Identification of an AP1-ZFP36 Regulatory Network Associated with Breast Cancer Prognosis.

It has been established that ZFP36 (also known as Tristetraprolin or TTP) promotes mRNA degradation of proteins involved in inflammation, proliferation and tumor invasiveness. In mammary epithelial cells ZFP36 expression is induced by STAT5 activation during lactogenesis, while in breast cancer ZFP36 expression is associated with lower grade and better prognosis. Here, we show that the AP-1 transcription factor components, i.e. JUN, JUNB, FOS, FOSB, in addition to DUSP1, EGR1, NR4A1, IER2 and BTG2, behave as a conserved co-regulated group of genes whose expression is associated to ZFP36 in cancer cells. In fact, a significant down-modulation of this gene network is observed in breast, liver, lung, kidney, and thyroid carcinomas compared to their normal counterparts. In breast cancer, the normal-like and Luminal A, show the highest expression of the ZFP36 gene network among the other intrinsic subtypes and patients with low expression of these genes display poor prognosis. It is also proposed that AP-1 regulates ZFP36 expression through responsive elements detected in the promoter region of this gene. Culture assays show that AP-1 activity induces ZFP36 expression in mammary cells in response to prolactin (PRL) treatment thorough ERK1/2 activation. These results suggest that JUN, JUNB, FOS and FOSB are not only co-expressed, but would also play a relevant role in regulating ZFP36 expression in mammary epithelial cells.

Involvement of PI3K/Akt and p38 MAPK in the induction of COX-2 expression by bacterial lipopolysaccharide in murine adrenocortical cells.

Previous studies from our laboratory demonstrated the involvement of COX-2 in the stimulation of steroid production by LPS in murine adrenocortical Y1 cells, as well as in the adrenal cortex of male Wistar rats. In this paper we analyzed signaling pathways involved in the induction of this key regulatory enzyme in adrenocortical cells and demonstrated that LPS triggers an increase in COX-2 mRNA levels by mechanisms involving the stimulation of reactive oxygen species (ROS) generation and the activation of p38 MAPK and Akt, in addition to the previously demonstrated increase in NFκB activity. In this sense we showed that: (1) inhibition of p38 MAPK or PI3K/Akt (pharmacological or molecular) prevented the increase in COX-2 protein levels by LPS, (2) LPS induced p38 MAPK and Akt phosphorylation, (3) antioxidant treatment blocked the effect of LPS on p38 MAPK phosphorylation and in COX-2 protein levels, (4) PI3K inhibition with LY294002 prevented p38 MAPK phosphorylation and, (5) the activity of an NFκB reporter was decreased by p38 MAPK or PI3K inhibition. These results suggest that activation of both p38 MAPK and PI3K/Akt pathways promote the stimulation of NFκB activity and that PI3K/Akt activity might regulate both p38 MAPK and NFκB signaling pathways. In summary, in this study we showed that in adrenal cells, LPS induces COX-2 expression by activating p38 MAPK and PI3K/Akt signaling pathways and that both pathways converge in the modulation of NFκB transcriptional activity.

Nitric oxide sets off an antioxidant response in adrenal cells: involvement of sGC and Nrf2 in HO-1 induction.

Induction of microsomal heme oxygenase 1 (HO-1) activity is considered a cytoprotective mechanism in different cell types. In adrenal cells, HO-1 induction by ACTH exerts a modulatory effect on steroid production as well. As nitric oxide (NO) has been also regarded as an autocrine/paracrine modulator of adrenal steroidogenesis we sought to study the effects of NO on the induction of HO-1 and the mechanism involved. We hereby analyzed the time and dose-dependent effect of a NO-donor (DETA/NO) on HO-1 induction in a murine adrenocortical cell line. We showed that this effect is mainly exerted at a transcriptional level as it is inhibited by actinomycin D and HO-1 mRNA degradation rates were not affected by DETA/NO treatment. HO-1 induction by NO does not appear to involve the generation of oxidative stress as it was not affected by antioxidant treatment. We also demonstrated that NO-treatment results in the nuclear translocation of the nuclear factor-erythroid 2-related factor (Nrf2), an effect that is attenuated by transfecting the cells with a dominant negative isoform of Nrf2. We finally show that the effects of the NO-donor are reproduced by a permeable analog of cGMP and that a soluble guanylate cyclase specific inhibitor blocked both the induction of HO-1 by NO and the nuclear translocation of Nrf2.

