the hypersensitive response caused by over‐expression of active Rops. In transgenic Arabidopsis, both CNF1 and toxin B inhibited Rop‐dependent polar morphogenesis of leaf epidermal cells. Toxin B expression also inhibited Rop‐dependent morphogenesis of root hairs and trichome branching, and resulted in root meristem enlargement and dwarf growth. Our results show that CNF1 and toxin B transgenes are effective tools in Rop GTPase signalling studies.     

Clostridium difficile toxin A binds colonocyte Src causing dephosphorylation of focal adhesion kinase and paxillin

Clostridium difficile toxin A impairs tight junction function of colonocytes by glucosylation of Rho family proteins causing actin filament disaggregation and cell rounding. We investigated the effect of toxin A on focal contact formation by assessing its action on focal adhesion kinase (FAK) and the adapter protein paxillin. Exposure of NCM460 human colonocytes to toxin A for 1 h resulted in complete dephosphorylation of FAK and paxillin, while protein tyrosine phosphatase activity was reduced. Blockage of toxin A-associated glucosyltransferase activity by co-incubation with UDP-2′3′ dialdehyde did not reduce toxin A-induced FAK and paxillin dephosphorylation. GST-pull down and in vitro kinase activity experiments demonstrated toxin A binding directly to the catalytic domain of Src with suppression of its kinase activity. Direct binding of toxin A to Src, independent of any effect on protein tyrosine phosphatase or Rho glucosylation, inhibits Src kinase activity followed by FAK/paxillin inactivation. These mechanisms may contribute to toxin A inhibition of colonocyte focal adhesion that occurs in human colonic epithelium exposed to toxin A.     

Aplidin induces JNK-dependent apoptosis in human breast cancer cells via alteration of glutathione homeostasis, Rac1 GTPase activation, and MKP-1 phosphatase downregulation

Aplidin is an antitumor agent in phase II clinical trials that induces apoptosis through the sustained activation of Jun N-terminal kinase (JNK). We report that Aplidin alters glutathione homeostasis increasing the ratio of oxidized to reduced forms (GSSG/GSH). Aplidin generates reactive oxygen species and disrupts the mitochondrial membrane potential. Exogenous GSH inhibits these effects and also JNK activation and cell death. We found two mechanisms by which Aplidin activates JNK: rapid activation of Rac1 small GTPase and downregulation of MKP-1 phosphatase. Rac1 activation was diminished by GSH and enhanced by L-buthionine (SR)-sulfoximine, which inhibits GSH synthesis. Downregulation of Rac1 by transfection of small interfering RNA (siRNA) duplexes or the use of a specific Rac1 inhibitor decreased Aplidin-induced JNK activation and cytotoxicity. Our results show that Aplidin induces apoptosis by increasing the GSSG/GSH ratio, a necessary step for induction of oxidative stress and sustained JNK activation through Rac1 activation and MKP-1 downregulation.     

An essential role for Rac/Cdc42 GTPases in cerebellar granule neuron survival

Rho family GTPases are critical molecular switches that regulate the actin cytoskeleton and cell function. In the current study, we investigated the involvement of Rho GTPases in regulating neuronal survival using primary cerebellar granule neurons. Clostridium difficile toxin B, a specific inhibitor of Rho, Rac, and Cdc42, induced apoptosis of granule neurons characterized by c-Jun phosphorylation, caspase-3 activation, and nuclear condensation. Serum and depolarization-dependent survival signals could not compensate for the loss of GTPase function. Unlike trophic factor withdrawal, toxin B did not affect the antiapoptotic kinase Akt or its target glycogen synthase kinase-3beta. The proapoptotic effects of toxin B were mimicked by Clostridium sordellii lethal toxin, a selective inhibitor of Rac/Cdc42. Although Rac/Cdc42 GTPase inhibition led to F-actin disruption, direct cytoskeletal disassembly with Clostridium botulinum C2 toxin was insufficient to induce c-Jun phosphorylation or apoptosis. Granule neurons expressed high basal JNK and low p38 mitogen-activated protein kinase activities that were unaffected by toxin B. However, pyridyl imidazole inhibitors of JNK/p38 attenuated c-Jun phosphorylation. Moreover, both pyridyl imidazoles and adenoviral dominant-negative c-Jun attenuated apoptosis, suggesting that JNK/c-Jun signaling was required for cell death. The results indicate that Rac/Cdc42 GTPases, in addition to trophic factors, are critical for survival of cerebellar granule neurons.     

