Neuronal nuclear organization is controlled by cyclin-dependent kinase 5 phosphorylation of Ras Guanine nucleotide releasing factor-1

RasGRF1 is a member of the Ras guanine nucleotide exchange factor (RasGEF) family of proteins which are directly responsible for the activation of Ras and Rac GTPases. Originally identified as a phosphoprotein, RasGRF1 has been shown to be phosphorylated by protein kinase A and more recently, by the non-receptor tyrosine kinases Ack1 and Src. In this report we show that RasGRF1 interacts with and is phosphorylated by Cdk5 on serine 731 to regulate its steady state levels in mammalian cells as well as in neurons. Phosphorylation on this site by Cdk5 leads to RasGRF1 degradation through a calpain-dependent mechanism. Additionally, cortical neurons from Cdk5 knockout mice have higher levels of RasGRF1 which are reduced when wild-type Cdk5 is transfected into these neurons. In mitotic cells, nuclei become disorganized when RasGRF1 is overexpressed and this is rescued when RasGRF1 is co-expressed with active Cdk5. When RasGRF1 levels are elevated in neurons through overexpression of either the wild-type RasGRF1, or the phosphorylation mutant of RasGRF1 and by the transfection of a dominant negative Cdk5 construct, nuclei appeared condensed and fragmented. On the other hand, a reduction of RasGRF1 levels through p35/Cdk5 overexpression also leads to nuclear condensation in neurons. These data show that phosphorylation of RasGRF1 by Cdk5 tightly regulates its levels, which is essential for proper cellular organization.     

Tiam1 as a Signaling Mediator of Nerve Growth Factor-Dependent Neurite Outgrowth

Nerve Growth Factor (NGF)-induced neuronal differentiation requires the activation of members of the Rho family of small GTPases. However, the molecular mechanisms through which NGF regulates cytoskeletal changes and neurite outgrowth are not totally understood. In this work, we identify the Rac1-specific guanine exchange factor (GEF) Tiam1 as a novel mediator of NGF/TrkA-dependent neurite elongation. In particular, we report that knockdown of Tiam1 causes a significant reduction in Rac1 activity and neurite outgrowth induced by NGF. Physical interaction between Tiam1 and active Ras (Ras-GTP), but not tyrosine phosphorylation of Tiam1, plays a central role in Rac1 activation by NGF. In addition, our findings indicate that Ras is required to associate Tiam1 with Rac1 and promote Rac1 activation upon NGF stimulation. Taken together, these findings define a novel molecular mechanism through which Tiam1 mediates TrkA signaling and neurite outgrowth induced by NGF.     

RasGRF suppresses Cdc42-mediated tumour cell movement, cytoskeletal dynamics and transformation

Individual tumour cells move in three-dimensional environments with either a rounded or an elongated 'mesenchymal' morphology. These two modes of movement are tightly regulated by Rho family GTPases: elongated movement requires activation of Rac1, whereas rounded/amoeboid movement engages specific Cdc42 and Rho signalling pathways. In siRNA screens targeting the genes encoding guanine nucleotide exchange factors (GEFs), we found that the Ras GEF RasGRF2 regulates conversion between elongated- and rounded-type movement. RasGRF2 suppresses rounded movement by inhibiting the activation of Cdc42 independently of its capacity to activate Ras. RasGRF2 and RasGRF1 directly bind to Cdc42, outcompeting Cdc42 GEFs, thereby preventing Cdc42 activation. By this mechanism, RasGRFs regulate other Cdc42-mediated cellular processes such as the formation of actin spikes, transformation and invasion in vitro and in vivo. These results demonstrate a role for RasGRF GEFs as negative regulators of Cdc42 activation.     

The guanine nucleotide exchange factor RasGRF1 directly binds microtubules via DHPH2-mediated interaction

RasGRF is a family of guanine nucleotide exchange factors with dual specificity for both Ras and Rac GTPases. In this study, using mouse brain extracts, we show that both RasGRF1 and RasGRF2 interact with microtubules in an in vitro microtubule assembly system and this binding is very tight. To characterize this association, recombinant purified proteins containing different regions of RasGRF1 were tested for their ability to bind microtubules preassembled from pure tubulin. Only the DHPH2 tandem directly associates with microtubules, whereas the isolated DH or PH2 domains do not, indicating that the entire DHPH2 region is required for this association. The interaction occurs with high affinity (Kd approximately = 2 microM) and with a stoichiometry, at saturating conditions, of one DHPH2 molecule for two tubulin dimers. Competition experiments support the hypothesis that the DHPH2 module is largely responsible for RasGRF1-microtubule interaction. In vivo colocalization of RasGRF1 and microtubules was also observed by fluorescence confocal microscopy in nonneuronal cells after stimulation with an oxidative stress agent and in highly differentiated neuron-like cells. Identification of microtubules as new binding partners of RasGRF1 may help to elucidate the signaling network in which RasGRF1 is involved.     

