Small GTPase Rin induces neurite outgrowth through Rac/Cdc42 and calmodulin in PC12 cells

The novel Ras-like small GTPase Rin is expressed prominently in adult neurons, and binds calmodulin (CaM) through its COOH-terminal–binding motif. It might be involved in calcium/CaM-mediated neuronal signaling, but Rin-mediated signal transduction pathways have not yet been elucidated. Here, we show that expression of Rin induces neurite outgrowth without nerve growth factor or mitogen-activated protein kinase activation in rat pheochromocytoma PC12 cells. Rin-induced neurite outgrowth was markedly inhibited by coexpression with dominant negative Rac/Cdc42 protein or CaM inhibitor treatment. We also found that expression of Rin elevated the endogenous Rac/Cdc42 activity. Rin mutant proteins, in which the mutation disrupted association with CaM, failed to induce neurite outgrowth irrespective of Rac/Cdc42 activation. Disruption of endogenous Rin function inhibited the neurite outgrowth stimulated by forskolin and extracellular calcium entry through voltage-dependent calcium channel evoked by KCl. These findings suggest that Rin-mediated neurite outgrowth signaling requires not only endogenous Rac/Cdc42 activation but also Rin–CaM association, and that endogenous Rin is involved in calcium/CaM-mediated neuronal signaling pathways.

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title: Structure and function of signal-transducing GTP-binding proteins.
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title: Increasing Complexity of the Ras Signaling Pathway*
abstract: Ras is a key regulator of cell growth in all eukaryotic cells. Genetic, biochemical, and molecular studies in Caenorhabditis elegans, Drosophila, and mammalian cells have positioned Ras centrally in signal transduction pathways that respond to diverse extracellular stimuli, including peptide growth factors, cytokines, and hormones. The biological activity of Ras is controlled by a regulated GDP/GTP cycle. Guanine nucleotide exchange factors (GEFs; RasGRF1/2 and Sos1/2) promote the formation of the active, GTP-bound form of Ras (1). GTPaseactivating proteins (GAPs; p120 GAP and NF1) accelerate the intrinsic GTP hydrolytic activity of Ras to promote formation of the inactive, GDP-bound form of Ras (1). Mutations in Ras at amino acids 12, 13, or 61 make Ras insensitive to GAP action and, hence, constitutively active in transforming mammalian cells (2, 3). These activating mutations in Ras are prevalent in a wide spectrum of human cancers. It has been estimated that 30% of all human tumors contain an activating mutation in Ras. The frequency of Ras mutations varies depending on tumor type, with the highest frequencies seen in lung, colon, thyroid, and pancreatic carcinomas (3). The frequency of Ras mutations is likely to be an underestimation of the contribution of aberrant signaling through the Ras pathway to human malignancies because chronic up-regulation of the Ras pathway can occur in the absence of mutations in Ras itself (4–6).

title: The Ras branch of small GTPases: Ras family members don't fall far from the tree.
abstract: The Ras branch of the Ras superfamily consists of small GTPases most closely related to Ras and include the R-Ras, Rap, Ral, Rheb, Rin and Rit proteins. Although our understanding of Ras signaling and biology is now considerable, recent observations suggest that Ras function is more complex than previously believed. First, the three Ras proteins may not be functionally identical. Second, Ras function involves functional cross-talk with their close relatives.

title: Rin, a Neuron-Specific and Calmodulin-Binding Small G-Protein, and Rit Define a Novel Subfamily of Ras Proteins
abstract: cDNAs encoding two novel 25 kDa Ras-like proteins, Rit and Rin, were isolated from mouse retina using a degenerate PCR-based cloning strategy. Using the expressed sequence tag database, human orthologs were also obtained and sequenced. The protein sequences of Rit and Rin, which are 64% identical, are more similar to each other than to any known Ras protein. Their closest homologs in the databases areMucor racemosus Ras2 and Ras3, to which they show ∼48% identity. Rit and Rin both bind GTP in vitro. An unusual feature of their structure is that they lack a known recognition signal for C-terminal lipidation, a modification that is generally necessary for plasma membrane association among the Ras subfamily of proteins. Nonetheless, transiently expressed Rit and Rin are plasma membrane-localized. Both proteins contain a C-terminal cluster of basic amino acids, which could provide a mechanism for membrane association. Deletion analysis suggested that this region is important for Rit membrane binding but is not necessary for Rin. Rit, like most Ras-related proteins, is ubiquitously expressed. Rin, however, is unusual in that it is expressed only in neurons. In addition, Rin binds calmodulin through a C-terminal binding motif. These results suggest that Rit and Rin define a novel subfamily of Ras-related proteins, perhaps using a new mechanism of membrane association, and that Rin may be involved in calcium-mediated signaling within neurons.