Glucosylation of Ras by Clostridium sordellii lethal toxin: consequences for effector loop conformations observed by NMR spectroscopy

The lethal toxin (LT) from Clostridium sordellii, which belongs to the family of large clostridial cytotoxins, acts as a monoglucosyltransferase for the Rho subfamily GTPase Rac and also modifies Ras. In the present study we investigated structural changes of H-Ras in its di- and triphosphate form that occur upon glucosylation of the effector domain amino acid threonine-35 by LT. (31)P NMR experiments recorded during the enzymatic glucosylation process, using UDP-glucose as a cosubstrate, show that the modification of the threonine side chain influences the chemical shifts of the phosphate groups of the bound nucleotides. In the diphosphate-bound form (Ras.GDP) glucosylation of Thr35 induces only small changes in the chemical environment of the active center. In the triphosphate form with the GTP analogue GppNHp bound (Ras.GppNHp) Ras shows at least two different conformations in the active center that exchange on a medium-range time scale (10 to 0.1 ms). Glucosylation selectively stabilizes one distinct conformation of the effector loop (state 1) with tyrosine-32 probably apart from the nucleotide and threonine-35 not involved in magnesium ion coordination. This conformation is known to have a low affinity to effector proteins such as Raf-1, AF-6, or Byr2 and thus prevents the transduction of the activation signal in the Ras-mediated pathway. NMR correlation spectra of Ras(T35glc).GDP and denaturation experiments with urea indicate that the glucose is bound in the alpha-anomeric form to the hydroxyl group of the threonine-35 side chain. Inhibition of the glucosylation reaction by 1,5-gluconolactone suggests a stereospecific reaction mechanism with a glucosyl oxonium ion transition state for the enzymatic activity of LT.     

A phosphatidylserine-binding site in the cytosolic fragment of Clostridium sordellii lethal toxin facilitates glucosylation of membrane-bound Rac and is required for cytotoxicity

Large clostridial toxins glucosylate some small G proteins on a threonine residue, thereby preventing their interactions with effector molecules and regulators. We show that the glucosyltransferase domain of lethal toxin from Clostridium sordellii (LT(cyt); amino acids 1-546), which is released into the cytosol during cell infection, binds preferentially to liposomes containing phosphatidylserine as compared with other anionic lipids. The binding of LT(cyt) to phosphatidylserine increases by two orders of magnitude the rate of glucosylation of liposome-bound geranyl-geranylated Rac-GDP. Limited proteolysis and deletion studies show that the binding site for phosphatidylserine lies within the first 18 N-terminal residues of LT(cyt). Deletion of these residues abolishes the effect of phosphatidylserine on the activity of LT(cyt) on liposome-bound geranyl-geranylated Rac-GDP and prevents the morphological effects induced by LT(cyt) microinjection into various cells, but it does not affect the intrinsic activity of LT(cyt) on non-geranyl-geranylated Rac-GDP in solution. We conclude that the avidity of LT(cyt) for phosphatidylserine facilitates its targeting to the cytosolic leaflet of cell membranes and, notably, the plasma membrane, where this anionic lipid is abundant and where several targets of lethal toxin reside.     