RasGRF1 mediates brain-derived neurotrophic factor-induced axonal growth in primary cultured cortical neurons

The appropriate development and regulation of neuronal morphology are important to establish functional neuronal circuits and enable higher brain function of the central nervous system. R-Ras, a member of the Ras family of small GTPases, plays crucial roles in the regulation of axonal morphology, including outgrowth, branching, and guidance. GTP-bound activated R-Ras reorganizes actin filaments and microtubules through interactions with its downstream effectors, leading to the precise control of axonal morphology. However, little is known about the upstream regulatory mechanisms for R-Ras activation in neurons. In this study, we found that brain-derived neurotrophic factor (BDNF) has a positive effect on endogenous R-Ras activation and promotes R-Ras-mediated axonal growth. RNA interference knockdown and overexpression experiments revealed that RasGRF1, a guanine nucleotide exchange factor (GEF) for R-Ras, is involved in BDNF-induced R-Ras activation and the promotion of axonal growth. Phosphorylation of RasGRF1 by protein kinase A at Ser916/898 is needed for the full activation of its GEF activity and to facilitate Ras signaling. We observed that BDNF treatment markedly increased this phosphorylation. Our results suggest that BDNF is one of the critical extrinsic regulators for R-Ras activation, and that RasGRF1 is an intrinsic key mediator for BDNF-induced R-Ras activation and R-Ras-mediated axonal morphological regulation.     

Clusterin interacts with SCLIP (SCG10-like protein) and promotes neurite outgrowth of PC12 cells

Clusterin has been known as a chaperone-like molecule capable of interacting with various proteins. In this study, we show that clusterin interacts with the microtubule-destabilizing stathmin family protein SCLIP by GST pull-down and co-immunoprecipitation assays. Interestingly, SCLIP interacts with 80 kDa mature form of clusterin in the cytosolic fraction of PC12 cells permeabilized by low concentration of a weak nonionic detergent digitonin, but not with intracellular variants of clusterin known as binding isoforms of Ku70 or TGF-beta receptors. Both clusterin and SCLIP are co-localized at the perinuclear region and growth cone of PC12 cells. In addition, we show that the minimal domains for the interaction are mapped to the C-terminal valine-rich region (367-447) of clusterin and the N-terminal palmitoylation and membrane attachment site (1-34) of SCLIP. Finally, we demonstrate that ectopic expression of clusterin in PC12 cells elongates neurite-formation triggered by NGF and induces spontaneous neurite outgrowth even in the absence of NGF. Taken together, these results suggest that the clusterin interacts with SCLIP and the interaction may act as an important modulator during neuronal differentiation.     

Structural and Spatial Determinants Regulating TC21 Activation by RasGRF Family Nucleotide Exchange Factors

RasGRF family guanine nucleotide exchange factors (GEFs) promote guanosine diphosphate (GDP)/guanosine triphosphate (GTP) exchange on several Ras GTPases, including H-Ras and TC21. Although the mechanisms controlling RasGRF function as an H-Ras exchange factor are relatively well characterized, little is known about how TC21 activation is regulated. Here, we have studied the structural and spatial requirements involved in RasGRF 1/2 exchange activity on TC21. We show that RasGRF GEFs can activate TC21 in all of its sublocalizations except at the Golgi complex. We also demonstrate that TC21 susceptibility to activation by RasGRF GEFs depends on its posttranslational modifications: farnesylated TC21 can be activated by both RasGRF1 and RasGRF2, whereas geranylgeranylated TC21 is unresponsive to RasGRF2. Importantly, we show that RasGRF GEFs ability to catalyze exchange on farnesylated TC21 resides in its pleckstrin homology 1 domain, by a mechanism independent of localization and of its ability to associate to membranes. Finally, our data indicate that Cdc42-GDP can inhibit TC21 activation by RasGRF GEFs, demonstrating that Cdc42 negatively affects the functions of RasGRF GEFs irrespective of the GTPase being targeted.     