title: RIC, a calmodulin‐binding Ras‐like GTPase.
abstract: Neuronal activity dramatically increases the concentration of cytosolic Ca2+, which then serves as a second messenger to direct diverse cellular responses. Calmodulin is a primary mediator of Ca2+ signals in the nervous system. In a screen for calmodulin‐binding proteins, we identified RIC, a protein related to the Ras subfamily of small GTPases. In addition to the ability to bind calmodulin, a number of unique features distinguished RIC from other Ras‐like GTPases, including the absence of a signal for prenylation and a distinct effector (G2) domain. Furthermore, we describe two human proteins, RIN and RIT, which were 71% and 66% identical to RIC respectively, shared related G2 domains with RIC, and lacked prenylation signals, suggesting that the RIC family is conserved from flies to humans. While Ric and RIT were widely expressed, expression of RIN was confined to the neuron system.

title: Biochemical characterization of the Ras-related GTPases Rit and Rin.
abstract: We report the biochemical characterization of Rit and Rin, two members of the Ras superfamily identified by expression cloning. Recombinant Rit and Rin bind GTP and exhibit intrinsic GTPase activity. Conversion of Gln to Leu at position 79 (for Rit) or 78 (for Rin) (equivalent to position 61 in Ras) resulted in a complete loss of GTPase activity. Surprisingly, significant differences were found when the guanine nucleotide dissociation constants of Rit and Rin were compared with the majority of Ras-related GTPases. Both proteins display higher k(off) values for GTP than GDP in the presence of 10 mM Mg(2+). These GTP dissociation rates are 5- to 10-fold faster than most Ras-like GTPases. Despite these unique biochemical properties, our data support the notion that both Rit and Rin function as nucleotide-dependent molecular switches. To begin to address whether these proteins act as regulators of distinct signaling pathways, we examined their interaction with a series of known Ras-binding proteins by yeast two-hybrid analysis. Although Rit, Rin, and Ras have highly related effector domain sequences, Rit and Rin were found to interact with the known Ras binding proteins RalGDS, Rlf, and AF-6/Canoe but not with the Raf kinases, RIN1, or the p110 subunit of phosphatidylinositol 3-kinase. These interactions were GTP and effector domain dependent and suggest that RalGDS, Rlf, and AF-6 are Rit and Rin effectors. Their biochemical properties and interaction with a subset of known Ras effector proteins suggest that Rit and Rin may play important roles in the regulation of signaling pathways and cellular processes distinct from those controlled by Ras.

title: The Ras-like small GTP-binding protein Rin is activated by growth factor stimulation.
abstract: A novel 25 K Ras-like protein, Rin, binds calmodulin in a Ca2+-dependent manner and is considered to participate in a calcium/calmodulin-mediated signaling pathway. However, little is known about Rin signaling mechanism. Here we examined the signal transduction pathway through Rin protein using pull down assay system. When we stimulated Rin-expressed Cos-7 cells with ionomycin, phorbol 12-myristate 13-acetate or epidermal growth factor (EGF), Rin protein was rapidly activated. Moreover, cells cotransfected with Rin and mSos showed a similar Rin activation profile, and Rin protein was coimmunoprecipitated with mSos protein in vivo. When cells were cotransfected with Rin and Ras-GTPase-activating proteins, basal Rin activity was decreased. Association with Rin and calmodulin was potentiated after stimulation and Rin activation was regulated by both calcium ion and calmodulin. These studies suggest that Rin may be involved in EGF receptor and mSos-mediated signaling pathway and may participate in calcium/calmodulin-mediated cellular processes.

title: Establishment of a noradrenergic clonal line of rat adrenal pheochromocytoma cells which respond to nerve growth factor.
abstract: A single cell clonal line which responds reversibly to nerve growth factor (NGF) has been established from a transplantable rat adrenal pheochromocytoma. This line, designated PC12, has a homogeneous and near-diploid chromosome number of 40. By 1 week's exposure to NGF, PC12 cells cease to multiply and begin to extend branching varicose processes similar to those produced by sympathetic neurons in primary cell culture. By several weeks of exposure to NGF, the PC12 processes reach 500-1000 mum in length. Removal of NGF is followed by degeneration of processes within 24 hr and by resumption of cell multiplication within 72 hr. PC12 cells grown with or without NGF contain dense core chromaffin-like granules up to 350 nm in diameter. The NGF-treated cells also contain small vesicles which accumulate in process varicosities and endings. PC12 cells synthesize and store the catecholamine neurotransmitters dopamine and norepinephrine. The levels (per mg of protein) of catecholamines and of the their synthetic enzymes in PC12 cells are comparable to or higher than those found in rat adrenals. NGF-treatment of PC12 cells results in no change in the levels of catecholamines or of their synthetic enzymes when expressed on a per cell basis, but does result in a 4- to 6-fold decrease in levels when expressed on a per mg of protein basis. PC12 cells do not synthesize epinephrine and cannot be induced to do so by treatment with dexamethasone. The PC12 cell line should be a useful model system for neurobiological and neurochemical studies.