A novel cytotoxin from Clostridium difficile serogroup F is a functional hybrid between two other large clostridial cytotoxins

The large clostridial cytotoxins (LCTs) constitute a group of high molecular weight clostridial cytotoxins that inactivate cellular small GTP-binding proteins. We demonstrate that a novel LCT (TcdB-1470) from Clostridium difficile strain 1470 is a functional hybrid between "reference" TcdB-10463 and Clostridium sordellii TcsL-1522. It bound to the same specific receptor as TcdB-10463 but glucosylated the same GTP-binding proteins as TcsL-1522. All three toxins had equal enzymatic potencies but were equally cytotoxic only when microinjected. When applied extracellularly TcdB-1470 and TcdB-10463 were considerably more potent cytotoxins than TcsL-1522. The small GTP-binding protein R-Ras was identified as a target for TcdB-1470 and also for TcsL-1522 but not for TcdB-10463. R-Ras is known to control integrin-extracellular matrix interactions from inside the cell. Its glucosylation may be a major determinant for the cell rounding and detachment induced by the two R-Ras-attacking toxins. In contrast, fibroblasts treated with TcdB-10463 were arborized and remained attached, with phosphotyrosine containing structures located at the cell-to-cell contacts and beta3-integrin remaining at the tips of cellular protrusions. These components were absent from cells treated with the R-Ras-inactivating toxins. The novel hybrid toxin will broaden the utility of the LCTs for clarifying the functions of several small GTPases, now including also R-Ras.     

Inositol hexakisphosphate-dependent processing of Clostridium sordellii lethal toxin and Clostridium novyi alpha-toxin

Clostridium sordellii lethal toxin and Clostridium novyi α-toxin, which are virulence factors involved in the toxic shock and gas gangrene syndromes, are members of the family of clostridial glucosylating toxins. The toxins inactivate Rho/Ras proteins by glucosylation or attachment of GlcNAc (α-toxin). Here, we studied the activation of the autoproteolytic processing of the toxins by inositol hexakisphosphate (InsP(6)) and compared it with the processing of Clostridium difficile toxin B. In the presence of low concentrations of InsP(6) (<1 μM), toxin fragments consisting of the N-terminal glucosyltransferase (or GlcNAc-transferase) domains and the cysteine protease domains (CPDs) of C. sordellii lethal toxin, C. novyi α-toxin, and C. difficile toxin B were autocatalytically processed. The cleavage sites of lethal toxin (Leu-543) and α-toxin (Leu-548) and the catalytic cysteine residues (Cys-698 of lethal toxin and Cys-707 of α-toxin) were identified. Affinity of the CPDs for binding InsP(6) was determined by isothermal titration calorimetry. In contrast to full-length toxin B and α-toxin, autocatalytic cleavage and InsP(6) binding of full-length lethal toxin depended on low pH (pH 5) conditions. The data indicate that C. sordellii lethal toxin and C. novyi α-toxin are InsP(6)-dependently processed. However, full-length lethal toxin, but not its short toxin fragments consisting of the glucosyltransferase domain and the CPD, requires a pH-sensitive conformational change to allow binding of InsP(6) and subsequent processing of the toxin.     

Human JIK, a novel member of the STE20 kinase family that inhibits JNK and is negatively regulated by epidermal growth factor

Mammalian members related to Saccharomyces cerevisiae serine/threonine kinase STE20 can be divided into two subfamilies based on their structure and function. The PAK subfamily is characterized by an N-terminal p21-binding domain (also known as CRIB domain), a C-terminal kinase domain, and is regulated by the small GTP-binding proteins Rac1 and Cdc42Hs. The second group is represented by the GCK-like members, which contain an N-terminal catalytic domain and lack the p21-binding domain. Some of them have been demonstrated to induce c-Jun N-terminal kinase/stress-activated protein kinase (JNK/SAPK) cascade, while others have been shown to be activated by a subset of stress conditions or apoptotic agents, although little is known about their specific function. Here, we have identified a novel human STE20-related serine/threonine kinase, belonging to the GCK-like subfamily. This kinase does not induce the JNK/SAPK pathway, but, instead, inhibits the basal activity of JNK/SAPK, and diminishes its activation in response to human epidermal growth factor (EGF). Therefore, we designated this molecule JIK for JNK/SAPK-inhibitory kinase. The inhibition of JNK/SAPK signaling pathway by JIK was found to occur between the EGF receptor and the small GTP-binding proteins Rac1 and Cdc42Hs. In contrast, JIK does not activate nor does it inhibit ERK2, ERK6, p38, or ERK5. Furthermore, JIK kinase activity is not modulated by any exogenous stimuli, but, interestingly, it is dramatically decreased upon EGF receptor activation. Thus, JIK might represent the first member of the STE20 kinase family whose activity can be negatively regulated by tyrosine kinase receptors, and whose downstream targets inhibit, rather than enhance, JNK/SAPK activation.     