Osmotic stress sensitizes naturally resistant cells to TNF-alpha-induced apoptosis.

Most cells are naturally resistant to TNF-alpha-induced cell death and become sensitized when NF-kappaB transactivation is blocked or in the presence of protein synthesis inhibitors that prevent the expression of anti-apoptotic genes. In this report we analyzed the role of osmotic stress on TNF-alpha-induced cell death. We found that it sensitizes the naturally resistant HeLa cells to TNF-alpha-induced apoptosis, with the involvement of an increase in the activity of several kinases, the inhibition of Bcl-2 expression, and a late increase on NF-kappaB activation. Cell death occurs regardless of the enhanced NF-kappaB activity, whose inhibition produces an increase in apoptosis. The inhibition of p38 kinase, also involved in NF-kappaB activation, significantly increases the effect of osmotic stress on TNF-alpha-induced cell death.

Signalling of the Ret receptor tyrosine kinase through the c-Jun NH2-terminal protein kinases (JNKS): evidence for a divergence of the ERKs and JNKs pathways induced by Ret.

The RET proto-oncogene encodes a functional receptor tyrosine kinase (Ret) for the Glial cell line Derived Neurotrophic Factor (GDNF). RET is involved in several neoplastic and non-neoplastic human diseases. Oncogenic activation of RET is detected in human papillary thyroid tumours and in multiple endocrine neoplasia type 2 syndromes. Inactivating mutations of RET have been associated to the congenital megacolon, i.e. Hirschprung's disease. In order to identify pathways that are relevant for Ret signalling to the nucleus, we have investigated its ability to induce the c-Jun NH2-terminal protein kinases (JNK). Here we show that triggering the endogenous Ret, expressed in PC12 cells, induces JNK activity; moreover, Ret is able to activate JNK either when transiently transfected in COS-1 cells or when stably expressed in NIH3T3 fibroblasts or in PC Cl 3 epithelial thyroid cells. JNK activation is dependent on the Ret kinase function, as a kinase-deficient RET mutant, associated with Hirschsprung's disease, fails to activate JNK. The pathway leading to the activation of JNK by RET is clearly divergent from that leading to the activation of ERK: substitution of the tyrosine 1062 of Ret, the Shc binding site, for phenylalanine abrogates ERK but not JNK activation. Experiments conducted with dominant negative mutants or with negative regulators demonstrate that JNK activation by Ret is mediated by Rho/Rac related small GTPases and, particularly, by Cdc42.

Characterization of Brx, a novel Dbl family member that modulates estrogen receptor action.

Regulation of gene activation by the estrogen receptor (ER) is complex and involves co-regulatory proteins, oncoproteins (such as Fos and Jun), and phosphorylation signaling pathways. Here we report the cloning and initial characterization of a novel protein, Brx, that contains a region of identity to the oncogenic Rho-guanine nucleotide exchange (Rho-GEF) protein Lbc, and a unique region capable of binding to nuclear hormone receptors, including the ER. Western and immunohistochemistry studies showed Brx to be expressed in estrogen-responsive reproductive tissues, including breast ductal epithelium. Brx bound specifically to the ER via an interaction that required distinct regions of ER and Brx. Furthermore, overexpression of Brx in transfection experiments using an estrogen-responsive reporter revealed that Brx augmented gene activation by the ER in an element-specific and ligand-dependent manner. Moreover, activation of ER by Brx could be specifically inhibited by a dominant-negative mutant of Cdc42Hs, but not by dominant negative mutants of RhoA or Rac1. Taken together, these data suggest that Brx represents a novel modular protein that may integrate cytoplasmic signaling pathways involving Rho family GTPases and nuclear hormone receptors.

G-protein-coupled receptor of Kaposi's sarcoma-associated herpesvirus is a viral oncogene and angiogenesis activator.