title: Neurotrophin signal transduction by the Trk receptor.
abstract: The initial event in the neuronal differentiation of PC12 cells is the binding of the neurotrophin nerve growth factor (NGF) to the Trk receptor. This interaction stimulates the intrinsic tyrosine kinase activity of Trk, initiating a signalling cascade involving the phosphorylation of intracellular proteins on tyrosine, serine, and threonine residues. These signals are then in turn propagated to other messengers, ultimately leading to differentiation, neurotrophin-dependent survival, and the loss of proliferative capacity. To transmit NGF signals, NGF-activated Trk rapidly associates with the cytoplasmic proteins, SHC, PI-3 kinase, and PLC-gamma 1. These proteins are involved in stimulating the formation of various second messenger molecules and activating the Ras signal transduction pathway. Studies with Trk mutants indicate that the activation of the Ras pathway is necessary for complete differentiation of PC12-derived cells and for the maintenance of the differentiated phenotype. Trk also induces the tyrosine phosphorylation of SNT, a specific target of neurotrophic factor activity in neuronal cells. This review will discuss the potential roles of Trk and the proteins of the Trk signalling pathways in NGF function, and summarize our attempts to understand the mechanisms used by Trk to generate the many phenotypic responses of PC12 cells to NGF.

title: Specificity of receptor tyrosine kinase signaling: Transient versus sustained extracellular signal-regulated kinase activation
abstract: A number of different intracellular signaling pathways have been shown to be activated by receptor tyrosine kinases. These activation events include the phosphoinositide 3-kinase, 70 kDa S6 kinase, mitogen-activated protein kinase (MAPK), phospholipase C-gamma, and the Jak/STAT pathways. The precise role of each of these pathways in cell signaling remains to be resolved, but studies on the differentiation of mammalian PC12 cells in tissue culture and the genetics of cell fate determination in Drosophila and Caenorhabditis suggest that the extracellular signal-regulated kinase (ERK-regulated) MAPK pathway may be sufficient for these cellular responses. Experiments with PC12 cells also suggest that the duration of ERK activation is critical for cell signaling decisions.

title: Regulation of Phosphorylation Pathways by p21 GTPases
abstract: The oncogenic Ras p21 GTPases regulate phosphorylation pathways that underlie a wealth of activities, including growth and differentiation, in organisms ranging from yeast to human. In metazoa, growth factors trigger conversion of Ras from an inactive GDP-bound form to an active GTP-bound form. This activation of Ras leads to activation of Raf. Raf is one of the initial kinases in the cytoplasmic mitogen-activated protein kinase (MAPK) cascade, involving extracellular-signal-regulated kinases (ERK), which culminates in nuclear transcription. The Ras-related subfamily of Rho p21s, including Rho, Rac and Cdc42 are similarly active in their GTP-bound forms. These p21s mediate growth-factor-induced morphological changes involving actin-based cellular structures. For example, in mammalian fibroblasts, Rho mediates the formation of cytoskeletal stress fibres induced by lysophosphatidic acid, while Rac mediates the formation of membrane ruffles induced by platelet-derived growth factor, and Cdc42 mediates the formation of peripheral filopodia by bradykinin. In some cases, factor-induced Rac activation results in Rho activation, and factor-induced Cdc42 activation leads to Rac activation, as determined by specific morphological changes. Although separate Cdc42/Rac and Rac/Rho hierarchies exist, these might not extend into a linear form (i.e. Cdc42→Rac→Rho) since Cdc42 and Rho activities may be competitive or even antagonistic. Thus Cdc42-mediated formation of filopodia is accompanied by loss of stress fibres (whose formation is mediated by Rho). Recently, mammalian kinases that bind to the GTP-bound forms of Rho p21s have been isolated. These kinases include the p21-activated serine/threonine kinase (PAK), which is stimulated by binding to Cdc42 and Rac, and the Rho-binding serine/threonine kinase (ROK), which is not as strongly stimulated by binding. These kinases act as effectors for their p21 partners since they can directly affect the reorganization of the relevant actin-containing structures. ROK promotes the formation of Rho-induced actin-containing stress fibres and focal-adhesion complexes, to which the ends of the stress fibres attach. PAK stimulates the disassembly of stress fibres, which has been shown to accompany formation of Cdc42-induced peripheral-actin-containing structures, including filopodia, which with Rac-induced membrane ruffles play a role in cell movement. PAK also fosters loss of focal-adhesion complexes. Thus, there is cooperation between different Rho p21s as well as antagonism, with their associated kinases having a role in the integration of the reorganization of the actin cytoskeleton. The similarity of PAK to the Saccharomyces cerevisiae kinase Ste20p, which initiates the yeast mating/pheromone MAPK cascade, led to experiments showing that Cdc42 regulates Ste20p in this MAPK pathway. This similarity has also led to the demonstration that mammalian Cdc42 and Rac can signal to the nucleus through MAPK pathways. However, c-Jun N-terminal kinase (JNK, stress-activated protein kinase) rather than ERK, is involved. PAK have been implicated in the JNK pathway, but their exact roles are uncertain. Thus members of the Rho subfamily, and kinases that bind to these p21s are intimately involved in immediate morphological processes as well as long-term transcriptional events.