The Ras-GRF1 exchange factor coordinates activation of H-Ras and Rac1 to control neuronal morphology

The Ras-GRF1 exchange factor has regulated guanine nucleotide exchange factor (GEF) activity for H-Ras and Rac1 through separate domains. Both H-Ras and Rac1 activation have been linked to synaptic plasticity and thus could contribute to the function of Ras-GRF1 in neuronal signal transduction pathways that underlie learning and memory. We defined the effects of Ras-GRF1 and truncation mutants that include only one of its GEF activities on the morphology of PC12 phaeochromocytoma cells. Ras-GRF1 required coexpression of H-Ras to induce morphological effects. Ras-GRF1 plus H-Ras induced a novel, expanded morphology in PC12 cells, which was characterized by a 10-fold increase in soma size and by neurite extension. A truncation mutant of Ras-GRF1 that included the Ras GEF domain, GRFdeltaN, plus H-Ras produced neurite extensions, but did not expand the soma. This neurite extension was blocked by inhibition of MAP kinase activation, but was independent of dominant-negative Rac1 or RhoA. A truncation mutant of Ras-GRF1 that included the Rac GEF domains, GRFdeltaC, produced the expanded phenotype in cotransfections with H-Ras. Cell expansion was inhibited by wortmannin or dominant-negative forms of Rac1 or Akt. GRFdeltaC binds H-Ras.GTP in both pulldown assays from bacterial lysates and by coimmunoprecipitation from HEK293 cells. These results suggest that coordinated activation of H-Ras and Rac1 by Ras-GRF1 may be a significant controller of neuronal cell size.     

The requirement of Ras and Rap1 for the activation of ERKs by cAMP, PACAP, and KCl in cerebellar granule cells

In cerebellar granule cells, the mitogen-activated protein kinase (MAPK) or extracellular signal-regulated kinase (ERK) cascade mediates multiple functions, including proliferation, differentiation, and survival. In these cells, ERKs are activated by diverse stimuli, including cyclic adenosine monophosphate (cAMP), pituitary adenylate cyclase activating protein (PACAP), depolarization induced by elevated extracellular potassium (KCl), and the neurotrophin brain-derived neurotrophic factor. Extensive studies in neuronal cell lines have implicated the small G proteins Ras and Rap1 in the activation of ERKs by cAMP, PACAP, and KCl. However, the requirement of Ras and Rap1 in these pathways in cerebellar granule cells has not been addressed. In this study, we utilize multiple biochemical assays to determine the mechanisms of action and requirement of Ras and Rap1 in cultured cerebellar granule cells. We show that both Ras and Rap1 can be activated by cAMP or PACAP via protein kinase (PKA)-dependent mechanisms. KCl activation of Ras also required PKA. Using both adenoviral and transgenic approaches, we show that Ras plays a major role in ERK activation by cAMP, PACAP, and KCl, while Rap1 also mediates activation of a selective membrane-associated pool of ERKs. Furthermore, Rap1, but not Ras, activation by PKA appears to require the action of Src family kinases.     

Novel RasGRF1-derived Tat-fused peptides inhibiting Ras-dependent proliferation and migration in mouse and human cancer cells

Mutations of RAS genes are critical events in the pathogenesis of different human tumors and Ras proteins represent a major clinical target for the development of specific inhibitors to use as anticancer agents. Here we present RasGRF1-derived peptides displaying both in vitro and in vivo Ras inhibitory properties. These peptides were designed on the basis of the down-sizing of dominant negative full-length RasGRF1 mutants. The over-expression of these peptides can revert the phenotype of K-RAS transformed mouse fibroblasts to wild type, as monitored by several independent biological readouts, including Ras-GTP intracellular levels, ERK activity, morphology, proliferative potential and anchorage independent growth. Fusion of the RasGRF1-derived peptides with the Tat protein transduction domain allows their uptake into mammalian cells. Chemically synthesized Tat-fused peptides, reduced to as small as 30 residues on the basis of structural constraints, retain Ras inhibitory activity. These small peptides interfere in vitro with the GEF catalyzed nucleotide dissociation and exchange on Ras, reduce cell proliferation of K-RAS transformed mouse fibroblasts, and strongly reduce Ras-dependent IGF-I-induced migration and invasion of human bladder cancer cells. These results support the use of RasGRF1-derived peptides as model compounds for the development of Ras inhibitory anticancer agents.     