The Kaposi's sarcoma-associated herpesvirus (KSHV/HHV8) is a gamma-2 herpesvirus that is implicated in the pathogenesis of Kaposi's sarcoma and of primary effusion B-cell lymphomas (PELs). KSHV infects malignant and progenitor cells of Kaposi's sarcoma and PEL, it encodes putative oncogenes and genes that may cause Kaposi's sarcoma pathogenesis by stimulating angiogenesis. The G-protein-coupled receptor encoded by an open reading frame (ORF 74) of KSHV is expressed in Kaposi's sarcoma lesions and in PEL and stimulates signalling pathways linked to cell proliferation in a constitutive (agonist-independent) way. Here we show that signalling by this KSHV G-protein-coupled receptor leads to cell transformation and tumorigenicity, and induces a switch to an angiogenic phenotype mediated by vascular endothelial growth factor, an angiogenesis and Kaposi's-spindle-cell growth factor. We find that this receptor can activate two protein kinases, JNK/SAPK and p38MAPK, by triggering signalling cascades like those induced by inflammatory cytokines that are angiogenesis activators and mitogens for Kaposi's sarcoma cells and B cells. We conclude that the KSHV G-protein-coupled receptor is a viral oncogene that can exploit cell signalling pathways to induce transformation and angiogenesis in KSHV-mediated oncogenesis.

The small GTP-binding protein Rho links G protein-coupled receptors and Galpha12 to the serum response element and to cellular transformation.

Receptors coupled to heterotrimeric G proteins can effectively stimulate growth promoting pathways in a large variety of cell types, and if persistently activated, these receptors can also behave as dominant-acting oncoproteins. Consistently, activating mutations for G proteins of the Galphas and Galphai2 families were found in human tumors; and members of the Galphaq and Galpha12 families are fully transforming when expressed in murine fibroblasts. In an effort aimed to elucidate the molecular events involved in proliferative signaling through heterotrimeric G proteins we have focused recently on gene expression regulation. Using NIH 3T3 fibroblasts expressing m1 muscarinic acetylcholine receptors as a model system, we have observed that activation of this transforming G protein-coupled receptors induces the rapid expression of a variety of early responsive genes, including the c-fos protooncogene. One of the c-fos promoter elements, the serum response element (SRE), plays a central regulatory role, and activation of SRE-dependent transcription has been found to be regulated by several proteins, including the serum response factor and the ternary complex factor. With the aid of reporter plasmids for gene expression, we observed here that stimulation of m1 muscarinic acetylcholine receptors potently induced SRE-driven reporter gene activity in NIH 3T3 cells. In these cells, only the Galpha12 family of heterotrimeric G protein alpha subunits strongly induced the SRE, while Gbeta1gamma2 dimers activated SRE to a more limited extent. Furthermore, our study provides strong evidence that m1, Galpha12 and the small GTP-binding protein RhoA are components of a novel signal transduction pathway that leads to the ternary complex factor-independent transcriptional activation of the SRE and to cellular transformation.

Signaling from G protein-coupled receptors to the c-jun promoter involves the MEF2 transcription factor. Evidence for a novel c-jun amino-terminal kinase-independent pathway.

The c-Jun amino-terminal kinases (JNKs) are a subfamily of mitogen-activated protein kinases that phosphorylate c-Jun and ATF2, and it has been postulated that phosphorylated c-Jun enhances its own expression through AP-1 sites on the c-jun promoter. In this study, we asked whether signals activating JNK regulate the c-jun promoter. Using NIH 3T3 cells expressing G protein-coupled m1 acetylcholine receptors as an experimental model, we have recently shown that the cholinergic agonist carbachol, but not platelet-derived growth factor, potently elevates JNK activity. Consistent with these findings, carbachol, but not platelet-derived growth factor, increased the activity of a c-jun promoter-driven reporter gene (for chloramphenicol acetyltransferase). However, coexpression of JNK kinase kinase (MEKK) effectively increased JNK activity, but resulted in surprisingly limited induction of the c-jun promoter. This raised the possibility that pathway(s) distinct from JNK control the c-jun promoter, and prompted us to explore which of its regulatory elements participate in transcriptional control. We observed that deletion of the 3' AP-1 site diminished chloramphenicol acetyltransferase activity in response to carbachol, but only to a limited extent. In contrast, deletion of a MEF2 site dramatically reduced expression, and deletion of both the MEF2 and 3' AP-1 sites abolished induction. Furthermore, cotransfection with MEF2C and MEF2D cDNAs potently enhanced the activity of the c-jun promoter in response to carbachol, and stimulation of m1 receptors, but not direct JNK activation, induced expression of a MEF2-responsive plasmid. Taken together, these data strongly suggest that MEF2 mediates c-jun promoter expression by G protein-coupled receptors through a yet to be identified pathway, distinct from that of JNK.