title: Sequence motifs for calmodulin recognition
abstract: Calmodulin (CaM) is recognized as a major calcium sensor and orchestrator of regulatory events through its interaction with a diverse group of cellular proteins. Many investigations have focused on defining the region of interaction between CaM and its cellular targets and the action of CaM on target protein function. Because CaM can bind with high affinity to a relatively small α‐helical region of many proteins, success in clearly defining the essential elements of CaM binding motifs seems feasible and should provide a means of identifying CaM binding proteins. Three recognition motifs for CaM interaction are discussed in the context of experimental investigations of a variety of CaM target proteins. A modified version of the IQ motif as a consensus for Ca2+‐independent binding and two related motifs for Ca2+‐dependent binding, termed 18‐14 and 1‐5‐10 based on the position of conserved hydrophobic residues, are proposed. Although considerable sequence diversity is observed among the different binding regions, these three classes of recognition motifs exist for many of the known CaM binding proteins.—Rhoads, A. R., Friedberg, F. Sequence motifs for calmodulin recognition. FASEB J. 11, 331‐340 (1997)

title: Transcriptional and post-transcriptional effects of nerve growth factor on expression of the three neurofilament subunits in PC-12 cells.
abstract: Nerve growth factor (NGF) is known to increase the levels of neurofilament proteins in PC-12 pheochromocytoma cells. In this report, we show that the three neurofilament subunits, NF-L, NF-M, and NF-H, are not induced coordinately. NF-H accumulated only after longer term NGF treatment than required for NF-L and NF-M. While NGF treatment resulted in 12- and 14-fold increases in NF-L and NF-M mRNA levels, respectively, over a 14-day period, no increase in the level of NF-H mRNA was observed. This indicated that in PC-12 cells, control of NF-H expression by NGF may occur at the post-transcriptional level. NGF appeared to have no effect on the stability of NF-L mRNA, although it increased the stability of NF-M mRNA relative to that in control PC-12 cells. Analysis of the effect of NGF on the transcription of neurofilament genes showed 4- and 5-fold increases in the rates of NF-L and NF-M gene transcription, respectively, and no increase in the rate of NF-H gene transcription. Taken together these results demonstrate that NGF stimulates the expression of individual neurofilament subunits at the transcriptional and/or post-transcriptional levels.

title: Characterization of Rac and Cdc42 Activation in Chemoattractant-stimulated Human Neutrophils Using a Novel Assay for Active GTPases*
abstract: A major function of Rac2 in neutrophils is the regulation of oxidant production important in bacterial killing. Rac and the related GTPase Cdc42 also regulate the dynamics of the actin cytoskeleton, necessary for leukocyte chemotaxis and phagocytosis of microorganisms. Although these GTPases appear to be critical downstream components of chemoattractant receptor signaling in human neutrophils, the pathways involved in direct control of Rac/Cdc42 activation remain to be determined. We describe an assay that measures the formation of Rac-GTP and Cdc42-GTP based on their specific binding to the p21-binding domain of p21-activated kinase 1. A p21-binding domain glutathione S-transferase fusion protein specifically binds Rac and Cdc42 in their GTP-bound forms both in vitro and in cell samples. Binding is selective for Rac and Cdc42 versusRhoA. Using this assay, we investigated Rac and Cdc42 activation in neutrophils and differentiated HL-60 cells. The chemoattractant fMet-Leu-Phe and the phorbol ester phorbol myristate acetate stimulate formation of Rac-GTP and Cdc42-GTP with distinct time courses that parallel cell activation. We also show that the signaling pathways leading to Rac and Cdc42 activation in HL-60 cells involve G proteins sensitive to pertussis toxin, as well as tyrosine kinase and phosphatidylinositol 3-kinase activities.

title: Naphthalenesulfonamides as calmodulin antagonists.
abstract: Publisher Summary This chapter describes the biopharmacological basis of naphthalenesulfonamides as calmodulin (CaM) antagonists and presents guidelines for the application of these agents to various biological systems. A comparison is made in the chapter between naphthalenesulfonamides and phenothiazines as CaM antagonists. N-(6-Aminohexyl)-[3H]-5-chloro-l-naphthalenesulfonamide (W-7) binds to purified CaM from the bovin aorta or brain in the presence of 0.1 mM CaCl2 as determined by equilibrium binding techniques; however, there is no binding in the absence of calcium ion. W-7 binds to CaM at two distinctly different types of sites: a high-affinity binding site and a low-affinity binding site. The chapter discusses a study in which the actions of naphthalenesulfonamides as CaM antagonists were shown to depend both on the chlorination of the naphthalene ring and on the length of the alkyl chain. The derivative with bromine was the most potent CaM antagonist and derivatives with chlorine or fluorine were intermediate in potency. The affinity of naphthalenesulfonamide, determined by the displacement of [3H]W-7 from purified CaM using an equilibrium-binding technique and the inhibition of Ca2+-dependent cyclic nucleotide phosphodiesterase, correlated well with their potency in the relaxation of vascular strips and in the inhibition of platelet aggregation. The compound with cyanogen was the least potent. In addition, the affinity of naphthalenesulfonamide correlated well with their hydrophobicity, determined as octanol-water partition coefficients.