Vav mediates Ras stimulation by direct activation of the GDP/GTP exchange factor Ras GRP1

Here we describe a new signaling cross-talk between the Vav/Rac1 and Ras pathways that is established through the stimulation of RasGRP1, an exchange factor for Ras subfamily GTPases. This interaction is crucial for Ras activation in lymphoid cells, since this GTPase cannot become activated in the absence of Vav proteins. The activation of RasGRP1 requires both the generation of diacylglycerol via phospho lipase C-gamma and the induction of actin polymerization, two responses induced by Vav and Rac1 that facilitate the translocation of RasGRP1 to juxtamembrane areas of the cell. Consistent with this, the cross-talk can be activated by tyrosine-phosphorylated wild-type Vav, oncogenic Vav and constitutively active Rac1. Conversely, Ras activation can be blocked in lymphocytes and ectopic systems using inhibitors affecting either phospholipase C-gamma or F-actin polymerization. These results indicate that a relay mechanism exists in lymphoid and other cells helping in the generation of robust signaling responses by the Rac/Rho and Ras pathways upon receptor engagement.     

Leukemia Inhibitory Factor and Ciliary Neurotrophic Factor Increase Activated Ras in a Neuroblastoma Cell Line and in Sympathetic Neuron Cultures

Abstract: The cytokines leukemia inhibitory factor (LIF) and ciliary neurotrophic factor (CNTF) have been implicated in determination of neuronal phenotype as well as promotion of neuronal survival. However, the intracellular mechanisms by which their signals are transduced remain poorly understood. We have previously studied the regulation of vasoactive intestinal polypeptide gene expression by LIF and CNTF in the NBFL neuroblastoma cell line. Because these cytokines induce tyrosine phosphorylation that may lead to Ras activation, we explored a possible role for Ras in LIF- and CNTF-induced signal transduction. In NBFL cells LIF increases activated Ras in a rapid, transient, and concentration-dependent manner. CNTF and a related cytokine, oncostatin M, produce similar increases. CNTF and LIF also increase activated Ras in neuron-enriched dissociated cultures of sympathetic ganglia. Moreover, these cytokines rapidly and transiently induce specific tyrosine-phosphorylated proteins, p165 and p195. The protein kinase inhibitors K252a and staurosporine block LIF-induced increases in tyrosine phosphorylation, activated Ras, and vasoactive intestinal polypeptide mRNA in NBFL cells. These data support a possible role for Ras in the cell differentiation effects of LIF and CNTF.     

Protein kinase C (PKC) betaII induces cell invasion through a Ras/Mek-, PKC iota/Rac 1-dependent signaling pathway

Protein kinase C betaII (PKCbetaII) promotes colon carcinogenesis. Expression of PKCbetaII in the colon of transgenic mice induces hyperproliferation and increased susceptibility to colon cancer. To determine molecular mechanisms by which PKCbetaII promotes colon cancer, we established rat intestinal epithelial (RIE) cells stably expressing PKCbetaII. Here we show that RIE/PKCbetaII cells acquire an invasive phenotype that is blocked by the PKCbeta inhibitor LY379196. Invasion is not observed in RIE cells expressing a kinase-deficient PKCbetaII, indicating that PKCbetaII activity is required for the invasive phenotype. PKCbetaII induces activation of K-Ras and the Ras effector, Rac1, in RIE/PKCbetaII cells. PKCbetaII-mediated invasion is blocked by the Mek inhibitor, U0126, and by expression of either dominant negative Rac1 or kinase-deficient atypical PKCiota. Expression of constitutively active Rac1 induces Mek activation and invasion in RIE cells, indicating that Rac1 is the critical downstream effector of PKCbetaII-mediated invasion. Taken together, our results define a novel PKCbetaII --> Ras --> PKCiota /Rac1 --> Mek signaling pathway that induces invasion in intestinal epithelial cells. This pathway provides a plausible mechanism by which PKCbetaII promotes colon carcinogenesis.     

Regulation of the protein kinase Raf-1 by oncogenic Ras through phosphatidylinositol 3-kinase, Cdc42/Rac and Pak