The pathway connecting m2 receptors to the nucleus involves small GTP-binding proteins acting on divergent MAP kinase cascades.

m1 and m2 receptors are traditionally linked to tissue specific functions performed by fully differentiated cells. However, these receptors have been also implicated in growth stimulation. The mechanisms whereby these receptors regulate proliferative signaling pathways are still poorly understood. Furthermore, pharmacological evidence suggest that many growth promoting agents act on Gi coupled receptors, but there is no formal proof that induction of DNA-synthesis results from decreased intracellular levels of cAMP. In our laboratory, we have used the expression of ml and m2 receptors as a model for studying proliferative signaling through G protein-coupled receptors. Currently available evidence suggest that these receptors signal to distinct members of the MAP kinase superfamily, MAP kinase and JNK, through betagamma subunits of heterotrimeric G proteins acting, respectively, on a Ras and Rac1 dependent pathway.

Selective activation of effector pathways by brain-specific G protein beta5.

While multiple G protein beta and gamma subunit isoforms have been identified, the implications of this potential diversity of betagamma heterodimers for signaling through betagamma-regulated effector pathways remains unclear. Furthermore the molecular mechanism(s) by which the betagamma complex modulates diverse mammalian effector molecules is unknown. Effector signaling by the structurally distinct brain-specific beta5 subunit was assessed by transient cotransfection with gamma2 in COS cells and compared with beta1. Transfection of either beta1 or beta5 with gamma2 stimulated the activity of cotransfected phospholipase C-beta2 (PLC-beta2), as previously reported. In contrast, cotransfection of beta1 but not beta5 with gamma2 stimulated the mitogen-activated protein kinase (MAPK) and c-Jun N-terminal kinase (JNK) pathways even though the expression of beta5 in COS cells was evident by immunoblotting. The G protein beta5 expressed in transfected COS cells was properly folded as its pattern of stable C-terminal proteolytic fragments was identical to that of native brain beta5. The inability of beta5 to activate the MAPK and JNK pathways was not overcome by cotransfection with three additional Ggamma isoforms. These results suggest it is the Gbeta subunit which determines the pattern of downstream signaling by the betagamma complex and imply that the structural features of the betagamma complex mediating effector regulation may differ among effectors.

Signaling from the small GTP-binding proteins Rac1 and Cdc42 to the c-Jun N-terminal kinase/stress-activated protein kinase pathway. A role for mixed lineage kinase 3/protein-tyrosine kinase 1, a novel member of the mixed lineage kinase family.

Certain small GTP-binding proteins control the enzymatic activity of a family of closely related serine-threonine kinases known as mitogen-activated protein kinases (MAPKs). In turn, these MAPKs, such as p44(mapk) and p42(mapk), referred to herein as MAPKs, and stress-activated protein kinases, also termed c-Jun N-terminal kinases (JNKs), phosphorylate and regulate the activity of key molecules that ultimately control the expression of genes essential for many cellular processes. Whereas Ras controls the activation of MAPK, we and others have recently observed that two members of the Rho family of small GTP-binding proteins, Rac1 and Cdc42, regulate the activity of JNKs. The identity of molecules communicating Rac1 and Cdc42 to JNK is still poorly understood. It has been suggested that Pak1 is the most upstream kinase connecting these GTPases to JNK; however, we have observed that coexpression of Pak1 with activated forms of Cdc42 or Rac1 diminishes rather than enhances JNK activation. This prompted us to explore the possibility that kinases other than Pak might participate in signaling from GTP-binding proteins to JNK. In this regard, a computer-assisted search for proteins containing areas of homology to that in Pak1 that is involved in binding to Rac1 and Cdc42 led to the identification of mixed lineage kinase 3 (MLK3), also known as protein-tyrosine kinase 1, as a potential candidate for this function. In this study, we found that MLK3 overexpression is sufficient to activate JNK potently without affecting the phosphorylating activity of MAPK or p38. Furthermore, we present evidence that MLK3 binds the GTP-binding proteins Cdc42 and Rac1 in vivo and that MLK3 mediates activation of MEKK-SEK-JNK kinase cascade by Rac1 and Cdc42. Taken together, these findings strongly suggest that members of the novel MLK family of highly related kinases link small GTP-binding proteins to the JNK signaling pathway.