title: Accumulation of GTP-bound RhoA during Cytokinesis and a Critical Role of ECT2 in This Accumulation*
abstract: We developed a new pull-down assay for GTP-Rho and examined its level during cell cycle. HeLa cells were arrested in the S phase by thymidine and were enriched in the prometaphase, metaphase, telophase, and G1 phase by collecting at 0, 45, 90, and 180 min after the release from the nocodazole arrest, respectively. The level of GTP-Rho did not change significantly from the S phase to the prometaphase, but increased thereafter, peaking in the telophase, and returned to the original level in the G1phase. The GDP-GTP exchange activity for Rho measured in cell lysates in parallel increased also during the mitosis with a peak in the metaphase. Using this system, we examined a role of ECT2, an exchanger for Rho GTPases, suggested to be involved in cytokinesis (Tatsumoto, T., Xie, X., Blumenthal, R., Okamoto, I., and Miki., T. (1999)J. ). Expression of the dominant negative form of ECT2 completely suppressed both the rise of GTP-Rho in the telophase and the increased GDP-GTP exchange activity in the mitotic cell extracts. These results suggest a critical role of ECT2 in Rho activation during cytokinesis.

title: Rho GTPases regulate distinct aspects of dendritic arbor growth in Xenopus central neurons in vivo
abstract: The development and structural plasticity of dendritic arbors are governed by several factors, including synaptic activity, neurotrophins and other growth-regulating molecules. The signal transduction pathways leading to dendritic structural changes are unknown, but likely include cytoskeleton regulatory components. To test whether GTPases regulate dendritic arbor development, we collected time-lapse images of single optic tectal neurons in albino Xenopus tadpoles expressing dominant negative or constitutively active forms of Rac, Cdc42 or RhoA. Analysis of images collected at two-hour intervals over eight hours indicated that enhanced Rac activity selectively increased branch additions and retractions, as did Cdc42 to a lesser extent. Activation of endogenous RhoA decreased branch extension without affecting branch additions and retractions, whereas dominant-negative RhoA increased branch extension. Finally, we provide data suggesting that RhoA mediates the promotion of normal dendritic arbor development by NMDA receptor activation.

title: Small GTPases Rac and Rho in the Maintenance of Dendritic Spines and Branches in Hippocampal Pyramidal Neurons
abstract: The shape of dendritic trees and the density of dendritic spines can undergo significant changes during the life of a neuron. We report here the function of the small GTPases Rac and Rho in the maintenance of dendritic structures. Maturing pyramidal neurons in rat hippocampal slice culture were biolistically transfected with dominant GTPase mutants. We found that expression of dominant-negative Rac1 results in a progressive elimination of dendritic spines, whereas hyperactivation of RhoA causes a drastic simplification of dendritic branch patterns that is dependent on the activity of a downstream kinase ROCK. Our results suggest that Rac and Rho play distinct functions in regulating dendritic spines and branches and are vital for the maintenance and reorganization of dendritic structures in maturing neurons.

title: Depolarization maintains neurites and priming of PC12 cells after nerve growth factor withdrawal
abstract: In contrast to its actions on certain neural populations, membrane depolarization by elevated K+ promotes neither the survival nor the differentiation of PC12 cells. We therefore employed this model system to examine directly the actions of elevated K+ on neurites. Here we report that elevated K+ prevents the degeneration of neurites that occurs when NGF is withdrawn from PC12 cell cultures. This effect is inhibited by the L-type Ca2+ channel blockers verapamil and nitrendipine. Although depolarization preserves preexisting neurites, unlike NGF, it does not promote neurite elongation. In addition to neurite stabilization, elevated K+ also maintains NGF-deprived cells in a “primed” state in which they can rapidly regenerate neurites when re- treated with NGF. Elevated K+ alone has no priming effect, nor is it neuritogenic on either naive or NGF-pretreated cells. To probe the molecular basis for these actions of depolarization, we examined several cytoskeletal proteins whose phosphorylations (beta-tubulin, MAP 1.2/1B, and 64, 72 and 80 kDa chartins) or levels (MAP 1.2/1B and peripherin) are regulated by NGF in parallel with neurite outgrowth. Elevated K+ alone does not mimic these effects of NGF. In all cases, NGF withdrawal leads to the return of these proteins to levels characteristic of naive cells; in contrast, with the exception of the 80 kDa chartins, depolarization of NGF-deprived cultures maintained these proteins at or near their NGF-stimulated states. Similar observations were obtained with the NILE/L1 glycoprotein. These findings suggest that elevated K+ preserves priming and preexisting neurites by maintaining NGF-induced changes in cell composition. Our experiments invoke the possibility that elevation of intraneuronal Ca2+ may lead to selective stabilization of preexisting axons or dendrites in the intact nervous system, especially under circumstances in which the supply of neurotrophic factors is absent or limiting.