Activation of the protein kinase Raf-1 is a complex process involving association with the GTP-bound form of Ras (Ras-GTP), membrane translocation and both serine/threonine and tyrosine phosphorylation (reviewed in [1]). We have reported previously that p21-activated kinase 3 (Pak3) upregulates Raf-1 through direct phosphorylation on Ser338 [2]. Here, we investigated the origin of the signal for Pak-mediated Raf-1 activation by examining the role of the small GTPase Cdc42, Rac and Ras, and of phosphatidylinositol (PI) 3-kinase. Pak3 acted synergistically with either Cdc42V12 or Rac1V12 to stimulate the activities of Raf-1, Raf-CX, a membrane-localized Raf-1 mutant, and Raf-1 mutants defective in Ras binding. Raf-1 mutants defective in Ras binding were also readily activated by RasV12. This indirect activation of Raf-1 by Ras was blocked by a dominant-negative mutant of Pak, implicating an alternative Ras effector pathway in Pak-mediated Raf-1 activation. Subsequently, we show that Pak-mediated Raf-1 activation is upregulated by both RasV12C40, a selective activator of PI 3-kinase, and p110-CX, a constitutively active PI 3-kinase. In addition, p85Delta, a mutant of the PI 3-kinase regulatory subunit, inhibited the stimulated activity of Raf-1. Pharmacological inhibitors of PI 3-kinase also blocked both activation and Ser338 phosphorylation of Raf-1 induced by epidermal growth factor (EGF). Thus, Raf-1 activation by Ras is achieved through a combination of both physical interaction and indirect mechanisms involving the activation of a second Ras effector, PI 3-kinase, which directs Pak-mediated regulatory phosphorylation of Raf-1.     

Regulatory proteins of R-Ras, TC21/R-Ras2, and M-Ras/R-Ras3

We studied the regulation of three closely related members of Ras family G proteins, R-Ras, TC21 (also known as R-Ras2), and M-Ras (R-Ras3). Guanine nucleotide exchange of R-Ras and TC21 was promoted by RasGRF, C3G, CalDAG-GEFI, CalDAG-GEFII (RasGRP), and CalDAG-GEFIII both in 293T cells and in vitro. By contrast, guanine nucleotide exchange of M-Ras was promoted by the guanine nucleotide exchange factors (GEFs) for the classical Ras (Ha-, K-, and N-), including mSos, RasGRF, CalDAG-GEFII, and CalDAG-GEFIII. GTPase-activating proteins (GAPs) for Ras, Gap1(m), p120 GAP, and NF-1 stimulated all of the R-Ras, TC21, and M-Ras proteins, whereas R-Ras GAP stimulated R-Ras and TC21 but not M-Ras. We did not find any remarkable difference in the subcellular localization of R-Ras, TC21, or M-Ras when these were expressed with a green fluorescent protein tag in 293T cells and MDCK cells. In conclusion, TC21 and R-Ras were regulated by the same GEFs and GAPs, whereas M-Ras was regulated as the classical Ras.     

CIIA functions as a molecular switch for the Rac1-specific GEF activity of SOS1

Son of sevenless 1 (SOS1) is a dual guanine nucleotide exchange factor (GEF) that activates the guanosine triphosphatases Rac1 and Ras, which mediate signaling initiated by peptide growth factors. In this paper, we show that CIIA is a new binding partner of SOS1. CIIA promoted the SOS1-Rac1 interaction and inhibited the SOS1-Ras interaction. Furthermore, CIIA promoted the formation of an SOS1-EPS8 complex and SOS1-mediated Rac1 activation, whereas it inhibited SOS1-mediated activation of Ras. Transforming growth factor β (TGF-β) up-regulated the expression of CIIA and thereby promoted the association between CIIA and SOS1 in A549 human lung adenocarcinoma cells. Depletion of CIIA in these cells by ribonucleic acid interference inhibited the TGF-β-induced interaction between SOS1 and EPS8, activation of Rac1, and cell migration. Together, these results suggest that CIIA mediates the TGF-β-induced activation of SOS1-Rac1 signaling and cell migration in A549 cells. They further show that CIIA functions as a molecular switch for the GEF activity of SOS1, directing this activity toward Rac1.     

The Ras guanine nucleotide exchange factor RasGRF1 promotes matrix metalloproteinase-3 production in rheumatoid arthritis synovial tissue

Introduction  

Fibroblast-like synoviocytes (FLS) from rheumatoid arthritis (RA) patients share many similarities with transformed cancer cells, including spontaneous production of matrix metalloproteinases (MMPs). Altered or chronic activation of proto-oncogenic Ras family GTPases is thought to contribute to inflammation and joint destruction in RA, and abrogation of Ras family signaling is therapeutic in animal models of RA. Recently, expression and post-translational modification of Ras guanine nucleotide releasing factor 1 (RasGRF1) was found to contribute to spontaneous MMP production in melanoma cancer cells. Here, we examine the potential relationship between RasGRF1 expression and MMP production in RA, reactive arthritis, and inflammatory osteoarthritis synovial tissue and FLS.