The small GTP-binding protein rho activates c-Jun N-terminal kinases/stress-activated protein kinases in human kidney 293T cells. Evidence for a Pak-independent signaling pathway.

Work from a number of laboratories has established a role for certain small GTP-binding proteins in controlling the enzymatic activity of a family of serine-threonine kinases known as mitogen-activated protein kinases (MAPKs). MAPKs have been classified into three subfamilies: extracellular signal-regulated kinases (ERKs), also known as MAPKs; c-Jun N-terminal kinases (JNKs); and p38 kinase. Whereas Ras controls the activation of MAPKs, we and others have recently observed that in certain cells, the small GTP-binding proteins Rac1 and Cdc42 but not Rho regulate the activity of JNKs. Furthermore, because Rac1 and Cdc42 but not Rho bind and activate a kinase known as Pak1, it has been suggested that Pak1 is the most upstream component of the pathway linking these GTPases to JNK. However, in both yeast and mammalian cells, Rho1p, a Rho homologue, and RhoA, respectively, directly interact with a number of proteins, including kinases related to protein kinase C. In addition, in yeast, Rho1p controls the activity of a MAPK cascade involved in bud formation. Considering this diversity of target molecules for small GTP-binding proteins, their likely tissue specific distribution, and the potential role for Rho in signaling to a kinase cascade, we decided to extend our initial analysis, exploring the ability of Ras and Rho-related GTP-binding proteins to activate MAPK or JNK in a variety of cell lines. We found that in the human kidney epithelial cell line, 293T, Cdc42 and all Rho proteins, RhoA, RhoB, and RhoC, but not Rac or Ras can induce activation of JNK. Furthermore, we provide evidence that signaling from Rho proteins to JNK in 293T cells does not involve Pak1. Taken together these findings demonstrate that Rho signals to JNK in a cell type-specific manner and suggest the existence of a novel, Pak1-independent signaling route communicating the Rho family of small GTP-binding proteins to the JNK pathway.

A C-terminal mutant of the G protein beta subunit deficient in the activation of phospholipase C-beta.

The molecular mechanism by which the G protein betagamma complex modulates multiple mammalian effector pathways is unknown. Homolog-scanning mutagenesis of the G protein beta subunit was employed to identify residues critical for the activation of phospholipase C-beta2 (PLC-beta2). A series of chimeras was made by introducing small segments of the Dictyostelium beta subunit into a background of mammalian beta1 and tested in COS cell cotransfection assays for their ability to activate PLC-beta2 and assemble with mammalian gamma2. A chimera that contained four Dictyostelium beta substitutions within the C-terminal 14 residues was unable to activate PLC-beta2 when cotransfected with gamma, despite its demonstrable expression in a gamma-dependent manner. Cotransfection of the mutant blocked m2 muscarinic receptor activation of PLC by a pertussis toxin-sensitive pathway. This C-terminal mutant retained the ability, however, to stimulate the mitogen-activated protein kinase pathway. These results imply that activation of different betagamma-responsive effectors is mediated by distinct domains.

Rac-1 dependent stimulation of the JNK/SAPK signaling pathway by Vav.