R-Ras glucosylation and transient RhoA activation determine the cytopathic effect produced by toxin B variants from toxin A-negative strains of Clostridium difficile

Clostridium difficile induces antibiotic-associated diarrhea through the production of toxin A and toxin B; the former toxin has been assumed to be responsible for the symptoms of the disease. Several toxin A-negative strains from C. difficile have recently been isolated from clinical cases and have been reported to produce toxin B variants eliciting an atypical cytopathic effect. Ultrastructural analysis indicated these toxins induce a rounding cytopathic effect and filopodia-like structures. Toxin B variants glucosylated R-Ras, and transfection with a constitutively active mutant of this GTPase protected cells against their cytopathic effect. Treatment of cells with toxin B variants induced detachment from the extracellular matrix and blockade of the epidermal growth factor-mediated phosphorylation of extracellular-regulated protein kinases, demonstrating a deleterious effect on the R-Ras-controlled avidity of integrins. Treatment with toxin B variants also induced a transient activation of RhoA probably because of inactivation of Rac1. Altogether, these data indicate that the cytopathic effect induced by toxin B variants is because of cell rounding and detachment mediated by R-Ras glucosylation, and the induction of filopodia-like structures is mediated by RhoA activation. Implications for the pathophysiology of C. difficile-induced diarrhea are discussed.     

EhRho1, a RhoA-like GTPase of Entamoeba histolytica, is modified by clostridial glucosylating cytotoxins

Clostridial glucosylating cytotoxins inactivate mammalian Rho GTPases by mono-O glucosylation of a conserved threonine residue located in the switch 1 region of the target protein. Here we report that EhRho1, a RhoA-like GTPase from the protozoan parasite Entamoeba histolytica, is glucosylated by clostridial cytotoxins. Recombinant glutathione S-transferase-EhRho1 and EhRho1 from cell lysate of Entamoeba histolytica were glucosylated by Clostridium difficile toxin B and Clostridium novyi alpha-toxin. In contrast, Clostridium difficile toxin A, which shares the same mammalian protein substrates with toxin B, did not modify EhRho1. Change of threonine 52 of EhRho1 to alanine prevented glucosylation by toxin B from Clostridium difficile and by alpha-toxin from Clostridium novyi, which suggests that the equivalent threonine residues are glucosylated in mammalian and Entamoeba Rho GTPases. Lethal toxin from Clostridium sordellii did not glucosylate EhRho1 but labeled several other substrate proteins in lysates from Entamoeba histolytica in the presence of UDP-[14C]glucose.     

Regulation of protein kinase C-related protein kinase 2 (PRK2) by an intermolecular PRK2-PRK2 interaction mediated by Its N-terminal domain

Protein kinase C-related protein kinases (PRKs) are effectors of the Rho family of small GTPases and play a role in the development of diseases such as prostate cancer and hepatitis C. Here we examined the mechanism underlying the regulation of PRK2 by its N-terminal region. We show that the N-terminal region of PRK2 prevents the interaction with its upstream kinase, the 3-phosphoinositide-dependent kinase 1 (PDK1), which phosphorylates the activation loop of PRK2. We confirm that the N-terminal region directly inhibits the kinase activity of PRK2. However, in contrast to previous models, our data indicate that this inhibition is mediated in trans through an intermolecular PRK2-PRK2 interaction. Our results also suggest that amino acids 487-501, located in the linker region between the N-terminal domains and the catalytic domain, contribute to the PRK2-PRK2 dimer formation. This dimerization is further supported by other N-terminal domains. Additionally, we provide evidence that the region C-terminal to the catalytic domain intramolecularly activates PRK2. Finally, we discovered that the catalytic domain mediates a cross-talk between the inhibitory N-terminal region and the activating C-terminal region. The results presented here describe a novel mechanism of regulation among AGC kinases and offer new insights into potential approaches to pharmacologically regulate PRK2.     