title: Stimulation of neurite outgrowth in PC12 cells by EGF and KCl depolarization: a Ca(2+)-independent phenomenon
abstract: MAP kinase activity is necessary for growth factor induction of neurite outgrowth in PC12 cells. Although NGF and EGF both stimulate MAP kinase activity, EGF does not stimulate neurite extension. We report that EGF, in combination with KCl, stimulates neurite outgrowth in PC12 cells. This phenomenon was independent of intracellular Ca2+ increases and not due to enhancement of MAP kinase activity over that seen with EGF alone. However, EGF plus KCl increased intracellular cAMP, and other cAMP elevating agents acted synergistically with EGF to promote neurite outgrowth. Stimulation of neurite outgrowth by cAMP and EGF was blocked by inhibitors of transcription suggesting that synergistic regulation of transcription by the cAMP and MAP kinase pathways may stimulate neurite growth.

title: Rit, a non-lipid-modified Ras-related protein, transforms NIH3T3 cells without activating the ERK, JNK, p38 MAPK or PI3K/Akt pathways
abstract: The biological functions of Rit (Ras-like protein in tissues) and Rin (Ras-like protein in neurons), members of a novel branch of Ras-related GTP-binding proteins that are ∼50% identical to Ras, have not been characterized. Therefore, we assessed their activity in growth control, transformation and signaling. NIH cells stably expressing a constitutively activated mutant of Rit [Rit(79L)] (analogous to the oncogenic mutant H-Ras(61L)) demonstrated strong growth transformation, proliferating rapidly in low serum and forming colonies in soft agar and tumors in nude mice. Although Rit(79L) alone did not promote morphologically transformed foci, it cooperated with both Raf and Rho A to form Rac/Rho-like foci. Rin [Rin(78L)] cooperated only with Raf. Rit(79L) but not Rin(78L) stimulated transcription from luciferase reporter constructs regulated by SRF, NF-κB, Elk-1 and Jun. However, neither activated ERK, JNK or p38, or PI3-K/Akt kinases in immune complex kinase assays. Interestingly, although Rit lacks any known recognition signal for C-terminal lipidation, Rit-transformed cell growth and survival in low serum is dependent on a farnesylated protein, as treatment with farnesyltransferase inhibitors caused apoptosis. Rin cooperated with Raf in focus assays but did not otherwise function in these assays, perhaps due to a lack of appropriate effector pathways in NIH3T3 fibroblasts for this neural-specific Ras family member. In summary, although Rit shares most core effector domain residues with Ras, our results suggest that Rit uses novel effector pathways to regulate proliferation and transformation.

title: Nerve Growth Factor-dependent Activation of the Small GTPase Rin*
abstract: The Rit and Rin proteins comprise a distinct and evolutionarily conserved subfamily of Ras-related small GTPases. Although we have defined a role for Rit-mediated signal transduction in the regulation of cell proliferation and transformation, the function of Rin remains largely unknown. Because we demonstrate that Rin is developmentally regulated and expressed in adult neurons, we examined its role in neuronal signaling. In this study, we show that stimulation of PC6 cells with either epidermal growth factor or nerve growth factor (NGF) results in rapid activation of Rin. This activation correlates with the onset of Ras activation, and dominant-negative Ras completely inhibits Rin activation induced by NGF. Further examination of Ras-mediated Rin activation suggests that this process is dependent upon neuronally expressed regulatory factors. Expression of mutationally activated H-Ras fails to activate Rin in non-neuronal cells, but results in potent stimulation of Rin-GTP levels in a variety of neuronal cell lines. Furthermore, although constitutively activated Rin does not induce neurite outgrowth on its own, both NGF-induced and oncogenic Ras-induced neurite outgrowth were inhibited by the expression of dominant-negative Rin. Together, these studies indicate that Rin activation is a direct downstream effect of growth factor-dependent signaling in neuronal cells and suggest that Rin may function to transduce signals within the mature nervous system.

title: 
 ras GTPase activating protein: Signal transmitter and signal terminator
abstract: Evidence that GAP is an effector of ras action can be summarized as follows: GAP interacts at a site on p21 defined genetically as the effector binding site. Regions of p21 that are nonessential for biological activity are nonessential for GAP interaction. GAP interacts with all known types of p21. (Upstream factors are expected to be specific for individual types). GAP interacts with p21 proteins (normal and mutant) in a GTP-dependent fashion. None of these constitute proof. It remains possible that GAP simply regulates p21-GTP levels, and binds to the same site as the true effector without transmitting a downstream signal. If indeed GAP mediates ras action, the question immediately arises as to the biochemical function of GAP itself. The requirement of ras proteins for membrane localization to exert their effects may be a valuable clue in the search for this function. Perhaps GAP is an enzyme (or is bound to an enzyme) that acts on membrane components in a p21-GTP-dependent manner and in doing so transmits signals to other downstream effectors. The ability of GAP to interact with many members of the ras family would allow many upstream signals to feed into this downstream pathway. Clearly, proof (or disproof) that GAP is downstream of ras is the next step toward clarification of this aspect of ras action; identification of biochemical activities associated with GAP (or the true ras effector) will, we hope, follow soon.