The protein product of the human vav oncogene, Vav exhibits a number of structural motifs suggestive of a role in signal transduction pathways, including a leucine-rich region, a plekstrin homology (PH) domain, a cysteine-rich domain, two SH3 regions, an SH2 domain, and a central Dbl homology (DH) domain. However, the transforming pathway(s) activated by Vav has not yet been elucidated. Interestingly, DH domains are frequently found in guanine nucleotide-exchange factors for small GTP-binding proteins of the Ras and Rho families, and it has been recently shown that, whereas Ras controls the activation of mitogen activated kinases (MAPKs), two members of the Rho family of small GTPases, Rac 1 and Cdc42, regulate activity of stress activated protein kinases (SAPKs), also termed c-jun N-terminal kinases (JNKs). The structural similarity between Vav and other guanine nucleotide exchange factors for small GTP-binding proteins, together with the recent identification of biochemical routes specific for members of the Ras and Rho family of GTPases, prompted us to explore whether MAPK or JNK are downstream components of the Vav signaling pathways. Using the COS-7 cell transient expression system, we have found that neither Vav nor the product of the vav proto-oncogene, proto-Vav, can enhance the enzymatic activity of a coexpressed, epitope tagged MAPK. On the other hand, we have observed that, whereas proto-Vav can slightly elevate JNK/SAPK activity, oncogenic Vav potently activates JNK/SAPK to an extent comparable to that elicited by two guanine-nucleotide exchange factors for Rho family members, Dbl and Ost. We also show that point mutations in conserved residues within the cysteine rich and DH domains of Vav both prevent its ability to activate JNK/SAPK and render Vav oncogenically inactive. In addition, we found that coexpression of the Rac-1 N17 dominant inhibitory mutant dramatically diminishes JNK/SAPK stimulation by Vav, as well as reduces the focus-forming ability of Vav in NIH3T3 murine fibroblasts. Taken together, these findings provide the first evidence that Rac-1 and JNK are integral components of the Vav signaling pathway.

Suppression of ceramide-mediated programmed cell death by sphingosine-1-phosphate.

Ceramide is an important regulatory participant of programmed cell death (apoptosis) induced by tumour-necrosis factor (TNF)-alpha and Fas ligand, members of the TNF superfamily. Conversely, sphingosine and sphingosine-1-phosphate, which are metabolites of ceramide, induce mitogenesis and have been implicated as second messengers in cellular proliferation induced by platelet-derived growth factor and serum. Here we report that sphingosine-1-phosphate prevents the appearance of the key features of apoptosis, namely intranucleosomal DNA fragmentation and morphological changes, which result from increased concentrations of ceramide. Furthermore, inhibition of ceramide-mediated apoptosis by activation of protein kinase C results from stimulation of sphingosine kinase and the concomitant increase in intracellular sphingosine-1-phosphate. Finally sphingosine-1-phosphate not only stimulates the extracellular signal-regulated kinase (ERK) pathway, it counteracts the ceramide-induced activation of stress-activated protein kinase (SAPK/JNK). Thus, the balance between the intracellular levels of ceramide and sphingosine-1-phosphate and their regulatory effects on different family members of mitogen-activated protein kinases determines the fate of the cell.

The PH domain of Ras-GAP is sufficient for in vitro binding to beta gamma subunits of heterotrimeric G proteins.

1. The noncatalytic domain of Ras-GAP can affect signaling through G protein-coupled receptors by a poorly understood mechanism. 2. In this study, fusion proteins containing elements of the noncatalytic domain of ras-GAP were examined for their ability to bind beta gamma subunits of heterotrimeric G proteins and phosphotyrosine-containing polypeptides. 3. Our results demonstrate that purified beta gamma dimers associated with bacterially expressed GAP proteins and that this association does not require SH2 or SH3 domains but is dependent on the presence of the GAP pleckstrin-homology (PH) domain. In contrast, only the SH2 domains are necessary for binding to tyrosine phosphorylated proteins. 4. These findings raise the possibility that heterotrimeric G proteins might affect functioning of ras-like proteins through beta gamma subunits acting on their regulatory molecules.

Signaling from G protein-coupled receptors to c-Jun kinase involves beta gamma subunits of heterotrimeric G proteins acting on a Ras and Rac1-dependent pathway.