A novel src homology 3 domain-containing adaptor protein, HIP-55, that interacts with hematopoietic progenitor kinase 1

Hematopoietic progenitor kinase 1 (HPK1) is a member of the mitogen-activated protein kinase kinase kinase kinase (MAP4K) family and an upstream activator of the c-Jun N-terminal kinase (JNK) signaling cascade. HPK1 interacts, through its proline-rich domains, with growth factor receptor-bound 2 (Grb2), CT10-regulated kinase (Crk), and Crk-like (CrkL) adaptor proteins. We identified a novel HPK1-interacting protein of 55 kDa (HIP-55), similar to the mouse SH3P7 protein, containing an N-terminal actin-binding domain and a C-terminal Src homology 3 domain. We found that HPK1 bound to HIP-55 both in vitro and in vivo. When co-transfected, HIP-55 increased HPK1's kinase activity as well as JNK1's kinase activity. A dominant-negative HPK1 mutant blocked activation of JNK1 by HIP-55 showing that HIP-55 activates the JNK1 signaling pathway via HPK1. Our results identify a novel protein, HIP-55, that binds to HPK1 and regulates the JNK1 signaling cascade.     

Inhibition of Rac GTPase triggers a c-Jun- and Bim-dependent mitochondrial apoptotic cascade in cerebellar granule neurons

Rho GTPases are key transducers of integrin/extracellular matrix and growth factor signaling. Although integrin-mediated adhesion and trophic support suppress neuronal apoptosis, the role of Rho GTPases in neuronal survival is unclear. Here, we have identified Rac as a critical pro-survival GTPase in cerebellar granule neurons (CGNs) and elucidated a death pathway triggered by its inactivation. GTP-loading of Rac1 was maintained in CGNs by integrin-mediated (RGD-dependent) cell attachment and trophic support. Clostridium difficile toxin B (ToxB), a specific Rho family inhibitor, induced a selective caspase-mediated degradation of Rac1 without affecting RhoA or Cdc42 protein levels. Both ToxB and dominant-negative N17Rac1 elicited CGN apoptosis, characterized by cytochrome c release and activation of caspase-9 and -3, whereas dominant-negative N19RhoA or N17Cdc42 did not cause significant cell death. ToxB stimulated mitochondrial translocation and conformational activation of Bax, c-Jun activation, and induction of the BH3-only protein Bim. Similarly, c-Jun activation and Bim induction were observed with N17Rac1. A c-jun N-terminal protein kinase (JNK)/p38 inhibitor, SB203580, and a JNK-specific inhibitor, SP600125, significantly decreased ToxB-induced Bim expression and blunted each subsequent step of the apoptotic cascade. These results indicate that Rac acts downstream of integrins and growth factors to promote neuronal survival by repressing c-Jun/Bim-mediated mitochondrial apoptosis.     

Inhibition of extracellular signal-regulated protein kinase or c-Jun N-terminal protein kinase cascade, differentially activated by cisplatin, sensitizes human ovarian cancer cell line.