title: pEF-BOS, a powerful mammalian expression vector.
abstract: Polypeptide chain elongation factor l a (EF-la) is an eukaryotic counterpart of E. coli EF-Tu which promotes the GTP-dependent binding of an aminoacyl-tRNA to ribosomes. EF-la is one of the most abundant proteins in eukaryotic cells, and expressed in almost all kinds of mammalian cells. Recently, we have isolated human chromosomal gene coding for EF-la, and shown that the promoter of EF-la chromosomal gene very efficiently stimulates the in vitro transcription (1). In this report, we have constructed a powerful mammalian expression vector, pEF-BOS, using the promoter of human EF-la chromosomal gene. As shown in Fig. 1, pEF-BOS carries the SV40 replication origin (311 bp of EcoRH G fragment), the promoter region of human EF-la chromosomal gene (1.2 kb), the stuffer fragment (450 bp) from CDM8 vector (2) and poly(A) adenylation signal from human G-CSF cDNA (700 bp EcoSll ~ EcoRl DNA fragment) (3) in HindUL-EcoRl site of pUC119. The promoter region of EF-la gene is from nucleotide position 373 to 1561 (1) which includes 203 bp 5' flanking region, 33 bp first exon, 943 bp first intron and 10 bp of the part of the second exon located at 20 bp upstream of the ATG initiation codon. The size of pEFBOS is 5.8 kb, and the cDNA to be expressed can be inserted at BstXl site using BstXl adapter, or Xbal site using Xbal linker. Human G-CSF cDNA (4) was inserted into BstXl site of pEFBOS or CDM8, or into BamhU site of pKCR vector containing SV40 early promoter (5). As shown in Table 1, when these plasmids were transfected into COS cells by DEAEdextran/chloroquine method, the construct in pEF-BOS has directed the synthesis of human G-CSF about 20 times more efficiently than the construct in CDM8, and 50 ~ 200 times more efficiently than the construct in pKCR. In addition, when E. coli chloramphenicol acetyltransferase (CAT) gene was inserted into pEF-BOS, the CAT activities observed with pEF-BOS-CAT were 1.5 ~ 50 times higher than that of pSV2-CAT or pRSV-CAT after transfection into various cell lines including murine L929, human HeLa, CHU-2 and simian COS cells (Table 2). The pEFBOS vector, therefore, will be used to produce a large amount of growth factors and proteins in mammalian cells, to express a high level of anti-sense RNA. Furthermore, the pEF-BOS-CAT will be an ideal positive control for CAT assay in various cell types.

title: A Conserved Negative Regulatory Region in αPAK: Inhibition of PAK Kinases Reveals Their Morphological Roles Downstream of Cdc42 and Rac1
abstract: αPAK in a constitutively active form can exert morphological effects (E. Manser, H.-Y. Huang, T.-H. Loo, X.-Q. Chen, J.-M. Dong, T. Leung, and L. Lim,  1997) resembling those of Cdc42G12V. PAK family kinases, conserved from yeasts to humans, are directly activated by Cdc42 or Rac1 through interaction with a conserved N-terminal motif (corresponding to residues 71 to 137 in αPAK). αPAK mutants with substitutions in this motif that resulted in severely reduced Cdc42 binding can be recruited normally to Cdc42G12V-driven focal complexes. Mutation of residues in the C-terminal portion of the motif (residues 101 to 137), though not affecting Cdc42 binding, produced a constitutively active kinase, suggesting this to be a negative regulatory region. Indeed, a 67-residue polypeptide encoding αPAK83-149 potently inhibited GTPγS-bound Cdc42-mediated kinase activation of both αPAK and βPAK. Coexpression of this PAK inhibitor with Cdc42G12V prevented the formation of peripheral actin microspikes and associated loss of stress fibers normally induced by the p21. Coexpression of PAK inhibitor with Rac1G12V also prevented loss of stress fibers but not ruffling induced by the p21. Coexpression of αPAK83-149 completely blocked the phenotypic effects of hyperactive αPAKL107F in promoting dissolution of focal adhesions and actin stress fibers. These results, coupled with previous observations with constitutively active PAK, demonstrate that these kinases play an important role downstream of Cdc42 and Rac1 in cytoskeletal reorganization.

title: Sending signals from the synapse to the nucleus: Possible roles for CaMK, Ras/ERK, and SAPK pathways in the regulation of synaptic plasticity and neuronal growth
abstract: The ability to learn and form memories depends on specific patterns of synaptic activity and is in part transcription dependent. However, the signal transduction pathways that connect signals generated at synapses with transcriptional responses in the nucleus are not well understood. In the present report, we discuss three signal transduction pathways: the Ca2+/calmodulin‐dependent kinase (CaMK) pathway, the Ras/ERK pathway, and the SAPK pathways that might function to couple synaptic activity to long‐term adaptive responses, in part through the regulation of new gene expression. Evidence suggests that these pathways become activated in response to stimuli that regulate synaptic function such as the influx of extracellular Ca2+ and certain neurotrophin growth factors such as brain‐derived neurotrophic factor. Once activated, the CaMK, Ras/ERK, and SAPK pathways lead to the phosphorylation and activation of transcription factors in the nucleus such as the cyclic AMP response element binding protein (CREB). Genes regulated by CREB or other transcription factor targets of the CaMK, Ras/ERK, and SAPK pathways could mediate important adaptive responses to changes in synaptic activity such as changes in synaptic strength and the regulation of neuronal survival and death.  1999.