Stimulation of a variety of cell surface receptors enhances the enzymatic activity of mitogen-activated protein kinases (MAPKs). MAPKs have been classified in three subfamilies: extracellular signal-regulated kinases (ERKs), stress-activated protein kinases or c-Jun NH2-terminal kinases (SAPKs/JNKs), and p38 kinase. Whereas the pathway linking cell surface receptors to ERKs has been partially elucidated, the mechanism of activation of JNKs is still poorly understood. Recently, we have shown that stimulation of G protein-coupled receptors can effectively induce JNK in NIH 3T3 cells (Coso, O. A., Chiariello, M., Kalinec, G., Kyriakis, J. M., Woodgett, J., and Gutkind, J. S. (1995) J. Biol. Chem. 270, 5620-5624). In the present study, we have used the transient expression in COS-7 cells of m1 and m2 muscarinic receptors (mAChRs) as a model system to study the signaling pathway linking G protein-coupled receptors to JNK. We show that stimulation of either muscarinic receptor subtype leads to JNK activation; however, this effect was not mimicked by expression of activated forms of alphas, alphai2, alphaq, or alpha13 G protein alpha subunits. In contrast, overexpression of Gbetagamma subunits potently induced JNK activity. Furthermore, we show that signaling from m1 and m2 mAChRs to JNK involves betagamma subunits of heterotrimeric G proteins, acting on a Ras and Rac1-dependent pathway.

Integrin function: molecular hierarchies of cytoskeletal and signaling molecules.

Integrin receptors play important roles in organizing the actin-containing cytoskeleton and in signal transduction from the extracellular matrix. The initial steps in integrin function can be analyzed experimentally using beads coated with ligands or anti-integrin antibodies to trigger rapid focal transmembrane responses. A hierarchy of transmembrane actions was identified in this study. Simple integrin aggregation triggered localized transmembrane accumulation of 20 signal transduction molecules, including RhoA, Rac1, Ras, Raf, MEK, ERK, and JNK. In contrast, out of eight cytoskeletal molecules tested, only tensin coaccumulated. Integrin aggregation alone was also sufficient to induce rapid activation of the JNK pathway, with kinetics of activation different from those of ERK. The tyrosine kinase inhibitors herbimycin A or genistein blocked both the accumulation of 19 out of 20 signal transduction molecules and JNK- and ERK-mediated signaling. Cytochalasin D had identical effects, whereas three other tyrosine kinase inhibitors did not. The sole exception among signaling molecules was the kinase pp125FAK which continued to coaggregate with alpha 5 beta 1 integrins even in the presence of these inhibitors. Tyrosine kinase inhibition also failed to block the ability of ligand occupancy plus integrin aggregation to trigger transmembrane accumulation of the three cytoskeletal molecules talin, alpha-actinin, and vinculin; these molecules accumulated even in the presence of cytochalasin D. However, it was necessary to fulfill all four conditions, i.e., integrin aggregation, integrin occupancy, tyrosine kinase activity, and actin cytoskeletal integrity, to achieve integrin-mediated focal accumulation of other cytoskeletal molecules including F-actin and paxillin. Integrins therefore mediate a transmembrane hierarchy of molecular responses.

The small GTP-binding proteins Rac1 and Cdc42 regulate the activity of the JNK/SAPK signaling pathway.

c-Jun amino-terminal kinases (JNKs) and mitogen-activated protein kinases (MAPKs) are closely related; however, they are independently regulated by a variety of environmental stimuli. Although molecules linking growth factor receptors to MAPKs have been recently identified, little is known about pathways controlling JNK activation. Here, we show that in COS-7 cells, activated Ras effectively stimulates MAPK but poorly induces JNK activity. In contrast, mutationally activated Rac1 and Cdc42 GTPases potently activate JNK without affecting MAPK, and oncogenic guanine nucleotide exchange factors for these Rho-like proteins selectively stimulate JNK activity. Furthermore, expression of inhibitory molecules for Rho-related GTPases and dominant negative mutants of Rac1 and Cdc42 block JNK activation by oncogenic exchange factors or after induction by inflammatory cytokines and growth factors. Taken together, these findings strongly support a critical role for Rac1 and Cdc42 in controlling the JNK signaling pathway.