We have studied the roles of c-Jun N-terminal protein kinase (JNK) and extracellular signal-regulated protein kinase (ERK) cascade in both the cisplatin-resistant Caov-3 and the cisplatin-sensitive A2780 human ovarian cancer cell lines. Treatment of both cells with cisplatin but not transplatin isomer activates JNK and ERK. Activation of JNK by cisplatin occurred at 30 min, reached a plateau at 3 h, and declined thereafter, whereas activation of ERK by cisplatin showed a biphasic pattern, indicating the different time frame. Activation of JNK by cisplatin was maximal at 1000 microM, whereas activation of ERK was maximal at 100 microM and was less at higher concentrations, indicating the different dose dependence. Cisplatin-induced JNK activation was neither extracellular and intracellular Ca(2+)- nor protein kinase C-dependent, whereas cisplatin-induced ERK activation was extracellular and intracellular Ca(2+)- dependent and protein kinase C-dependent. A mitogen-activated protein kinase/extracellular signal-regulated kinase kinase inhibitor, PD98059, had no effect on the cisplatin-induced JNK activity, suggesting an absence of cross-talk between the ERK and JNK cascades. We further examined the effect of each cascade on the viability following cisplatin treatment. Either exogenous expression of dominant negative c-Jun or the treatment by PD98059 induced sensitivity to cisplatin in both cells. Our findings suggest that cisplatin-induced DNA damage differentially activates JNK and ERK cascades and that inhibition of either of these cascades sensitizes ovarian cancer cells to cisplatin.     

Pertussis toxin activates protein kinase C and a tyrosine protein kinase in the human T cell line Jurkat

Pertussis toxin activates T lymphocytes by a mechanism that is independent of its ADP-ribosylation activity. The toxin stimulates increases in diacylglycerol and intracellular calcium apparently by interacting with a cell surface receptor. Consistent with the production of these second messengers we have found that pertussis toxin activates protein kinase C in the Jurkat cell line. The toxin was also found to activate a tyrosine protein kinase in these cells in a manner similar to that observed with phytohemagglutinin. These results provide evidence that the mechanism of activation of T cells by pertussis toxin involves stimulating the activity of protein kinase C and a tyrosine protein kinase.     

Differential activation of mitogen-activated protein kinases in AGS gastric epithelial cells by cag+ and cag- Helicobacter pylori.

The aim of this study was to determine whether Helicobacter pylori activates mitogen-activated protein (MAP) kinases in gastric epithelial cells. Infection of AGS cells with an H. pylori cag+ strain rapidly (5 min) induced a dose-dependent activation of extracellular signal-regulated kinases (ERK), p38, and c-Jun N-terminal kinase (JNK) MAP kinases, as determined by Western blot analysis and in vitro kinase assay. Compared with cag+ strains, cag- clinical isolates were less potent in inducing MAP kinase, particularly JNK and p38, activation. Isogenic inactivation of the picB region of the cag pathogenicity island resulted in a similar loss of JNK and p38 MAP kinase activation. The specific MAP kinase inhibitors, PD98059 (25 microM; MAP kinase kinase (MEK-1) inhibitor) and SB203580 (10 microM; p38 inhibitor), reduced H. pylori-induced IL-8 production in AGS cells by 78 and 82%, respectively (p < 0.01 for each). Both inhibitors together completely blocked IL-8 production (p < 0.001). However, the MAP kinase inhibitors did not prevent H. pylori-induced IkappaBalpha degradation or NF-kappaB activation. Thus, H. pylori rapidly activates ERK, p38, and JNK MAP kinases in gastric epithelial cells; cag+ isolates are more potent than cag- strains in inducing MAP kinase phosphorylation and gene products of the cag pathogenicity island are required for maximal MAP kinase activation. p38 and MEK-1 activity are required for H. pylori-induced IL-8 production, but do not appear to be essential for H. pylori-induced NF-kappaB activation. Since MAP kinases regulate cell proliferation, differentiation, programmed death, stress, and inflammatory responses, activation of gastric epithelial cell MAP kinases by H. pylori cag+ strains may be instrumental in inducing gastroduodenal inflammation, ulceration, and neoplasia.     