title: IQGAP1, a Rac- and Cdc42-binding Protein, Directly Binds and Cross-links Microfilaments
abstract: Activated forms of the GTPases, Rac and Cdc42, are known to stimulate formation of microfilament-rich lamellipodia and filopodia, respectively, but the underlying mechanisms have remained obscure. We now report the purification and characterization of a protein, IQGAP1, which is likely to mediate effects of these GTPases on microfilaments. Native IQGAP1 purified from bovine adrenal comprises two ∼190-kD subunits per molecule plus substoichiometric calmodulin. Purified IQGAP1 bound directly to F-actin and cross-linked the actin filaments into irregular, interconnected bundles that exhibited gel-like properties. Exogenous calmodulin partially inhibited binding of IQGAP1 to F-actin, and was more effective in the absence, than in the presence of calcium. Immunofluorescence microscopy demonstrated cytochalasin D–sensitive colocalization of IQGAP1 with cortical microfilaments. These results, in conjunction with prior evidence that IQGAP1 binds directly to activated Rac and Cdc42, suggest that IQGAP1 serves as a direct molecular link between these GTPases and the actin cytoskeleton, and that the actin-binding activity of IQGAP1 is regulated by calmodulin.

title: RHO GTPASES in neuronal morphogenesis
abstract: The Rho family of small GTPases act as intracellular molecular switches that transduce signals from extracellular stimuli to the actin cytoskeleton and the nucleus. Recent evidence implicates Rho GTPases in the regulation of neuronal morphogenesis, including migration, polarity, axon growth and guidance, dendrite elaboration and plasticity, and synapse formation. Signalling pathways from membrane receptors to Rho GTPases and from Rho GTPases to the actin cytoskeleton are beginning to be discovered. Mutations in these signalling pathways have been reported in human neurological diseases, which underscores their importance in the development and function of the nervous system.

title: Calcium-Stimulated Adenylyl Cyclase Activity Is Critical for Hippocampus-Dependent Long-Term Memory and Late Phase LTP
abstract: It is hypothesized that Ca2+ stimulation of calmodulin (CaM)-activated adenylyl cyclases (AC1 or AC8) generates cAMP signals critical for late phase LTP (L-LTP) and long-term memory (LTM). However, mice lacking either AC1 or AC8 exhibit normal L-LTP and LTM. Here, we report that mice lacking both enzymes (DKO) do not exhibit L-LTP or LTM. To determine if these defects are due to a loss of cAMP increases in the hippocampus, DKO mice were unilaterally cannulated to deliver forskolin. Administration of forskolin to area CA1 before training restored normal LTM. We conclude that Ca2+-stimulated adenylyl cyclase activity is essential for L-LTP and LTM and that AC1 or AC8 can produce the necessary cAMP signal.

title: Role of Ca2+-stimulated adenylyl cyclases in LTP and memory formation
abstract: Studies with invertebrates and vertebrates have strongly implicated the CREB/CRE transcriptional pathway in long‐term memory (LTM) and transcriptionally‐dependent L‐LTP. It is hypothesized that LTM and L‐LTP are both dependent upon a Ca2+ signal generated through activation of NMDA receptors. This review discusses evidence that Ca2+ signals generated through activation of NMDA receptors coactivate the Erk/MAP kinase and cAMP signal transduction pathways. It is hypothesized that activation of these two regulatory pathways increases the transcription of a family of genes through the CREB/CRE transcriptional pathway. Gene disruption studies have shown that Ca2+ activated adenylyl cyclases play a critical role in generating the cAMP signal required for LTM and L‐LTP. Although cAMP may be required for several events in this complex signal transduction cascade, one of the major roles of cAMP may be to support nuclear translocation of Erk/MAP kinase in hippocampal neurons.

title: Duplexes of 21-nucleotide RNAs mediate RNA interference in cultured mammalian cells
abstract: RNA interference (RNAi) is the process of sequence-specific, post-transcriptional gene silencing in animals and plants, initiated by double-stranded RNA (dsRNA) that is homologous in sequence to the silenced gene. The mediators of sequence-specific messenger RNA degradation are 21- and 22-nucleotide small interfering RNAs (siRNAs) generated by ribonuclease III cleavage from longer dsRNAs. Here we show that 21-nucleotide siRNA duplexes specifically suppress expression of endogenous and heterologous genes in different mammalian cell lines, including human embryonic kidney (293) and HeLa cells. Therefore, 21-nucleotide siRNA duplexes provide a new tool for studying gene function in mammalian cells and may eventually be used as gene-specific therapeutics.