MEKK4 stimulation of p38 and JNK activity is negatively regulated by GSK3beta

The MAPK kinase kinase MEKK4 is required for neurulation and skeletal patterning during mouse development. MEKK4 phosphorylates and activates MKK4/MKK7 and MKK3/MKK6 leading to the activation of JNK and p38, respectively. MEKK4 is believed to be auto-inhibited, and its interaction with other proteins controls its dimerization and activation. TRAF4, GADD45, and Axin each bind and activate MEKK4, with TRAF4 and Axin binding to the kinase domain and GADD45 binding within the N-terminal regulatory domain. Here we show that similar to the interaction with TRAF4 and Axin, the kinase domain of MEKK4 interacts with the multifunctional serine/threonine kinase GSK3beta. GSK3beta binding to MEKK4 blocks MEKK4 dimerization that is required for MEKK4 activation, effectively inhibiting MEKK4 stimulation of the JNK and p38 MAPK pathways. Inhibition of GSK3beta kinase activity with SB216763 results in enhanced MEKK4 kinase activity and increased JNK and p38 activation, indicating that an active state of GSK3beta is required for binding and inhibition of MEKK4 dimerization. Furthermore, GSK3beta phosphorylates specific serines and threonines in the N terminus of MEKK4. Together, these findings demonstrate that GSK3beta binds to the kinase domain of MEKK4 and regulates MEKK4 dimerization. However, unlike TRAF4, Axin, and GADD45, GSK3beta inhibits MEKK4 activity and prevents its activation of JNK and p38. Thus, control of MEKK4 dimerization is regulated both positively and negatively by its interaction with specific proteins.     

Ras mediates the cAMP-dependent activation of extracellular signal-regulated kinases (ERKs) in melanocytes

In melanocytes and melanoma cells, cAMP activates extracellular signal-regulated kinases (ERKs) and MEK-1 by an unknown mechanism. We demonstrate that B-Raf is activated by cAMP in melanocytes. A dominant-negative mutant of B-Raf, but not of Raf-1, blocked the cAMP-induced activation of ERK, indicating that B-Raf is the MEK-1 upstream regulator mediating this cAMP effect. Studies using Clostridium sordelii lethal toxin and Clostridium difficile toxin B have suggested that Rap-1 or Ras might transduce cAMP action. We show that Ras, but not Rap-1, is activated cell-specifically and mediates the cAMP-dependent activation of ERKs, while Rap-1 is not involved in this process in melanocytes. Our results suggest a novel, cell-specific mechanism involving Ras small GTPase and B-Raf kinase as mediators of ERK activation by cAMP. Also, in melanocytes, Ras or ERK activation by cAMP is not mediated through protein kinase A activation. Neither the Ras exchange factor, Son of sevenless (SOS), nor the cAMP-responsive Rap-1 exchange factor, Epac, participate in the cAMP-dependent activation of Ras. These findings suggest the existence of a melanocyte-specific Ras exchange factor directly regulated by cAMP.     

Clostridium difficile glucosyltransferase toxin B-essential amino acids for substrate binding

Recently the crystal structure of the catalytic domain of Clostridium difficile toxin B was solved ( Reinert, D. J., Jank, T., Aktories, K., and Schulz, G. E. (2005) J. Mol. Biol. 351, 973-981 ). On the basis of this structure, we studied the functional role of several amino acids located in the catalytic center of toxin B. Besides the (286)DXD(288) motif and Trp(102), which were shown to be necessary for Mn(2+) and UDP binding, respectively, we identified by alanine scanning Asp(270), Arg(273), Tyr(284), Asn(384), and Trp(520) as being important for enzyme activity. The amino acids Arg(455), Asp(461), Lys(463), and Glu(472) and residues of helix alpha17 (e.g. Glu(449)) of toxin B are essential for enzyme-protein substrate recognition. Introduction of helix alpha17 of toxin B into Clostridium sordellii lethal toxin inhibited modification of Ras subfamily proteins but enabled glucosylation of RhoA, indicating that helix alpha17 is involved in RhoA recognition by toxin B. The data allow the design of a model of the interaction of the glucosyltransferase domain of toxin B with its protein substrate RhoA.